Assessment of a novel multi-array normalization method based on spike-in control probes suitable for microRNA datasets with global decreases in expression.

PubMed

Sewer, Alain; Gubian, Sylvain; Kogel, Ulrike; Veljkovic, Emilija; Han, Wanjiang; Hengstermann, Arnd; Peitsch, Manuel C; Hoeng, Julia

2014-05-17

High-quality expression data are required to investigate the biological effects of microRNAs (miRNAs). The goal of this study was, first, to assess the quality of miRNA expression data based on microarray technologies and, second, to consolidate it by applying a novel normalization method. Indeed, because of significant differences in platform designs, miRNA raw data cannot be normalized blindly with standard methods developed for gene expression. This fundamental observation motivated the development of a novel multi-array normalization method based on controllable assumptions, which uses the spike-in control probes to adjust the measured intensities across arrays. Raw expression data were obtained with the Exiqon dual-channel miRCURY LNA™ platform in the "common reference design" and processed as "pseudo-single-channel". They were used to apply several quality metrics based on the coefficient of variation and to test the novel spike-in controls based normalization method. Most of the considerations presented here could be applied to raw data obtained with other platforms. To assess the normalization method, it was compared with 13 other available approaches from both data quality and biological outcome perspectives. The results showed that the novel multi-array normalization method reduced the data variability in the most consistent way. Further, the reliability of the obtained differential expression values was confirmed based on a quantitative reverse transcription-polymerase chain reaction experiment performed for a subset of miRNAs. The results reported here support the applicability of the novel normalization method, in particular to datasets that display global decreases in miRNA expression similarly to the cigarette smoke-exposed mouse lung dataset considered in this study. Quality metrics to assess between-array variability were used to confirm that the novel spike-in controls based normalization method provided high-quality miRNA expression data suitable for reliable downstream analysis. The multi-array miRNA raw data normalization method was implemented in an R software package called ExiMiR and deposited in the Bioconductor repository.

Assessment of a novel multi-array normalization method based on spike-in control probes suitable for microRNA datasets with global decreases in expression

PubMed Central

2014-01-01

Background High-quality expression data are required to investigate the biological effects of microRNAs (miRNAs). The goal of this study was, first, to assess the quality of miRNA expression data based on microarray technologies and, second, to consolidate it by applying a novel normalization method. Indeed, because of significant differences in platform designs, miRNA raw data cannot be normalized blindly with standard methods developed for gene expression. This fundamental observation motivated the development of a novel multi-array normalization method based on controllable assumptions, which uses the spike-in control probes to adjust the measured intensities across arrays. Results Raw expression data were obtained with the Exiqon dual-channel miRCURY LNA™ platform in the “common reference design” and processed as “pseudo-single-channel”. They were used to apply several quality metrics based on the coefficient of variation and to test the novel spike-in controls based normalization method. Most of the considerations presented here could be applied to raw data obtained with other platforms. To assess the normalization method, it was compared with 13 other available approaches from both data quality and biological outcome perspectives. The results showed that the novel multi-array normalization method reduced the data variability in the most consistent way. Further, the reliability of the obtained differential expression values was confirmed based on a quantitative reverse transcription–polymerase chain reaction experiment performed for a subset of miRNAs. The results reported here support the applicability of the novel normalization method, in particular to datasets that display global decreases in miRNA expression similarly to the cigarette smoke-exposed mouse lung dataset considered in this study. Conclusions Quality metrics to assess between-array variability were used to confirm that the novel spike-in controls based normalization method provided high-quality miRNA expression data suitable for reliable downstream analysis. The multi-array miRNA raw data normalization method was implemented in an R software package called ExiMiR and deposited in the Bioconductor repository. PMID:24886675

MicroRNA Expression in Formalin-fixed Paraffin-embedded Cancer Tissue: Identifying Reference MicroRNAs and Variability.

PubMed

Boisen, Mogens Karsbøl; Dehlendorff, Christian; Linnemann, Dorte; Schultz, Nicolai Aagaard; Jensen, Benny Vittrup; Høgdall, Estrid Vilma Solyom; Johansen, Julia Sidenius

2015-12-29

Archival formalin-fixed paraffin-embedded (FFPE) cancer tissue samples are a readily available resource for microRNA (miRNA) biomarker identification. No established standard for reference miRNAs in FFPE tissue exists. We sought to identify stable reference miRNAs for normalization of miRNA expression in FFPE tissue samples from patients with colorectal (CRC) and pancreatic (PC) cancer and to quantify the variability associated with sample age and fixation. High-throughput miRNA profiling results from 203 CRC and 256 PC FFPE samples as well as from 37 paired frozen/FFPE samples from nine other CRC tumors (methodological samples) were used. Candidate reference miRNAs were identified by their correlation with global mean expression. The stability of reference genes was analyzed according to published methods. The association between sample age and global mean miRNA expression was tested using linear regression. Variability was described using correlation coefficients and linear mixed effects models. Normalization effects were determined by changes in standard deviation and by hierarchical clustering. We created lists of 20 miRNAs with the best correlation to global mean expression in each cancer type. Nine of these miRNAs were present in both lists, and miR-103a-3p was the most stable reference miRNA for both CRC and PC FFPE tissue. The optimal number of reference miRNAs was 4 in CRC and 10 in PC. Sample age had a significant effect on global miRNA expression in PC (50% reduction over 20 years) but not in CRC. Formalin fixation for 2-6 days decreased miRNA expression 30-65%. Normalization using global mean expression reduced variability for technical and biological replicates while normalization using the expression of the identified reference miRNAs reduced variability only for biological replicates. Normalization only had a minor impact on clustering results. We identified suitable reference miRNAs for future miRNA expression experiments using CRC- and PC FFPE tissue samples. Formalin fixation decreased miRNA expression considerably, while the effect of increasing sample age was estimated to be negligible in a clinical setting.

Multiclass cancer classification using a feature subset-based ensemble from microRNA expression profiles.

PubMed

Piao, Yongjun; Piao, Minghao; Ryu, Keun Ho

2017-01-01

Cancer classification has been a crucial topic of research in cancer treatment. In the last decade, messenger RNA (mRNA) expression profiles have been widely used to classify different types of cancers. With the discovery of a new class of small non-coding RNAs; known as microRNAs (miRNAs), various studies have shown that the expression patterns of miRNA can also accurately classify human cancers. Therefore, there is a great demand for the development of machine learning approaches to accurately classify various types of cancers using miRNA expression data. In this article, we propose a feature subset-based ensemble method in which each model is learned from a different projection of the original feature space to classify multiple cancers. In our method, the feature relevance and redundancy are considered to generate multiple feature subsets, the base classifiers are learned from each independent miRNA subset, and the average posterior probability is used to combine the base classifiers. To test the performance of our method, we used bead-based and sequence-based miRNA expression datasets and conducted 10-fold and leave-one-out cross validations. The experimental results show that the proposed method yields good results and has higher prediction accuracy than popular ensemble methods. The Java program and source code of the proposed method and the datasets in the experiments are freely available at https://sourceforge.net/projects/mirna-ensemble/. Copyright © 2016 Elsevier Ltd. All rights reserved.

A Novel Real-Time PCR Assay of microRNAs Using S-Poly(T), a Specific Oligo(dT) Reverse Transcription Primer with Excellent Sensitivity and Specificity

PubMed Central

Kang, Kang; Zhang, Xiaoying; Liu, Hongtao; Wang, Zhiwei; Zhong, Jiasheng; Huang, Zhenting; Peng, Xiao; Zeng, Yan; Wang, Yuna; Yang, Yi; Luo, Jun; Gou, Deming

2012-01-01

Background MicroRNAs (miRNAs) are small, non-coding RNAs capable of postranscriptionally regulating gene expression. Accurate expression profiling is crucial for understanding the biological roles of miRNAs, and exploring them as biomarkers of diseases. Methodology/Principal Findings A novel, highly sensitive, and reliable miRNA quantification approach,termed S-Poly(T) miRNA assay, is designed. In this assay, miRNAs are subjected to polyadenylation and reverse transcription with a S-Poly(T) primer that contains a universal reverse primer, a universal Taqman probe, an oligo(dT)11 sequence and six miRNA-specific bases. Individual miRNAs are then amplified by a specific forward primer and a universal reverse primer, and the PCR products are detected by a universal Taqman probe. The S-Poly(T) assay showed a minimum of 4-fold increase in sensitivity as compared with the stem-loop or poly(A)-based methods. A remarkable specificity in discriminating among miRNAs with high sequence similarity was also obtained with this approach. Using this method, we profiled miRNAs in human pulmonary arterial smooth muscle cells (HPASMC) and identified 9 differentially expressed miRNAs associated with hypoxia treatment. Due to its outstanding sensitivity, the number of circulating miRNAs from normal human serum was significantly expanded from 368 to 518. Conclusions/Significance With excellent sensitivity, specificity, and high-throughput, the S-Poly(T) method provides a powerful tool for miRNAs quantification and identification of tissue- or disease-specific miRNA biomarkers. PMID:23152780

Biological mechanism analysis of acute renal allograft rejection: integrated of mRNA and microRNA expression profiles

PubMed Central

Huang, Shi-Ming; Zhao, Xia; Zhao, Xue-Mei; Wang, Xiao-Ying; Li, Shan-Shan; Zhu, Yu-Hui

2014-01-01

Objectives: Renal transplantation is the preferred method for most patients with end-stage renal disease, however, acute renal allograft rejection is still a major risk factor for recipients leading to renal injury. To improve the early diagnosis and treatment of acute rejection, study on the molecular mechanism of it is urgent. Methods: MicroRNA (miRNA) expression profile and mRNA expression profile of acute renal allograft rejection and well-functioning allograft downloaded from ArrayExpress database were applied to identify differentially expressed (DE) miRNAs and DE mRNAs. DE miRNAs targets were predicted by combining five algorithm. By overlapping the DE mRNAs and DE miRNAs targets, common genes were obtained. Differentially co-expressed genes (DCGs) were identified by differential co-expression profile (DCp) and differential co-expression enrichment (DCe) methods in Differentially Co-expressed Genes and Links (DCGL) package. Then, co-expression network of DCGs and the cluster analysis were performed. Functional enrichment analysis for DCGs was undergone. Results: A total of 1270 miRNA targets were predicted and 698 DE mRNAs were obtained. While overlapping miRNA targets and DE mRNAs, 59 common genes were gained. We obtained 103 DCGs and 5 transcription factors (TFs) based on regulatory impact factors (RIF), then built the regulation network of miRNA targets and DE mRNAs. By clustering the co-expression network, 5 modules were obtained. Thereinto, module 1 had the highest degree and module 2 showed the most number of DCGs and common genes. TF CEBPB and several common genes, such as RXRA, BASP1 and AKAP10, were mapped on the co-expression network. C1R showed the highest degree in the network. These genes might be associated with human acute renal allograft rejection. Conclusions: We conducted biological analysis on integration of DE mRNA and DE miRNA in acute renal allograft rejection, displayed gene expression patterns and screened out genes and TFs that may be related to acute renal allograft rejection. PMID:25664019

miRNA-15a, miRNA-15b, and miRNA-499 are Reduced in Erythrocytes of Pre-Diabetic African-American Adults

PubMed Central

Fluitt, Maurice B.; Kumari, Namita; Nunlee-Bland, Gail; Nekhai, Sergei; Gambhir, Kanwal K.

2017-01-01

Aims The use of circulatory miRNAs as biomarkers and therapeutic targets for T2DM is an explosive area of study. However, no study has investigated circulatory miRNA expression exclusively in African-American adults. The aim of this study was to identify the expression of nine selected miRNAs in erythrocytes of pre-diabetic and type 2 diabetic African-American adults. Main Methods Patients were recruited from the Howard University Hospital Diabetes Treatment Center following an 8 to 10 hour overnight fast. Expression of the nine selected miRNAs (miRNA-499, miRNA-146, miRNA-126, miRNA-223, miRNA-15a, miRNA-15b, miRNA-224, miRNA-326, and miRNA-375) was evaluated using quantitative real time PCR. Key Findings miRNA-15a, miRNA-15b, and miRNA-499 were significantly reduced in erythrocytes of pre-diabetic African-American adults. In the T2DM group, we found significant correlations between miRNA-15a and BMI (r=0.59, p=0.04), miRNA-15a and weight (r=0.52, p=0.01), and miRNA-15b and diastolic blood pressure (r=−0.52, p=0.02). In the pre-diabetic group, we found significant correlations between miRNA-15b and weight (r=0.90, p=0.02) and miRNA-499 and HbA1c (r=−0.89, p=0.01). Significance To our knowledge, this is the first study investigating miRNA expression in erythrocytes of non-diabetic high-risk obese--pre-diabetic and type 2 diabetic African-American adults. The findings of this study are consistent with previous reports of reduced expression of miRNA-15a, miRNA-15b, and miRNA-499 in human plasma or serum and in animal models. The current findings support the use of circulating miRNA-15a, miRNA-15b, and miRNA-499 as potential biomarkers for T2DM in African-American adults. PMID:29399662

An Assessment of Database-Validated microRNA Target Genes in Normal Colonic Mucosa: Implications for Pathway Analysis.

PubMed

Slattery, Martha L; Herrick, Jennifer S; Stevens, John R; Wolff, Roger K; Mullany, Lila E

2017-01-01

Determination of functional pathways regulated by microRNAs (miRNAs), while an essential step in developing therapeutics, is challenging. Some miRNAs have been studied extensively; others have limited information. In this study, we focus on 254 miRNAs previously identified as being associated with colorectal cancer and their database-identified validated target genes. We use RNA-Seq data to evaluate messenger RNA (mRNA) expression for 157 subjects who also had miRNA expression data. In the replication phase of the study, we replicated associations between 254 miRNAs associated with colorectal cancer and mRNA expression of database-identified target genes in normal colonic mucosa. In the discovery phase of the study, we evaluated expression of 18 miR-NAs (those with 20 or fewer database-identified target genes along with miR-21-5p, miR-215-5p, and miR-124-3p which have more than 500 database-identified target genes) with expression of 17 434 mRNAs to identify new targets in colon tissue. Seed region matches between miRNA and newly identified targeted mRNA were used to help determine direct miRNA-mRNA associations. From the replication of the 121 miRNAs that had at least 1 database-identified target gene using mRNA expression methods, 97.9% were expressed in normal colonic mucosa. Of the 8622 target miRNA-mRNA associations identified in the database, 2658 (30.2%) were associated with gene expression in normal colonic mucosa after adjusting for multiple comparisons. Of the 133 miRNAs with database-identified target genes by non-mRNA expression methods, 97.2% were expressed in normal colonic mucosa. After adjustment for multiple comparisons, 2416 miRNA-mRNA associations remained significant (19.8%). Results from the discovery phase based on detailed examination of 18 miRNAs identified more than 80 000 miRNA-mRNA associations that had not previously linked to the miRNA. Of these miRNA-mRNA associations, 15.6% and 14.8% had seed matches for CRCh38 and CRCh37, respectively. Our data suggest that miRNA target gene databases are incomplete; pathways derived from these databases have similar deficiencies. Although we know a lot about several miRNAs, little is known about other miRNAs in terms of their targeted genes. We encourage others to use their data to continue to further identify and validate miRNA-targeted genes.

Paper-Based MicroRNA Expression Profiling from Plasma and Circulating Tumor Cells.

PubMed

Leong, Sai Mun; Tan, Karen Mei-Ling; Chua, Hui Wen; Huang, Mo-Chao; Cheong, Wai Chye; Li, Mo-Huang; Tucker, Steven; Koay, Evelyn Siew-Chuan

2017-03-01

Molecular characterization of circulating tumor cells (CTCs) holds great promise for monitoring metastatic progression and characterizing metastatic disease. However, leukocyte and red blood cell contamination of routinely isolated CTCs makes CTC-specific molecular characterization extremely challenging. Here we report the use of a paper-based medium for efficient extraction of microRNAs (miRNAs) from limited amounts of biological samples such as rare CTCs harvested from cancer patient blood. Specifically, we devised a workflow involving the use of Flinders Technology Associates (FTA) ® Elute Card with a digital PCR-inspired "partitioning" method to extract and purify miRNAs from plasma and CTCs. We demonstrated the sensitivity of this method to detect miRNA expression from as few as 3 cancer cells spiked into human blood. Using this method, background miRNA expression was excluded from contaminating blood cells, and CTC-specific miRNA expression profiles were derived from breast and colorectal cancer patients. Plasma separated out during purification of CTCs could likewise be processed using the same paper-based method for miRNA detection, thereby maximizing the amount of patient-specific information that can be derived from a single blood draw. Overall, this paper-based extraction method enables an efficient, cost-effective workflow for maximized recovery of small RNAs from limited biological samples for downstream molecular analyses. © 2016 American Association for Clinical Chemistry.

[Expression profiles of miRNA-182 and Clock mRNA in the pineal gland of neonatal rats with hypoxic-ischemic brain damage].

PubMed

Han, Xing; Ding, Xin; Xu, Li-Xiao; Liu, Ming-Hua; Feng, Xing

2016-03-01

To study the changes of miRNA expression in the pineal gland of neonatal rats with hypoxic-ischemic brain damage (HIBD) and the possible roles of miRNA in the pathogenesis of circadian rhythm disturbance after HIBD. Seven-day-old Sprague-Dawley (SD) rats were randomly divided into 2 groups: HIBD and sham-operated. HIBD was induced according to the Rice-Vannucci method. The pineal glands were obtained 24 hours after the HIBD event. The expression profiles of miRNAs were determined using GeneChip technigue and quantitative real-time PCR (RT-PCR). Then the miRNA which was highly expressed was selected. The expression levels of the chosen miRNA were detected in different tissues (lungs, intestines, stomach, kidneys, cerebral cortex, pineal gland). RT-PCR analysis was performed to measure the expression profiles of the chosen miRNA and the targeted gene Clock mRNA in the pineal gland at 0, 24, 48 and 72 hours after HIBD. miRNA-182 that met the criteria was selected by GeneChip and RT-PCR. miRNA-182 was highly expressed in the pineal gland. Compared with the sham-operated group, the expression of miRNA-182 was significantly up-regulated in the pineal gland at 24 and 48 hours after HIBD (P<0.05). Compared with the sham-operated group, Clock mRNA expression in the HIBD group increased at 0 hour after HIBD, decreased at 48 hours after HIBD and increased at 72 hours after HIBD (P<0.05). miRNA-182 may be involved in the pathogenesis of circadian rhythm disturbance after HIBD.

Regulatory network involving miRNAs and genes in serous ovarian carcinoma

PubMed Central

Zhao, Haiyan; Xu, Hao; Xue, Luchen

2017-01-01

Serous ovarian carcinoma (SOC) is one of the most life-threatening types of gynecological malignancy, but the pathogenesis of SOC remains unknown. Previous studies have indicated that differentially expressed genes and microRNAs (miRNAs) serve important functions in SOC. However, genes and miRNAs are identified in a disperse form, and limited information is known about the regulatory association between miRNAs and genes in SOC. In the present study, three regulatory networks were hierarchically constructed, including a differentially-expressed network, a related network and a global network to reveal associations between each factor. In each network, there were three types of factors, which were genes, miRNAs and transcription factors that interact with each other. Focus was placed on the differentially-expressed network, in which all genes and miRNAs were differentially expressed and therefore may have affected the development of SOC. Following the comparison and analysis between the three networks, a number of signaling pathways which demonstrated differentially expressed elements were highlighted. Subsequently, the upstream and downstream elements of differentially expressed miRNAs and genes were listed, and a number of key elements (differentially expressed miRNAs, genes and TFs predicted using the P-match method) were analyzed. The differentially expressed network partially illuminated the pathogenesis of SOC. It was hypothesized that if there was no differential expression of miRNAs and genes, SOC may be prevented and treatment may be identified. The present study provided a theoretical foundation for gene therapy for SOC. PMID:29113276

Analysis of microRNA expression and function.

PubMed

Van Wynsberghe, Priscilla M; Chan, Shih-Peng; Slack, Frank J; Pasquinelli, Amy E

2011-01-01

Originally discovered in C. elegans, microRNAs (miRNAs) are small RNAs that regulate fundamental cellular processes in diverse organisms. MiRNAs are encoded within the genome and are initially transcribed as primary transcripts that can be several kilobases in length. Primary transcripts are successively cleaved by two RNase III enzymes, Drosha in the nucleus and Dicer in the cytoplasm, to produce ∼70 nucleotide (nt) long precursor miRNAs and 22 nt long mature miRNAs, respectively. Mature miRNAs regulate gene expression post-transcriptionally by imperfectly binding target mRNAs in association with the multiprotein RNA induced silencing complex (RISC). The conserved sequence, expression pattern, and function of some miRNAs across distinct species as well as the importance of specific miRNAs in many biological pathways have led to an explosion in the study of miRNA biogenesis, miRNA target identification, and miRNA target regulation. Many advances in our understanding of miRNA biology have come from studies in the powerful model organism C. elegans. This chapter reviews the current methods used in C. elegans to study miRNA biogenesis, small RNA populations, miRNA-protein complexes, and miRNA target regulation. Copyright © 2011 Elsevier Inc. All rights reserved.

Towards a new standardized method for circulating miRNAs profiling in clinical studies: Interest of the exogenous normalization to improve miRNA signature accuracy.

PubMed

Vigneron, Nicolas; Meryet-Figuière, Matthieu; Guttin, Audrey; Issartel, Jean-Paul; Lambert, Bernard; Briand, Mélanie; Louis, Marie-Hélène; Vernon, Mégane; Lebailly, Pierre; Lecluse, Yannick; Joly, Florence; Krieger, Sophie; Lheureux, Stéphanie; Clarisse, Bénédicte; Leconte, Alexandra; Gauduchon, Pascal; Poulain, Laurent; Denoyelle, Christophe

2016-08-01

Circulating miRNAs are promising biomarkers in oncology but have not yet been implemented in the clinic given the lack of concordance across studies. In order to increase the cross-studies reliability, we attempted to reduce and to control the circulating miRNA expression variability between patients. First, to maximize profiling signals and to reduce miRNA expression variability, three isolation kits were compared and the NucleoSpin(®) kit provided higher miRNA concentrations than the other widely used kits. Second, to control inter-sample variability during the profiling step, the exogenous miRNAs normalization method commonly used for RT-qPCR validation step was adapted to microarray experiments. Importantly, exogenous miRNAs presented two-fold lower inter-sample variability than the widely used endogenous miR-16-5p reflecting that the latter is subject to both biological and technical variability. Although Caenorhabditis elegans miRNAs isolation yields were heterogeneous, they correlated to each other and to their geometrical mean across samples. The normalization based on the geometrical mean of three exogenous miRNAs increased the correlation up-to 0.97 between the microarrays and individual RT-qPCR steps of circulating miRNAs expression. Overall, this new strategy open new avenue to identify reliable circulating miRNA signatures for translation into clinical practice. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Characterization of circulating microRNA expression in patients with a ventricular septal defect.

PubMed

Li, Dong; Ji, Long; Liu, Lianbo; Liu, Yizhi; Hou, Haifeng; Yu, Kunkun; Sun, Qiang; Zhao, Zhongtang

2014-01-01

Ventricular septal defect (VSD), one of the most common types of congenital heart disease (CHD), results from a combination of environmental and genetic factors. Recent studies demonstrated that microRNAs (miRNAs) are involved in development of CHD. This study was to characterize the expression of miRNAs that might be involved in the development or reflect the consequences of VSD. MiRNA microarray analysis and reverse transcription-polymerase chain reaction (RT-PCR) were employed to determine the miRNA expression profile from 3 patients with VSD and 3 VSD-free controls. 3 target gene databases were employed to predict the target genes of differentially expressed miRNAs. miRNAs that were generally consensus across the three databases were selected and then independently validated using real time PCR in plasma samples from 20 VSD patients and 15 VSD-free controls. Target genes of validated 8 miRNAs were predicted using bioinformatic methods. 36 differentially expressed miRNAs were found in the patients with VSD and the VSD-free controls. Compared with VSD-free controls, expression of 15 miRNAs were up-regulated and 21 miRNAs were downregulated in the VSD group. 15 miRNAs were selected based on database analysis results and expression levels of 8 miRNAs were validated. The results of the real time PCR were consistent with those of the microarray analysis. Gene ontology analysis indicated that the top target genes were mainly related to cardiac right ventricle morphogenesis. NOTCH1, HAND1, ZFPM2, and GATA3 were predicted as targets of hsa-let-7e-5p, hsa-miR-222-3p and hsa-miR-433. We report for the first time the circulating miRNA profile for patients with VSD and showed that 7 miRNAs were downregulated and 1 upregulated when matched to VSD-free controls. Analysis revealed target genes involved in cardiac development were probably regulated by these miRNAs.

Identification and validation of Asteraceae miRNAs by the expressed sequence tag analysis.

PubMed

Monavar Feshani, Aboozar; Mohammadi, Saeed; Frazier, Taylor P; Abbasi, Abbas; Abedini, Raha; Karimi Farsad, Laleh; Ehya, Farveh; Salekdeh, Ghasem Hosseini; Mardi, Mohsen

2012-02-10

MicroRNAs (miRNAs) are small non-coding RNA molecules that play a vital role in the regulation of gene expression. Despite their identification in hundreds of plant species, few miRNAs have been identified in the Asteraceae, a large family that comprises approximately one tenth of all flowering plants. In this study, we used the expressed sequence tag (EST) analysis to identify potential conserved miRNAs and their putative target genes in the Asteraceae. We applied quantitative Real-Time PCR (qRT-PCR) to confirm the expression of eight potential miRNAs in Carthamus tinctorius and Helianthus annuus. We also performed qRT-PCR analysis to investigate the differential expression pattern of five newly identified miRNAs during five different cotyledon growth stages in safflower. Using these methods, we successfully identified and characterized 151 potentially conserved miRNAs, belonging to 26 miRNA families, in 11 genus of Asteraceae. EST analysis predicted that the newly identified conserved Asteraceae miRNAs target 130 total protein-coding ESTs in sunflower and safflower, as well as 433 additional target genes in other plant species. We experimentally confirmed the existence of seven predicted miRNAs, (miR156, miR159, miR160, miR162, miR166, miR396, and miR398) in safflower and sunflower seedlings. We also observed that five out of eight miRNAs are differentially expressed during cotyledon development. Our results indicate that miRNAs may be involved in the regulation of gene expression during seed germination and the formation of the cotyledons in the Asteraceae. The findings of this study might ultimately help in the understanding of miRNA-mediated gene regulation in important crop species. Copyright © 2011 Elsevier B.V. All rights reserved.

miRNA Temporal Analyzer (mirnaTA): a bioinformatics tool for identifying differentially expressed microRNAs in temporal studies using normal quantile transformation.

PubMed

Cer, Regina Z; Herrera-Galeano, J Enrique; Anderson, Joseph J; Bishop-Lilly, Kimberly A; Mokashi, Vishwesh P

2014-01-01

Understanding the biological roles of microRNAs (miRNAs) is a an active area of research that has produced a surge of publications in PubMed, particularly in cancer research. Along with this increasing interest, many open-source bioinformatics tools to identify existing and/or discover novel miRNAs in next-generation sequencing (NGS) reads become available. While miRNA identification and discovery tools are significantly improved, the development of miRNA differential expression analysis tools, especially in temporal studies, remains substantially challenging. Further, the installation of currently available software is non-trivial and steps of testing with example datasets, trying with one's own dataset, and interpreting the results require notable expertise and time. Subsequently, there is a strong need for a tool that allows scientists to normalize raw data, perform statistical analyses, and provide intuitive results without having to invest significant efforts. We have developed miRNA Temporal Analyzer (mirnaTA), a bioinformatics package to identify differentially expressed miRNAs in temporal studies. mirnaTA is written in Perl and R (Version 2.13.0 or later) and can be run across multiple platforms, such as Linux, Mac and Windows. In the current version, mirnaTA requires users to provide a simple, tab-delimited, matrix file containing miRNA name and count data from a minimum of two to a maximum of 20 time points and three replicates. To recalibrate data and remove technical variability, raw data is normalized using Normal Quantile Transformation (NQT), and linear regression model is used to locate any miRNAs which are differentially expressed in a linear pattern. Subsequently, remaining miRNAs which do not fit a linear model are further analyzed in two different non-linear methods 1) cumulative distribution function (CDF) or 2) analysis of variances (ANOVA). After both linear and non-linear analyses are completed, statistically significant miRNAs (P < 0.05) are plotted as heat maps using hierarchical cluster analysis and Euclidean distance matrix computation methods. mirnaTA is an open-source, bioinformatics tool to aid scientists in identifying differentially expressed miRNAs which could be further mined for biological significance. It is expected to provide researchers with a means of interpreting raw data to statistical summaries in a fast and intuitive manner.

[Expression profiles of the exosomal miRNAs in the chronic hepatitis B patients with persistently normal ALT].

PubMed

Li, Ronghua; Fu, Xiaoyu; Tang, Yujing; Fu, Lei; Tan, Deming; Ouyang, Yi; Peng, Shifang

2018-05-28

To investigate expression profiles of the plasma exosomal miRNAs of the chronic hepatitis B (CHB) patients with persistently normal alamine aminotransferase (PNALT) for the first time and try to find exosomal miRNAs which could reflect liver inflammation better. 
 Methods: Five CHB patients with liver tissue inflammation grade ≥A2 of PNALT and 5 CHB patients with liver tissue inflammation grade

Mirna biogenesis pathway is differentially regulated during adipose derived stromal/stem cell differentiation.

PubMed

Martin, E C; Qureshi, A T; Llamas, C B; Burow, M E; King, A G; Lee, O C; Dasa, V; Freitas, M A; Forsberg, J A; Elster, E A; Davis, T A; Gimble, J M

2018-02-07

Stromal/stem cell differentiation is controlled by a vast array of regulatory mechanisms. Included within these are methods of mRNA gene regulation that occur at the level of epigenetic, transcriptional, and/or posttranscriptional modifications. Current studies that evaluate the posttranscriptional regulation of mRNA demonstrate microRNAs (miRNAs) as key mediators of stem cell differentiation through the inhibition of mRNA translation. miRNA expression is enhanced during both adipogenic and osteogenic differentiation; however, the mechanism by which miRNA expression is altered during stem cell differentiation is less understood. Here we demonstrate for the first time that adipose-derived stromal/stem cells (ASCs) induced to an adipogenic or osteogenic lineage have differences in strand preference (-3p and -5p) for miRNAs originating from the same primary transcript. Furthermore, evaluation of miRNA expression in ASCs demonstrates alterations in both miRNA strand preference and 5'seed site heterogeneity. Additionally, we show that during stem cell differentiation there are alterations in expression of genes associated with the miRNA biogenesis pathway. Quantitative RT-PCR demonstrated changes in the Argonautes (AGO1-4), Drosha, and Dicer at intervals of ASC adipogenic and osteogenic differentiation compared to untreated ASCs. Specifically, we demonstrated altered expression of the AGOs occurring during both adipogenesis and osteogenesis, with osteogenesis increasing AGO1-4 expression and adipogenesis decreasing AGO1 gene and protein expression. These data demonstrate changes to components of the miRNA biogenesis pathway during stromal/stem cell differentiation. Identifying regulatory mechanisms for miRNA processing during ASC differentiation may lead to novel mechanisms for the manipulation of lineage differentiation of the ASC through the global regulation of miRNA as opposed to singular regulatory mechanisms.

A Graphene-enhanced imaging of microRNA with enzyme-free signal amplification of catalyzed hairpin assembly in living cells.

PubMed

Liu, Haiyun; Tian, Tian; Ji, Dandan; Ren, Na; Ge, Shenguang; Yan, Mei; Yu, Jinghua

2016-11-15

In situ imaging of miRNA in living cells could help us to monitor the miRNA expression in real time and obtain accurate information for studying miRNA related bioprocesses and disease. Given the low-level expression of miRNA, amplification strategies for intracellular miRNA are imperative. Here, we propose an amplification strategy with a non-destructive enzyme-free manner in living cells using catalyzed hairpin assembly (CHA) based on graphene oxide (GO) for cellular miRNA imaging. The enzyme-free CHA exhibits stringent recognition and excellent signal amplification of miRNA in the living cells. GO is a good candidate as a fluorescence quencher and cellular carrier. Taking the advantages of the CHA and GO, we can monitor the miRNA at low level in living cells with a simple, sensitive and real-time manner. Finally, imaging of miRNAs in the different expression cells is realized. The novel method could supply an effective tool to visualize intracellular low-level miRNAs and help us to further understand the role of miRNAs in cellular processes. Copyright © 2016 Elsevier B.V. All rights reserved.

Determination of absolute expression profiles using multiplexed miRNA analysis

PubMed Central

Song, Jee Hoon; Cheng, Yulan; Saeui, Christopher T.; Cheung, Douglas G.; Croce, Carlo M.; Yarema, Kevin J.; Meltzer, Stephen J.; Liu, Kelvin J.; Wang, Tza-Huei

2017-01-01

Accurate measurement of miRNA expression is critical to understanding their role in gene expression as well as their application as disease biomarkers. Correct identification of changes in miRNA expression rests on reliable normalization to account for biological and technological variance between samples. Ligo-miR is a multiplex assay designed to rapidly measure absolute miRNA copy numbers, thus reducing dependence on biological controls. It uses a simple 2-step ligation process to generate length coded products that can be quantified using a variety of DNA sizing methods. We demonstrate Ligo-miR’s ability to quantify miRNA expression down to 20 copies per cell sensitivity, accurately discriminate between closely related miRNA, and reliably measure differential changes as small as 1.2-fold. Then, benchmarking studies were performed to show the high correlation between Ligo-miR, microarray, and TaqMan qRT-PCR. Finally, Ligo-miR was used to determine copy number profiles in a number of breast, esophageal, and pancreatic cell lines and to demonstrate the utility of copy number analysis for providing layered insight into expression profile changes. PMID:28704432

Expression profile analysis of circulating microRNAs and their effects on ion channels in Chinese atrial fibrillation patients

PubMed Central

Lu, Yingmin; Hou, Shuxin; Huang, Damin; Luo, Xiaohan; Zhang, Jinchun; Chen, Jian; Xu, Weiping

2015-01-01

Objective: To investigate the changes in expression profile of circulating microRNAs (miRNAs) and the regulatory effect of atrial fibrilation (AF)-related miRNAs on ion channels. Methods: 112 patients with AF were assigned into observation group, and another 112 non-AF people were assigned into control group. Total plasma RNAs were extracted from patients’ blood samples. Differentially expressed miRNA-1s were transfected into primary-cultured neonatal rat cardiac myocytes. Results: Compared with control group, significant differences were observed in 15 kinds of miRNAs in observation group. Down-regulation of the expression of miRNAs included hsa-miR-328, hsa-miR-145, hsa-miR-222, hsa-miR-1, hsa-miR-162, hsa-miR-432, and hsa-miR-493b; Up-regulation of the expression included hsa-miR634, hsa-miR-664, hsa-miR-9, hsa-miR-152, hsa-miR-19, hsa-miR-454, hsa-miR-146, and hsa-miR-374a. The expression level of CACNB2 protein in miRNA-1 group was significantly lower than that in blank control group, negative control group, MTmiRNA-1 group, AMO-1 group and miRNA-1+AMO-1 cotransfection group (P < 0.05), while in AMO-1 group, the expression level of CACNB2 protein was significantly higher than that in other groups (P < 0.05). These results indicated that transfected miRNA-1 could significantly inhibit the expression of CACNB2 protein. Conclusions: Circulating miRNAs can be used in studies concerning on the regulation mechanism of the occurrence and development of AF. MiRNA-1 can decrease the intracellular Ca2+ concentration and prevent the AF. PMID:25785065

Functional characterization of three MicroRNAs of the Asian Tiger Mosquito, Aedes albopictus

PubMed Central

2013-01-01

Background Temporal and stage specific expression of microRNAs (miRNAs) in embryos, larvae, pupae and adults of Aedes albopictus showed differential expression levels across the four developmental stages, indicating their potential regulatory roles in mosquito development. The functional characterization of these miRNAs was not known. Accordingly our study evaluated the functional characterization of three miRNAs, which are temporally up-regulated in the various developmental stages of Ae. albopictus mosquitoes. Methods miRNA mimics, inhibitors and negative controls were designed and their knock-in and knock-down efficiency were analyzed by qRT-PCR after transfecting the mosquito cell lines C6/36, and also by injecting in their specific developmental stages. The functional role of each individual miRNA was analyzed with various parameters of development such as, hatching rate and hatching time in embryos, eclosion rate in larvae, longevity and fecundity in the adult mosquitoes. Results The knock-in with the specifically designed miRNA mimics showed increased levels of expression of miRNA compared with their normal controls. We confirmed these findings using qRT-PCR, both by in vitro expression in C6/36 mosquito cell lines after transfection as well as in in vivo expression in developmental stages of mosquitoes by microinjection. The knock-down of expression with the corresponding inhibitors showed a considerable decrease in the expression levels of these miRNAs and obvious functional effects in Ae. albopictus development, detected by a decrease in the hatching rate of embryos and eclosion rate in larvae and a marked reduction in longevity and fecundity in adults. Conclusion This study carried out by knock-in and knock-down of specifically and temporally expressed miRNAs in Ae. albopictus by microinjection is a novel study to delineate the importance of the miRNA expression in regulating mosquito development. The knock-down and loss of function of endogenously expressed miRNAs by the miRNA inhibitors in specific developmental stages had considerable effects on development, but enhancement of their gain of function was not observed on knock-in of these specific miRNAs. Hence, our study indicates that an optimal level of endogenous expression of miRNA is indispensable for the normal development and maintenance of the vectorial population density and pathogen transmissibility of this mosquito vector. PMID:23924583

Biological mechanism analysis of acute renal allograft rejection: integrated of mRNA and microRNA expression profiles.

PubMed

Huang, Shi-Ming; Zhao, Xia; Zhao, Xue-Mei; Wang, Xiao-Ying; Li, Shan-Shan; Zhu, Yu-Hui

2014-01-01

Renal transplantation is the preferred method for most patients with end-stage renal disease, however, acute renal allograft rejection is still a major risk factor for recipients leading to renal injury. To improve the early diagnosis and treatment of acute rejection, study on the molecular mechanism of it is urgent. MicroRNA (miRNA) expression profile and mRNA expression profile of acute renal allograft rejection and well-functioning allograft downloaded from ArrayExpress database were applied to identify differentially expressed (DE) miRNAs and DE mRNAs. DE miRNAs targets were predicted by combining five algorithm. By overlapping the DE mRNAs and DE miRNAs targets, common genes were obtained. Differentially co-expressed genes (DCGs) were identified by differential co-expression profile (DCp) and differential co-expression enrichment (DCe) methods in Differentially Co-expressed Genes and Links (DCGL) package. Then, co-expression network of DCGs and the cluster analysis were performed. Functional enrichment analysis for DCGs was undergone. A total of 1270 miRNA targets were predicted and 698 DE mRNAs were obtained. While overlapping miRNA targets and DE mRNAs, 59 common genes were gained. We obtained 103 DCGs and 5 transcription factors (TFs) based on regulatory impact factors (RIF), then built the regulation network of miRNA targets and DE mRNAs. By clustering the co-expression network, 5 modules were obtained. Thereinto, module 1 had the highest degree and module 2 showed the most number of DCGs and common genes. TF CEBPB and several common genes, such as RXRA, BASP1 and AKAP10, were mapped on the co-expression network. C1R showed the highest degree in the network. These genes might be associated with human acute renal allograft rejection. We conducted biological analysis on integration of DE mRNA and DE miRNA in acute renal allograft rejection, displayed gene expression patterns and screened out genes and TFs that may be related to acute renal allograft rejection.

Bioinformatic identification and experimental validation of miRNAs from foxtail millet (Setaria italica).

PubMed

Han, Jun; Xie, Hao; Sun, Qingpeng; Wang, Jun; Lu, Min; Wang, Weixiang; Guo, Erhu; Pan, Jinbao

2014-08-10

MiRNAs are a novel group of non-coding small RNAs that negatively regulate gene expression. Many miRNAs have been identified and investigated extensively in plant species with sequenced genomes. However, few miRNAs have been identified in foxtail millet (Setaria italica), which is an ancient cereal crop of great importance for dry land agriculture. In this study, 271 foxtail millet miRNAs belonging to 44 families were identified using a bioinformatics approach. Twenty-three pairs of sense/antisense miRNAs belonging to 13 families, and 18 miRNA clusters containing members of 8 families were discovered in foxtail millet. We identified 432 potential targets for 38 miRNA families, most of which were predicted to be involved in plant development, signal transduction, metabolic pathways, disease resistance, and environmental stress responses. Gene ontology (GO) analysis revealed that 101, 56, and 23 target genes were involved in molecular functions, biological processes, and cellular components, respectively. We investigated the expression patterns of 43 selected miRNAs using qRT-PCR analysis. All of the miRNAs were expressed ubiquitously with many exhibiting different expression levels in different tissues. We validated five predicted targets of four miRNAs using the RNA ligase mediated rapid amplification of cDNA end (5'-RLM-RACE) method. Copyright © 2014 Elsevier B.V. All rights reserved.

MicroRNA Expression in Alpha and Beta Cells of Human Pancreatic Islets

PubMed Central

Vargas, Nancy; Rosero, Samuel; Piroso, Julieta; Ichii, Hirohito; Umland, Oliver; Zhijie, Jiang; Tsinoremas, Nicholas; Ricordi, Camillo; Inverardi, Luca; Domínguez-Bendala, Juan; Pastori, Ricardo L.

2013-01-01

microRNAs (miRNAs) play an important role in pancreatic development and adult β-cell physiology. Our hypothesis is based on the assumption that each islet cell type has a specific pattern of miRNA expression. We sought to determine the profile of miRNA expression in α-and β-cells, the main components of pancreatic islets, because this analysis may lead to a better understanding of islet gene regulatory pathways. Highly enriched (>98%) subsets of human α-and β-cells were obtained by flow cytometric sorting after intracellular staining with c-peptide and glucagon antibody. The method of sorting based on intracellular staining is possible because miRNAs are stable after fixation. MiRNA expression levels were determined by quantitative high throughput PCR-based miRNA array platform screening. Most of the miRNAs were preferentially expressed in β-cells. From the total of 667 miRNAs screened, the Significant Analysis of Microarray identified 141 miRNAs, of which only 7 were expressed more in α-cells (α-miRNAs) and 134 were expressed more in β-cells (β-miRNAs). Bioinformatic analysis identified potential targets of β-miRNAs analyzing the Beta Cell Gene Atlas, described in the T1Dbase, the web platform, supporting the type 1 diabetes (T1D) community. cMaf, a transcription factor regulating glucagon expression expressed selectively in α-cells (TFα) is targeted by β-miRNAs; miR-200c, miR-125b and miR-182. Min6 cells treated with inhibitors of these miRNAs show an increased expression of cMaf RNA. Conversely, over expression of miR-200c, miR-125b or miR-182 in the mouse alpha cell line αTC6 decreases the level of cMAF mRNA and protein. MiR-200c also inhibits the expression of Zfpm2, a TFα that inhibits the PI3K signaling pathway, at both RNA and protein levels. In conclusion, we identified miRNAs differentially expressed in pancreatic α- and β-cells and their potential transcription factor targets that could add new insights into different aspects of islet biology and pathophysiology. PMID:23383059

MicroRNA expression in alpha and beta cells of human pancreatic islets.

PubMed

Klein, Dagmar; Misawa, Ryosuke; Bravo-Egana, Valia; Vargas, Nancy; Rosero, Samuel; Piroso, Julieta; Ichii, Hirohito; Umland, Oliver; Zhijie, Jiang; Tsinoremas, Nicholas; Ricordi, Camillo; Inverardi, Luca; Domínguez-Bendala, Juan; Pastori, Ricardo L

2013-01-01

microRNAs (miRNAs) play an important role in pancreatic development and adult β-cell physiology. Our hypothesis is based on the assumption that each islet cell type has a specific pattern of miRNA expression. We sought to determine the profile of miRNA expression in α-and β-cells, the main components of pancreatic islets, because this analysis may lead to a better understanding of islet gene regulatory pathways. Highly enriched (>98%) subsets of human α-and β-cells were obtained by flow cytometric sorting after intracellular staining with c-peptide and glucagon antibody. The method of sorting based on intracellular staining is possible because miRNAs are stable after fixation. MiRNA expression levels were determined by quantitative high throughput PCR-based miRNA array platform screening. Most of the miRNAs were preferentially expressed in β-cells. From the total of 667 miRNAs screened, the Significant Analysis of Microarray identified 141 miRNAs, of which only 7 were expressed more in α-cells (α-miRNAs) and 134 were expressed more in β-cells (β-miRNAs). Bioinformatic analysis identified potential targets of β-miRNAs analyzing the Beta Cell Gene Atlas, described in the T1Dbase, the web platform, supporting the type 1 diabetes (T1D) community. cMaf, a transcription factor regulating glucagon expression expressed selectively in α-cells (TFα) is targeted by β-miRNAs; miR-200c, miR-125b and miR-182. Min6 cells treated with inhibitors of these miRNAs show an increased expression of cMaf RNA. Conversely, over expression of miR-200c, miR-125b or miR-182 in the mouse alpha cell line αTC6 decreases the level of cMAF mRNA and protein. MiR-200c also inhibits the expression of Zfpm2, a TFα that inhibits the PI3K signaling pathway, at both RNA and protein levels.In conclusion, we identified miRNAs differentially expressed in pancreatic α- and β-cells and their potential transcription factor targets that could add new insights into different aspects of islet biology and pathophysiology.

Validation of miRNA genes suitable as reference genes in qPCR analyses of miRNA gene expression in Atlantic salmon (Salmo salar).

PubMed

Johansen, Ilona; Andreassen, Rune

2014-12-23

MicroRNAs (miRNAs) are an abundant class of endogenous small RNA molecules that downregulate gene expression at the post-transcriptional level. They play important roles by regulating genes that control multiple biological processes, and recent years there has been an increased interest in studying miRNA genes and miRNA gene expression. The most common method applied to study gene expression of single genes is quantitative PCR (qPCR). However, before expression of mature miRNAs can be studied robust qPCR methods (miRNA-qPCR) must be developed. This includes identification and validation of suitable reference genes. We are particularly interested in Atlantic salmon (Salmo salar). This is an economically important aquaculture species, but no reference genes dedicated for use in miRNA-qPCR methods has been validated for this species. Our aim was, therefore, to identify suitable reference genes for miRNA-qPCR methods in Salmo salar. We used a systematic approach where we utilized similar studies in other species, some biological criteria, results from deep sequencing of small RNAs and, finally, experimental validation of candidate reference genes by qPCR to identify the most suitable reference genes. Ssa-miR-25-3p was identified as most suitable single reference gene. The best combinations of two reference genes were ssa-miR-25-3p and ssa-miR-455-5p. These two genes were constitutively and stably expressed across many different tissues. Furthermore, infectious salmon anaemia did not seem to affect their expression levels. These genes were amplified with high specificity, good efficiency and the qPCR assays showed a good linearity when applying a simple cybergreen miRNA-PCR method using miRNA gene specific forward primers. We have identified suitable reference genes for miRNA-qPCR in Atlantic salmon. These results will greatly facilitate further studies on miRNA genes in this species. The reference genes identified are conserved genes that are identical in their mature sequence in many aquaculture species. Therefore, they may also be suitable as reference genes in other teleosts. Finally, the systematic approach used in our study successfully identified suitable reference genes, suggesting that this may be a useful strategy to apply in similar validation studies in other aquaculture species.

Genome-wide discovery of novel and conserved microRNAs in white shrimp (Litopenaeus vannamei).

PubMed

Xi, Qian-Yun; Xiong, Yuan-Yan; Wang, Yuan-Mei; Cheng, Xiao; Qi, Qi-En; Shu, Gang; Wang, Song-Bo; Wang, Li-Na; Gao, Ping; Zhu, Xiao-Tong; Jiang, Qing-Yan; Zhang, Yong-Liang; Liu, Li

2015-01-01

Of late years, a large amount of conserved and species-specific microRNAs (miRNAs) have been performed on identification from species which are economically important but lack a full genome sequence. In this study, Solexa deep sequencing and cross-species miRNA microarray were used to detect miRNAs in white shrimp. We identified 239 conserved miRNAs, 14 miRNA* sequences and 20 novel miRNAs by bioinformatics analysis from 7,561,406 high-quality reads representing 325,370 distinct sequences. The all 20 novel miRNAs were species-specific in white shrimp and not homologous in other species. Using the conserved miRNAs from the miRBase database as a query set to search for homologs from shrimp expressed sequence tags (ESTs), 32 conserved computationally predicted miRNAs were discovered in shrimp. In addition, using microarray analysis in the shrimp fed with Panax ginseng polysaccharide complex, 151 conserved miRNAs were identified, 18 of which were significant up-expression, while 49 miRNAs were significant down-expression. In particular, qRT-PCR analysis was also performed for nine miRNAs in three shrimp tissues such as muscle, gill and hepatopancreas. Results showed that these miRNAs expression are tissue specific. Combining results of the three methods, we detected 20 novel and 394 conserved miRNAs. Verification with quantitative reverse transcription (qRT-PCR) and Northern blot showed a high confidentiality of data. The study provides the first comprehensive specific miRNA profile of white shrimp, which includes useful information for future investigations into the function of miRNAs in regulation of shrimp development and immunology.

miRNA signature associated with outcome of gastric cancer patients following chemotherapy

PubMed Central

2011-01-01

Background Identification of patients who likely will or will not benefit from cytotoxic chemotherapy through the use of biomarkers could greatly improve clinical management by better defining appropriate treatment options for patients. microRNAs may be potentially useful biomarkers that help guide individualized therapy for cancer because microRNA expression is dysregulated in cancer. In order to identify miRNA signatures for gastric cancer and for predicting clinical resistance to cisplatin/fluorouracil (CF) chemotherapy, a comprehensive miRNA microarray analysis was performed using endoscopic biopsy samples. Methods Biopsy samples were collected prior to chemotherapy from 90 gastric cancer patients treated with CF and from 34 healthy volunteers. At the time of disease progression, post-treatment samples were additionally collected from 8 clinical responders. miRNA expression was determined using a custom-designed Agilent microarray. In order to identify a miRNA signature for chemotherapy resistance, we correlated miRNA expression levels with the time to progression (TTP) of disease after CF therapy. Results A miRNA signature distinguishing gastric cancer from normal stomach epithelium was identified. 30 miRNAs were significantly inversely correlated with TTP whereas 28 miRNAs were significantly positively correlated with TTP of 82 cancer patients (P<0.05). Prominent among the upregulated miRNAs associated with chemosensitivity were miRNAs known to regulate apoptosis, including let-7g, miR-342, miR-16, miR-181, miR-1, and miR-34. When this 58-miRNA predictor was applied to a separate set of pre- and post-treatment tumor samples from the 8 clinical responders, all of the 8 pre-treatment samples were correctly predicted as low-risk, whereas samples from the post-treatment tumors that developed chemoresistance were predicted to be in the high-risk category by the 58 miRNA signature, suggesting that selection for the expression of these miRNAs occurred as chemoresistance arose. Conclusions We have identified 1) a miRNA expression signature that distinguishes gastric cancer from normal stomach epithelium from healthy volunteers, and 2) a chemoreresistance miRNA expression signature that is correlated with TTP after CF therapy. The chemoresistance miRNA expression signature includes several miRNAs previously shown to regulate apoptosis in vitro, and warrants further validation. PMID:22112324

Evidence for Alteration of Gene Regulatory Networks through MicroRNAs of the HIV-infected brain: novel analysis of retrospective cases.

PubMed

Tatro, Erick T; Scott, Erick R; Nguyen, Timothy B; Salaria, Shahid; Banerjee, Sugato; Moore, David J; Masliah, Eliezer; Achim, Cristian L; Everall, Ian P

2010-04-26

HIV infection disturbs the central nervous system (CNS) through inflammation and glial activation. Evidence suggests roles for microRNA (miRNA) in host defense and neuronal homeostasis, though little is known about miRNAs' role in HIV CNS infection. MiRNAs are non-coding RNAs that regulate gene translation through post-transcriptional mechanisms. Messenger-RNA profiling alone is insufficient to elucidate the dynamic dance of molecular expression of the genome. We sought to clarify RNA alterations in the frontal cortex (FC) of HIV-infected individuals and those concurrently infected and diagnosed with major depressive disorder (MDD). This report is the first published study of large-scale miRNA profiling from human HIV-infected FC. The goals of this study were to: 1. Identify changes in miRNA expression that occurred in the frontal cortex (FC) of HIV individuals, 2. Determine whether miRNA expression profiles of the FC could differentiate HIV from HIV/MDD, and 3. Adapt a method to meaningfully integrate gene expression data and miRNA expression data in clinical samples. We isolated RNA from the FC (n = 3) of three separate groups (uninfected controls, HIV, and HIV/MDD) and then pooled the RNA within each group for use in large-scale miRNA profiling. RNA from HIV and HIV/MDD patients (n = 4 per group) were also used for non-pooled mRNA analysis on Affymetrix U133 Plus 2.0 arrays. We then utilized a method for integrating the two datasets in a Target Bias Analysis. We found miRNAs of three types: A) Those with many dysregulated mRNA targets of less stringent statistical significance, B) Fewer dysregulated target-genes of highly stringent statistical significance, and C) unclear bias. In HIV/MDD, more miRNAs were downregulated than in HIV alone. Specific miRNA families at targeted chromosomal loci were dysregulated. The dysregulated miRNAs clustered on Chromosomes 14, 17, 19, and X. A small subset of dysregulated genes had many 3' untranslated region (3'UTR) target-sites for dysregulated miRNAs. We provide evidence that certain miRNAs serve as key elements in gene regulatory networks in HIV-infected FC and may be implicated in neurobehavioral disorder. Finally, our data indicates that some genes may serve as hubs of miRNA activity.

MicroRNA profiling of the murine hematopoietic system

PubMed Central

Monticelli, Silvia; Ansel, K Mark; Xiao, Changchun; Socci, Nicholas D; Krichevsky, Anna M; Thai, To-Ha; Rajewsky, Nikolaus; Marks, Debora S; Sander, Chris; Rajewsky, Klaus; Rao, Anjana; Kosik, Kenneth S

2005-01-01

Background MicroRNAs (miRNAs) are a class of recently discovered noncoding RNA genes that post-transcriptionally regulate gene expression. It is becoming clear that miRNAs play an important role in the regulation of gene expression during development. However, in mammals, expression data are principally based on whole tissue analysis and are still very incomplete. Results We used oligonucleotide arrays to analyze miRNA expression in the murine hematopoietic system. Complementary oligonucleotides capable of hybridizing to 181 miRNAs were immobilized on a membrane and probed with radiolabeled RNA derived from low molecular weight fractions of total RNA from several different hematopoietic and neuronal cells. This method allowed us to analyze cell type-specific patterns of miRNA expression and to identify miRNAs that might be important for cell lineage specification and/or cell effector functions. Conclusion This is the first report of systematic miRNA gene profiling in cells of the hematopoietic system. As expected, miRNA expression patterns were very different between hematopoietic and non-hematopoietic cells, with further subtle differences observed within the hematopoietic group. Interestingly, the most pronounced similarities were observed among fully differentiated effector cells (Th1 and Th2 lymphocytes and mast cells) and precursors at comparable stages of differentiation (double negative thymocytes and pro-B cells), suggesting that in addition to regulating the process of commitment to particular cellular lineages, miRNAs might have an important general role in the mechanism of cell differentiation and maintenance of cell identity. PMID:16086853

Large-scale identification and comparative analysis of miRNA expression profile in the respiratory tree of the sea cucumber Apostichopus japonicus during aestivation.

PubMed

Chen, Muyan; Storey, Kenneth B

2014-02-01

The sea cucumber Apostichopus japonicus withstands high water temperatures in the summer by suppressing its metabolic rate and entering a state of aestivation. We hypothesized that changes in the expression of miRNAs could provide important post-transcriptional regulation of gene expression during hypometabolism via control over mRNA translation. The present study analyzed profiles of miRNA expression in the sea cucumber respiratory tree using Solexa deep sequencing technology. We identified 279 sea cucumber miRNAs, including 15 novel miRNAs specific to sea cucumber. Animals sampled during deep aestivation (DA; after at least 15 days of continuous torpor) were compared with animals from a non-aestivation (NA) state (animals that had passed through aestivation and returned to an active state). We identified 30 differentially expressed miRNAs ([RPM (reads per million) >10, |FC| (|fold change|)≥1, FDR (false discovery rate)<0.01]) during aestivation, which were validated by two other miRNA profiling methods: miRNA microarray and real-time PCR. Among the most prominent miRNA species, miR-124, miR-124-3p, miR-79, miR-9 and miR-2010 were significantly over-expressed during deep aestivation compared with non-aestivation animals, suggesting that these miRNAs may play important roles in metabolic rate suppression during aestivation. High-throughput sequencing data and microarray data have been submitted to the GEO database with accession number: 16902695. Copyright © 2014 Elsevier B.V. All rights reserved.

Effector and regulatory dendritic cells display distinct patterns of miRNA expression

PubMed Central

Luce, Sonia; Moussu, Hélène; Morizur, Lise; Gueguen, Claire; Neukirch, Catherine; Chollet‐Martin, Sylvie; Mascarell, Laurent; Aubier, Michel; Baron‐Bodo, Véronique; Moingeon, Philippe

2017-01-01

Abstract Introduction MicroRNAs (miRNAs) contribute to the regulation of dendritic cell (DC) polarization, thereby influencing the balance of adaptive immune responses. Herein, we studied the expression of miRNAs in polarized DCs and analyzed whether expression of these miRNAs could be associated with allergic rhinitis and allergen immunotherapy (AIT) outcome. Method Using specific culture conditions, we differentiated immature human monocyte‐derived DCs into DC1, DC2, and DCreg subsets (supporting the differentiation of TH1, TH2 or regulatory T cells, respectively). Profiling of miRNA expression was performed in these DC subpopulations using microarrays. Levels of miRNAs specific for polarized DCs were then evaluated in a cohort of 58 patients with allergic rhinitis and 25 non‐allergic controls, as well as in samples from 30 subjects treated with sublingual grass pollen tablets or placebo for four months. Results We successfully identified 16 miRNAs differentially regulated between immature DCs, DC1, DC2, and DCreg cells. In allergic rhinoconjunctivitis patients, the expression of two of those miRNAs (miR‐132 and miR‐155), was down‐regulated compared to non‐allergic individuals. However, the levels of these miRNAs were not significantly modified following four months of grass pollen immunotherapy. Conclusions Studying polarized DCs and clinical samples from subjects with or without allergic rhinoconjunctivitis, we demonstrated that the expression of two miRNAs linked to effector DCs (i.e., DC1 and/or DC2 cells), was reduced in the blood of patients with allergic rhinoconjunctivitis. Nevertheless, these miRNAs did not represent relevant biomarkers to predict or follow‐up AIT efficacy. PMID:28497578

Identification and profiling of novel microRNAs in the Brassica rapa genome based on small RNA deep sequencing

PubMed Central

2012-01-01

Background MicroRNAs (miRNAs) are one of the functional non-coding small RNAs involved in the epigenetic control of the plant genome. Although plants contain both evolutionary conserved miRNAs and species-specific miRNAs within their genomes, computational methods often only identify evolutionary conserved miRNAs. The recent sequencing of the Brassica rapa genome enables us to identify miRNAs and their putative target genes. In this study, we sought to provide a more comprehensive prediction of B. rapa miRNAs based on high throughput small RNA deep sequencing. Results We sequenced small RNAs from five types of tissue: seedlings, roots, petioles, leaves, and flowers. By analyzing 2.75 million unique reads that mapped to the B. rapa genome, we identified 216 novel and 196 conserved miRNAs that were predicted to target approximately 20% of the genome’s protein coding genes. Quantitative analysis of miRNAs from the five types of tissue revealed that novel miRNAs were expressed in diverse tissues but their expression levels were lower than those of the conserved miRNAs. Comparative analysis of the miRNAs between the B. rapa and Arabidopsis thaliana genomes demonstrated that redundant copies of conserved miRNAs in the B. rapa genome may have been deleted after whole genome triplication. Novel miRNA members seemed to have spontaneously arisen from the B. rapa and A. thaliana genomes, suggesting the species-specific expansion of miRNAs. We have made this data publicly available in a miRNA database of B. rapa called BraMRs. The database allows the user to retrieve miRNA sequences, their expression profiles, and a description of their target genes from the five tissue types investigated here. Conclusions This is the first report to identify novel miRNAs from Brassica crops using genome-wide high throughput techniques. The combination of computational methods and small RNA deep sequencing provides robust predictions of miRNAs in the genome. The finding of numerous novel miRNAs, many with few target genes and low expression levels, suggests the rapid evolution of miRNA genes. The development of a miRNA database, BraMRs, enables us to integrate miRNA identification, target prediction, and functional annotation of target genes. BraMRs will represent a valuable public resource with which to study the epigenetic control of B. rapa and other closely related Brassica species. The database is available at the following link: http://bramrs.rna.kr [1]. PMID:23163954

A Customized Quantitative PCR MicroRNA Panel Provides a Technically Robust Context for Studying Neurodegenerative Disease Biomarkers and Indicates a High Correlation Between Cerebrospinal Fluid and Choroid Plexus MicroRNA Expression.

PubMed

Wang, Wang-Xia; Fardo, David W; Jicha, Gregory A; Nelson, Peter T

2017-12-01

MicroRNA (miRNA) expression varies in association with different tissue types and in diseases. Having been found in body fluids including blood and cerebrospinal fluid (CSF), miRNAs constitute potential biomarkers. CSF miRNAs have been proposed as biomarkers for neurodegenerative diseases; however, there is a lack of consensus about the best candidate miRNA biomarkers and there has been variability in results from different research centers, perhaps due to technical factors. Here, we sought to optimize technical parameters for CSF miRNA studies. We examined different RNA isolation methods and performed miRNA expression profiling with TaqMan® miRNA Arrays. More specifically, we developed a customized CSF-miRNA low-density array (TLDA) panel that contains 47 targets: miRNAs shown previously to be relevant to neurodegenerative disease, miRNAs that are abundant in CSF, data normalizers, and controls for potential blood and tissue contamination. The advantages of using this CSF-miRNA TLDA panel include specificity, sensitivity, fast processing and data analysis, and cost effectiveness. We optimized technical parameters for this assay. Further, the TLDA panel can be tailored to other specific purposes. We tested whether the profile of miRNAs in the CSF resembled miRNAs isolated from brain tissue (hippocampus or cerebellum), blood, or the choroid plexus. We found that the CSF miRNA expression profile most closely resembles that of choroid plexus tissue, underscoring the potential importance of choroid plexus-derived signaling through CSF miRNAs. In summary, the TLDA miRNA array panel will enable evaluation and discovery of CSF miRNA biomarkers and can potentially be utilized in clinical diagnosis and disease stage monitoring.

A microRNA detection system based on padlock probes and rolling circle amplification

PubMed Central

Jonstrup, Søren Peter; Koch, Jørn; Kjems, Jørgen

2006-01-01

The differential expression and the regulatory roles of microRNAs (miRNAs) are being studied intensively these years. Their minute size of only 19–24 nucleotides and strong sequence similarity among related species call for enhanced methods for reliable detection and quantification. Moreover, miRNA expression is generally restricted to a limited number of specific cells within an organism and therefore requires highly sensitive detection methods. Here we present a simple and reliable miRNA detection protocol based on padlock probes and rolling circle amplification. It can be performed without specialized equipment and is capable of measuring the content of specific miRNAs in a few nanograms of total RNA. PMID:16888321

A microRNA detection system based on padlock probes and rolling circle amplification.

PubMed

Jonstrup, Søren Peter; Koch, Jørn; Kjems, Jørgen

2006-09-01

The differential expression and the regulatory roles of microRNAs (miRNAs) are being studied intensively these years. Their minute size of only 19-24 nucleotides and strong sequence similarity among related species call for enhanced methods for reliable detection and quantification. Moreover, miRNA expression is generally restricted to a limited number of specific cells within an organism and therefore requires highly sensitive detection methods. Here we present a simple and reliable miRNA detection protocol based on padlock probes and rolling circle amplification. It can be performed without specialized equipment and is capable of measuring the content of specific miRNAs in a few nanograms of total RNA.

High-Throughput Sequencing Reveals Hypothalamic MicroRNAs as Novel Partners Involved in Timing the Rapid Development of Chicken (Gallus gallus) Gonads.

PubMed

Han, Wei; Zou, Jianmin; Wang, Kehua; Su, Yijun; Zhu, Yunfen; Song, Chi; Li, Guohui; Qu, Liang; Zhang, Huiyong; Liu, Honglin

2015-01-01

Onset of the rapid gonad growth is a milestone in sexual development that comprises many genes and regulatory factors. The observations in model organisms and mammals including humans have shown a potential link between miRNAs and development timing. To determine whether miRNAs play roles in this process in the chicken (Gallus gallus), the Solexa deep sequencing was performed to analyze the profiles of miRNA expression in the hypothalamus of hens from two different pubertal stages, before onset of the rapid gonad development (BO) and after onset of the rapid gonad development (AO). 374 conserved and 46 novel miRNAs were identified as hypothalamus-expressed miRNAs in the chicken. 144 conserved miRNAs were showed to be differentially expressed (reads > 10, P < 0.05) during the transition from BO to AO. Five differentially expressed miRNAs were validated by real-time quantitative RT-PCR (qRT-PCR) method. 2013 putative genes were predicted as the targets of the 15 most differentially expressed miRNAs (fold-change > 4.0, P < 0.01). Of these genes, 7 putative circadian clock genes, Per2, Bmal1/2, Clock, Cry1/2, and Star were found to be targeted multiple times by the miRNAs. qRT-PCR revealed the basic transcription levels of these clock genes were much higher (P < 0.01) in AO than in BO. Further functional analysis suggested that these 15 miRNAs play important roles in transcriptional regulation and signal transduction pathways. The results provide new insights into miRNAs functions in timing the rapid development of chicken gonads. Considering the characteristics of miRNA functional conservation, the results will contribute to the research on puberty onset in humans.

Synchronous detection of miRNAs, their targets and downstream proteins in transferred FFPE sections: applications in clinical and basic research.

PubMed

Zhao, Jin-yao; Liu, Chun-qing; Zhao, He-nan; Ding, Yan-Fang; Bi, Tie; Wang, Bo; Lin, Xing-chi; Guo, Gordon; Cui, Shi-ying

2012-10-01

After discovering new miRNAs, it is often difficult to determine their targets and effects on downstream protein expression. In situ hybridization (ISH) and immunohistochemistry (IHC) are two commonly used methods for clinical diagnosis and basic research. We used an optimized technique that simultaneously detects miRNAs, their binding targets and corresponding proteins on transferred serial formalin fixed paraffin embedded (FFPE) sections from patients. Combined with bioinformatics, this method was used to validate the reciprocal expression of specific miRNAs and targets that were detected by ISH, as well as the expression of downstream proteins that were detected by IHC. A complete analysis was performed using a limited number of transferred serial FFPE sections that had been stored for 1-4 years at room temperature. Some sections had even been previously stained with H&E. We identified a miRNA that regulates epithelial ovarian cancer, along with its candidate target and related downstream protein. These findings were directly validated using sub-cellular components obtained from the same patient sample. In addition, the expression of Nephrin (a podocyte marker) and Stmn1 (a recently identified marker related to glomerular development) were confirmed in transferred FFPE sections of mouse kidney. This procedure may be adapted for clinical diagnosis and basic research, providing a qualitative and efficient method to dissect the detailed spatial expression patterns of miRNA pathways in FFPE tissue, especially in cases where only a small biopsy sample can be obtained. Copyright © 2012 Elsevier Inc. All rights reserved.

High-Throughput Sequencing of microRNAs in Peripheral Blood Mononuclear Cells: Identification of Potential Weight Loss Biomarkers

PubMed Central

Milagro, Fermín I.; Miranda, Jonatan; Portillo, María P.; Fernandez-Quintela, Alfredo; Campión, Javier; Martínez, J. Alfredo

2013-01-01

Introduction MicroRNAs (miRNAs) are being increasingly studied in relation to energy metabolism and body composition homeostasis. Indeed, the quantitative analysis of miRNAs expression in different adiposity conditions may contribute to understand the intimate mechanisms participating in body weight control and to find new biomarkers with diagnostic or prognostic value in obesity management. Objective The aim of this study was the search for miRNAs in blood cells whose expression could be used as prognostic biomarkers of weight loss. Methods Ten Caucasian obese women were selected among the participants in a weight-loss trial that consisted in following an energy-restricted treatment. Weight loss was considered unsuccessful when <5% of initial body weight (non-responders) and successful when >5% (responders). At baseline, total miRNA isolated from peripheral blood mononuclear cells (PBMC) was sequenced with SOLiD v4. The miRNA sequencing data were validated by RT-PCR. Results Differential baseline expression of several miRNAs was found between responders and non-responders. Two miRNAs were up-regulated in the non-responder group (mir-935 and mir-4772) and three others were down-regulated (mir-223, mir-224 and mir-376b). Both mir-935 and mir-4772 showed relevant associations with the magnitude of weight loss, although the expression of other transcripts (mir-874, mir-199b, mir-766, mir-589 and mir-148b) also correlated with weight loss. Conclusions This research addresses the use of high-throughput sequencing technologies in the search for miRNA expression biomarkers in obesity, by determining the miRNA transcriptome of PBMC. Basal expression of different miRNAs, particularly mir-935 and mir-4772, could be prognostic biomarkers and may forecast the response to a hypocaloric diet. PMID:23335998

MicroRNAs at the human 14q32 locus have prognostic significance in osteosarcoma

PubMed Central

2013-01-01

Background Deregulation of microRNA (miRNA) transcript levels has been observed in many types of tumors including osteosarcoma. Molecular pathways regulated by differentially expressed miRNAs may contribute to the heterogeneous tumor behaviors observed in naturally occurring cancers. Thus, tumor-associated miRNA expression may provide informative biomarkers for disease outcome and metastatic potential in osteosarcoma patients. We showed previously that clusters of miRNAs at the 14q32 locus are downregulated in human osteosarcoma. Methods Human and canine osteosarcoma patient’s samples with clinical follow-up data were used in this study. We used bioinformatics and comparative genomics approaches to identify miRNA based prognostic biomarkers in osteosarcoma. Kaplan-Meier survival curves and Whitney Mann U tests were conducted for validating the statistical significance. Results Here we show that an inverse correlation exists between aggressive tumor behavior (increased metastatic potential and accelerated time to death) and the residual expression of 14q32 miRNAs (using miR-382 as a representative of 14q32 miRNAs) in a series of clinically annotated samples from human osteosarcoma patients. We also show a comparable decrease in expression of orthologous 14q32 miRNAs in canine osteosarcoma samples, with conservation of the inverse correlation between aggressive behavior and expression of orthologous miRNA miR-134 and miR-544. Conclusions We conclude that downregulation of 14q32 miRNA expression is an evolutionarily conserved mechanism that contributes to the biological behavior of osteosarcoma, and that quantification of representative transcripts from this family, such as miR-382, miR-134, and miR-544, provide prognostic and predictive markers that can assist in the management of patients with this disease. PMID:23311495

miRNA regulation of cytotoxic effects in mouse Sertoli cells exposed to nonylphenol

PubMed Central

2011-01-01

Background It is known that some environmental chemicals affect the human endocrine system. The harmful effects of endocrine disrupting chemical (EDC) nonylphenol (NP) have been studied since the 1980s. It is known that NP adversely affects physiological functions by mimicking the natural hormone 17 beta-estradiol. In the present study, we analyzed the expression of miRNAs and their target genes in mouse Sertoli TM4 cells to better understand the regulatory roles of miRNAs on Sertoli cells after NP exposure. Methods Mouse TM4 Sertoli cells were treated with NP for 3 or 24 h, and global gene and miRNA expression were analyzed using Agilent mouse whole genome and mouse miRNA v13 arrays. Results We identified genes that were > 2-fold differentially expressed in NP-treated cells and control cells (P < 0.05) and analyzed their functions through Gene Ontology analysis. We also identified miRNAs that were differentially expressed in NP-treated and control cells. Of the 186 miRNAs the expression of which differed between NP-treated and control cells, 59 and 147 miRNAs exhibited 1.3-fold increased or decreased expression at 3 and 24 h, respectively. Network analysis of deregulated miRNAs suggested that Ppara may regulate the expression of certain miRNAs, including miR-378, miR-125a-3p miR-20a, miR-203, and miR-101a, after exposure to NP. Additionally, comprehensive analysis of predicted target genes for miRNAs showed that the expression of genes with roles in cell proliferation, the cell cycle, and cell death were regulated by miRNA in NP-treated TM4 cells. Levels of expression of the miRNAs miR-135a* and miR-199a-5p were validated by qRT-PCR. Finally, miR-135a* target gene analysis suggests that the generation of reactive oxygen species (ROS) following exposure to NP exposure may be mediated by miR-135a* through regulation of the Wnt/beta-catenin signaling pathway. Conclusions Collectively, these data help to determine NP's actions on mouse TM4 Sertoli cells and increase our understanding of the molecular mechanisms underlying the adverse effects of xenoestrogens on the reproductive system. PMID:21914226

Relationship between microRNA-146a expression and plasma renalase levels in hemodialyzed patients

PubMed Central

Koch, Wojciech; Kukula-Koch, Wirginia; Gaweł, Kinga; Bednarek-Skublewska, Anna; Małecka-Massalska, Teresa; Milanowski, Janusz; Petkowicz, Beata; Solski, Janusz

2017-01-01

Background microRNA (miRNA) belongs to the non-coding RNAs family responsible for the regulation of gene expression. Renalase is a protein composed of 342 amino acids, secreted by the kidneys and possibly plays an important role in the regulation of sympathetic tone and blood pressure. The aim of the present study was to investigate plasma renalase concentration, and explore the relationship between miRNA-146a-5p expression and plasma renalase levels in hemodialyzed patients. Methods The study population comprised 55 subjects who succumbed to various cardiac events, 27 women and 28 men, aged 65–70 years. The total RNA including miRNA fraction was isolated using QiagenmiRNEasy Serum/Plasma kit according to the manufacturer’s protocol. The isolated miRNAs were analyzed using a quantitative polymerase chain reaction (qRT-PCR) technique. The plasma renalase levels were measured using a commercial ELISA kit. Results In the group of patients with high levels of renalase, higher miRNA-146a expression was found, compared with those with low concentration of renalase. Patients with simultaneous low miRNA-146a expression and high level of renalase were confirmed to deliver a significantly longer survival time compared with other patients. Conclusions miRNA-146a and plasma renalase levels were estimated as independent prognostic factors of hemodialyzed patients’ survival time. Patients with low miRNA-146a expression demonstrated a significantly longer survival time in contrast to the patients with a high expression level of miRNA-146a. Moreover, a significantly longer survival time was found in patients with high renalase activity compared with patients with low activity of the enzyme. PMID:28614373

High-Throughput Sequencing Reveals Differential Expression of miRNAs in Intestine from Sea Cucumber during Aestivation

PubMed Central

Chen, Muyan; Zhang, Xiumei; Liu, Jianning; Storey, Kenneth B.

2013-01-01

The regulatory role of miRNA in gene expression is an emerging hot new topic in the control of hypometabolism. Sea cucumber aestivation is a complicated physiological process that includes obvious hypometabolism as evidenced by a decrease in the rates of oxygen consumption and ammonia nitrogen excretion, as well as a serious degeneration of the intestine into a very tiny filament. To determine whether miRNAs play regulatory roles in this process, the present study analyzed profiles of miRNA expression in the intestine of the sea cucumber (Apostichopus japonicus), using Solexa deep sequencing technology. We identified 308 sea cucumber miRNAs, including 18 novel miRNAs specific to sea cucumber. Animals sampled during deep aestivation (DA) after at least 15 days of continuous torpor, were compared with animals from a non-aestivation (NA) state (animals that had passed through aestivation and returned to the active state). We identified 42 differentially expressed miRNAs [RPM (reads per million) >10, |FC| (|fold change|) ≥1, FDR (false discovery rate) <0.01] during aestivation, which were validated by two other miRNA profiling methods: miRNA microarray and real-time PCR. Among the most prominent miRNA species, miR-200-3p, miR-2004, miR-2010, miR-22, miR-252a, miR-252a-3p and miR-92 were significantly over-expressed during deep aestivation compared with non-aestivation animals. Preliminary analyses of their putative target genes and GO analysis suggest that these miRNAs could play important roles in global transcriptional depression and cell differentiation during aestivation. High-throughput sequencing data and microarray data have been submitted to GEO database. PMID:24143179

A BAP1 Mutation-specific MicroRNA Signature Predicts Clinical Outcomes in Clear Cell Renal Cell Carcinoma Patients with Wild-type BAP1

PubMed Central

Ge, Yu-Zheng; Xu, Lu-Wei; Zhou, Chang-Cheng; Lu, Tian-Ze; Yao, Wen-Tao; Wu, Ran; Zhao, You-Cai; Xu, Xiao; Hu, Zhi-Kai; Wang, Min; Yang, Xiao-Bing; Zhou, Liu-Hua; Zhong, Bing; Xu, Zheng; Li, Wen-Cheng; Zhu, Jia-Geng; Jia, Rui-Peng

2017-01-01

Background: Clear cell renal cell carcinoma (ccRCC) is the most prevalent histologic subtype of kidney cancers in adults, which could be divided into two distinct subgroups according to the BRCA1 associated protein-1 (BAP1) mutation status. In the current study, we comprehensively analyzed the genome-wide microRNA (miRNA) expression profiles in ccRCC, with the aim to identify the differentially expressed miRNAs between BAP1 mutant and wild-type tumors, and generate a BAP1 mutation-specific miRNA signature for ccRCC patients with wild-type BAP1. Methods: The BAP1 mutation status and miRNA profiles in BAP1 mutant and wild-type tumors were analyzed. Subsequently, the association of the differentially expressed miRNAs with patient survival was examined, and a BAP1 mutation-specific miRNA signature was generated and examined with Kaplan-Meier survival, univariate and multivariate Cox regression analyses. Finally, the bioinformatics methods were adopted for the target prediction of selected miRNAs and functional annotation analyses. Results: A total of 350 treatment-naïve primary ccRCC patients were selected from The Cancer Genome Atlas project, among which 35 (10.0%) subjects carried mutant BAP1 and had a shorter overall survival (OS) time. Furthermore, 33 miRNAs were found to be differentially expressed between BAP1 mutant and wild-type tumors, among which 11 (miR-149, miR-29b-2, miR-182, miR-183, miR-21, miR-365-2, miR-671, miR-365-1, miR-10b, miR-139, and miR-181a-2) were significantly associated with OS in ccRCC patients with wild-type BAP1. Finally, a BAP1 mutation-specific miRNA signature consisting of 11 miRNAs was generated and validated as an independent prognostic parameter. Conclusions: In summary, our study identified a total of 33 miRNAs differentially expressed between BAP1 mutant and wild-type tumors, and generated a BAP1 mutation-specific miRNA signature including eleven miRNAs, which could serve as a novel prognostic biomarker for ccRCC patients with wild-type BAP1. PMID:28900502

A path-based measurement for human miRNA functional similarities using miRNA-disease associations

NASA Astrophysics Data System (ADS)

Ding, Pingjian; Luo, Jiawei; Xiao, Qiu; Chen, Xiangtao

2016-09-01

Compared with the sequence and expression similarity, miRNA functional similarity is so important for biology researches and many applications such as miRNA clustering, miRNA function prediction, miRNA synergism identification and disease miRNA prioritization. However, the existing methods always utilized the predicted miRNA target which has high false positive and false negative to calculate the miRNA functional similarity. Meanwhile, it is difficult to achieve high reliability of miRNA functional similarity with miRNA-disease associations. Therefore, it is increasingly needed to improve the measurement of miRNA functional similarity. In this study, we develop a novel path-based calculation method of miRNA functional similarity based on miRNA-disease associations, called MFSP. Compared with other methods, our method obtains higher average functional similarity of intra-family and intra-cluster selected groups. Meanwhile, the lower average functional similarity of inter-family and inter-cluster miRNA pair is obtained. In addition, the smaller p-value is achieved, while applying Wilcoxon rank-sum test and Kruskal-Wallis test to different miRNA groups. The relationship between miRNA functional similarity and other information sources is exhibited. Furthermore, the constructed miRNA functional network based on MFSP is a scale-free and small-world network. Moreover, the higher AUC for miRNA-disease prediction indicates the ability of MFSP uncovering miRNA functional similarity.

Development of a low-cost detection method for miRNA microarray.

PubMed

Li, Wei; Zhao, Botao; Jin, Youxin; Ruan, Kangcheng

2010-04-01

MicroRNA (miRNA) microarray is a powerful tool to explore the expression profiling of miRNA. The current detection method used in miRNA microarray is mainly fluorescence based, which usually requires costly detection system such as laser confocal scanner of tens of thousands of dollars. Recently, we developed a low-cost yet sensitive detection method for miRNA microarray based on enzyme-linked assay. In this approach, the biotinylated miRNAs were captured by the corresponding oligonucleotide probes immobilized on microarray slide; and then the biotinylated miRNAs would capture streptavidin-conjugated alkaline phosphatase. A purple-black precipitation on each biotinylated miRNA spot was produced by the enzyme catalytic reaction. It could be easily detected by a charge-coupled device digital camera mounted on a microscope, which lowers the detection cost more than 100 fold compared with that of fluorescence method. Our data showed that signal intensity of the spot correlates well with the biotinylated miRNA concentration and the detection limit for miRNAs is at least 0.4 fmol and the detection dynamic range spans about 2.5 orders of magnitude, which is comparable to that of fluorescence method.

A high-throughput microRNA expression profiling system.

PubMed

Guo, Yanwen; Mastriano, Stephen; Lu, Jun

2014-01-01

As small noncoding RNAs, microRNAs (miRNAs) regulate diverse biological functions, including physiological and pathological processes. The expression and deregulation of miRNA levels contain rich information with diagnostic and prognostic relevance and can reflect pharmacological responses. The increasing interest in miRNA-related research demands global miRNA expression profiling on large numbers of samples. We describe here a robust protocol that supports high-throughput sample labeling and detection on hundreds of samples simultaneously. This method employs 96-well-based miRNA capturing from total RNA samples and on-site biochemical reactions, coupled with bead-based detection in 96-well format for hundreds of miRNAs per sample. With low-cost, high-throughput, high detection specificity, and flexibility to profile both small and large numbers of samples, this protocol can be adapted in a wide range of laboratory settings.

An Integrated Analysis of miRNA and mRNA Expressions in Non-Small Cell Lung Cancers

PubMed Central

Ma, Lina; Huang, Yanyan; Zhu, Wangyu; Zhou, Shiquan; Zhou, Jihang; Zeng, Fang; Liu, Xiaoguang; Zhang, Yongkui; Yu, Jun

2011-01-01

Using DNA microarrays, we generated both mRNA and miRNA expression data from 6 non-small cell lung cancer (NSCLC) tissues and their matching normal control from adjacent tissues to identify potential miRNA markers for diagnostics. We demonstrated that hsa-miR-96 is significantly and consistently up-regulated in all 6 NSCLCs. We validated this result in an independent set of 35 paired tumors and their adjacent normal tissues, as well as their sera that are collected before surgical resection or chemotherapy, and the results suggested that hsa-miR-96 may play an important role in NSCLC development and has great potential to be used as a noninvasive marker for diagnosing NSCLC. We predicted potential miRNA target mRNAs based on different methods (TargetScan and miRanda). Further classification of miRNA regulated genes based on their relationship with miRNAs revealed that hsa-miR-96 and certain other miRNAs tend to down-regulate their target mRNAs in NSCLC development, which have expression levels permissive to direct interaction between miRNAs and their target mRNAs. In addition, we identified a significant correlation of miRNA regulation with genes coincide with high density of CpG islands, which suggests that miRNA may represent a primary regulatory mechanism governing basic cellular functions and cell differentiations, and such mechanism may be complementary to DNA methylation in repressing or activating gene expression. PMID:22046296

Pathway analysis from lists of microRNAs: common pitfalls and alternative strategy

PubMed Central

Godard, Patrice; van Eyll, Jonathan

2015-01-01

MicroRNAs (miRNAs) are involved in the regulation of gene expression at a post-transcriptional level. As such, monitoring miRNA expression has been increasingly used to assess their role in regulatory mechanisms of biological processes. In large scale studies, once miRNAs of interest have been identified, the target genes they regulate are often inferred using algorithms or databases. A pathway analysis is then often performed in order to generate hypotheses about the relevant biological functions controlled by the miRNA signature. Here we show that the method widely used in scientific literature to identify these pathways is biased and leads to inaccurate results. In addition to describing the bias and its origin we present an alternative strategy to identify potential biological functions specifically impacted by a miRNA signature. More generally, our study exemplifies the crucial need of relevant negative controls when developing, and using, bioinformatics methods. PMID:25800743

Extract of Stellerachamaejasme L(ESC) inhibits growth and metastasis of human hepatocellular carcinoma via regulating microRNA expression.

PubMed

Liu, Xiaoni; Wang, Shuang; Xu, Jianji; Kou, Buxin; Chen, Dexi; Wang, Yajie; Zhu, Xiaoxin

2018-03-20

MicroRNAs(miRNAs)are involved in the initiation and progression of hepatocellular carcinoma. ESC, an extract of Stellerachamaejasme L, had been confirmed as a potential anti-tumor extract of Traditional Chinese Medicine. In light of the important role of miRNAs in hepatocellular carcinoma, we questioned whether the inhibitory effects of ESC on hepatocellular carcinoma (HCC) were associated with miRNAs. The proliferation inhibition of ESC on HCC cells was measured with MTT assay. The migration inhibition of ESC on HCC cells was measured with transwell assay. The influences of ESC on growth and metastasis inhibition were evaluated with xenograft tumor model of HCC. Protein expressions were measured with western blot and immunofluorescence methods and miRNA profiles were detected with miRNA array. Differential miRNA and target mRNAs were verified with real-time PCR. The results showed that ESC could inhibit proliferation and epithelial mesenchymal transition (EMT) in HCC cells in vitro and tumor growth and metastasis in xenograft models in vivo. miRNA array results showed that 69 differential miRNAs in total of 429 ones were obtained in MHCC97H cells treated by ESC. hsa-miR-107, hsa-miR-638, hsa-miR-106b-5p were selected to be validated with real-time PCR method in HepG2 and MHCC97H cells. Expressions of hsa-miR-107 and hsa-miR-638 increased obviously in HCC cells treated by ESC. Target genes of three miRNAs were also validated with real-time PCR. Interestingly, only target genes of hsa-miR-107 changed greatly. ESC downregulated the MCL1, SALL4 and BCL2 gene expressions significantly but did not influence the expression of CACNA2D1. The findings suggested ESC regressed growth and metastasis of human hepatocellular carcinoma via regulating microRNAs expression and their corresponding target genes.

Colorectal tumor molecular phenotype and miRNA: expression profiles and prognosis.

PubMed

Slattery, Martha L; Herrick, Jennifer S; Mullany, Lila E; Wolff, Erica; Hoffman, Michael D; Pellatt, Daniel F; Stevens, John R; Wolff, Roger K

2016-08-01

MiRNAs regulate gene expression by post-transcriptionally suppressing mRNA translation or by causing mRNA degradation. It has been proposed that unique miRNAs influence specific tumor molecular phenotype. In this paper, we test the hypotheses that miRNA expression differs by tumor molecular phenotype and that those differences may influence prognosis. Data come from population-based studies of colorectal cancer conducted in Utah and the Northern California Kaiser Permanente Medical Care Program. A total of 1893 carcinoma samples were run on the Agilent Human miRNA Microarray V19.0 containing 2006 miRNAs. We assessed differences in miRNA expression between TP53-mutated and non-mutated, KRAS-mutated and non-mutated, BRAF-mutated and non-mutated, CpG island methylator phenotype (CIMP) high and CIMP low, and microsatellite instability (MSI) and microsatellite stable (MSS) colon and rectal tumors. Using a Cox proportional hazard model we evaluated if those miRNAs differentially expressed by tumor phenotype influenced survival after adjusting for age, sex, and AJCC stage. There were 22 differentially expressed miRNAs for TP53-mutated colon tumors and 5 for TP53-mutated rectal tumors with a fold change of >1.49 (or <0.67). Additionally, 13 miRNAS were differentially expressed for KRAS-mutated rectal tumors, 8 differentially expressed miRNAs for colon CIMP high tumors, and 2 differentially expressed miRNAs for BRAF-mutated colon tumors. The majority of differentially expressed miRNAS were observed between MSI and MSS tumors (94 differentially expressed miRNAs for colon; 41 differentially expressed miRNAs for rectal tumors). Of these miRNAs differentially expressed between MSI and MSS tumors, the majority were downregulated. Ten of the differentially expressed miRNAs were associated with survival; after adjustment for MSI status, five miRNAS, miR-196b-5p, miR-31-5p, miR-99b-5p, miR-636, and miR-192-3p, were significantly associated with survival. In summary, it appears that the majority of miRNAs that are differentially expressed by tumor molecular phenotype are MSI tumors. However, these miRNAs appear to have minimal effect on prognosis.

miRNA 206 and miRNA 574-5p are highly expression in coronary artery disease

PubMed Central

Zhou, Jianqing; Shao, Guofeng; Chen, Xiaoliang; Yang, Xi; Huang, Xiaoyan; Peng, Ping; Ba, Yanna; Zhang, Lin; Jehangir, Tashina; Bu, Shizhong; Liu, Ningsheng; Lian, Jiangfang

2015-01-01

Coronary artery disease (CAD) is the leading cause of human morbidity and mortality worldwide. Innovative diagnostic biomarkers are a pressing need for this disease. miRNAs profiling is an innovative method of identifying biomarkers for many diseases and could be proven as a powerful tool in the diagnosis and treatment of CAD. We performed miRNA microarray analysis from the plasma of three CAD patients and three healthy controls. Subsequently, we performed quantitative real-time PCR (qRT-PCR) analysis of miRNA expression in plasma of another 67 CAD patients and 67 healthy controls. We identified two miRNAs (miR-206 and miR-574-5p) that were significantly up-regulated in CAD patients as compared with healthy controls (P<0.05). The receiver operating characteristic (ROC) curves indicated these two miRNAs had great potential to provide sensitive and specific diagnostic value for CAD. PMID:26685009

Curcumin inhibits the proliferation and invasion of human osteosarcoma cell line MG-63 by regulating miR-138

PubMed Central

Yu, Dazhi; An, Fengmei; He, Xu; Cao, Xuecheng

2015-01-01

Objective: In this study, we screened the different human osteosarcoma cell line MG-63 miRNAs after the treatment of curcumin and explored the effects of curcumin on MG-63 cells and its mechanism. Methods: Affemitrix miRNA chip was used to detect the changes of miRNA expression profile in MG-63 cells before and after curcumin treatment, and screen different expression of miRNAs. The target gene of miRNA was analyzed by bioinformatics. The expression levels of miRNA-138 target genes Smad4, NFκB p65 and cyclin D3 were detected. MTT and Transwell Cell invasion assays were used to observe the effects of curcumin on MG-63 cells. Results: Curcumin could significantly inhibit the proliferation of MG-63 cells and the expression levels of miRNA-138 target genes Smad4, NFκB p65 and cyclin D3 in MG-63 cells (P<0.05); overexpression of hsa-miR-138 down-regulated the expression levels of Smad4, NFκB p65 and cyclin D3 compared with the treatment of curcumin, while inhibition of hsa-miR-138 up-regulated the expression levels of Smad4, NFκB p65 and cyclin D3. Conclusions: Curcumin could increase the expression of hsa-miR-138, hsa-miR-138 inhibited cell proliferation and invasive ability by inhibition of its target genes. PMID:26823826

Nano metal-organic framework (NMOF)-based strategies for multiplexed microRNA detection in solution and living cancer cells

NASA Astrophysics Data System (ADS)

Wu, Yafeng; Han, Jianyu; Xue, Peng; Xu, Rong; Kang, Yuejun

2015-01-01

MiRNAs are an emerging type of biomarker for diagnostics and prognostics. A reliable sensing strategy that can monitor miRNA expression in living cancer cells would be critical in view of its extensive advantages for fundamental research related to miRNA-associated bioprocesses and biomedical applications. Conventional miRNA sensing methods include northern blot, microarrays and real-time quantitative PCR. However, none of them is able to monitor miRNA levels expressed in living cancer cells in a real-time fashion. Some fluorescennt biosensors developed recently from carbon nanomaterials, such as single-walled carbon nanotubes (SWNTs), graphene oxide (GO), and carbon nanoparticles, have been successfully used for assaying miRNA in vitro; however the preparation processes are often expensive, complicated and time-consuming, which have motivated the research on other substitute and novel materials. Herein we present a novel sensing strategy based on peptide nucleic acid (PNA) probes labeled with fluorophores and conjugated with an NMOF vehicle to monitor multiplexed miRNAs in living cancer cells. The NMOF works as a fluorescence quencher of the labelled PNA that is firmly bound with the metal center. In the presence of a target miRNA, PNA is hybridized and released from the NMOF leading to the recovery of fluorescence. This miRNA sensor not only enables the quantitative and highly specific detection of multiplexed miRNAs in living cancer cells, but it also allows the precise and in situ monitoring of the spatiotemporal changes of miRNA expression.MiRNAs are an emerging type of biomarker for diagnostics and prognostics. A reliable sensing strategy that can monitor miRNA expression in living cancer cells would be critical in view of its extensive advantages for fundamental research related to miRNA-associated bioprocesses and biomedical applications. Conventional miRNA sensing methods include northern blot, microarrays and real-time quantitative PCR. However, none of them is able to monitor miRNA levels expressed in living cancer cells in a real-time fashion. Some fluorescennt biosensors developed recently from carbon nanomaterials, such as single-walled carbon nanotubes (SWNTs), graphene oxide (GO), and carbon nanoparticles, have been successfully used for assaying miRNA in vitro; however the preparation processes are often expensive, complicated and time-consuming, which have motivated the research on other substitute and novel materials. Herein we present a novel sensing strategy based on peptide nucleic acid (PNA) probes labeled with fluorophores and conjugated with an NMOF vehicle to monitor multiplexed miRNAs in living cancer cells. The NMOF works as a fluorescence quencher of the labelled PNA that is firmly bound with the metal center. In the presence of a target miRNA, PNA is hybridized and released from the NMOF leading to the recovery of fluorescence. This miRNA sensor not only enables the quantitative and highly specific detection of multiplexed miRNAs in living cancer cells, but it also allows the precise and in situ monitoring of the spatiotemporal changes of miRNA expression. Electronic supplementary information (ESI) available: Extra figures and tables. See DOI: 10.1039/c4nr05447d

Circulating Plasma Levels of MicroRNA-21 and MicroRNA-221 Are Potential Diagnostic Markers for Primary Intrahepatic Cholangiocarcinoma

PubMed Central

Kemeny, Nancy; Kingham, T. Peter; Allen, Peter J.; D’Angelica, Michael I.; DeMatteo, Ronald P.; Betel, Doron; Klimstra, David; Jarnagin, William R.; Ventura, Andrea

2016-01-01

Background MicroRNAs (miRNAs) are potential biomarkers in various malignancies. We aim to characterize miRNA expression in intrahepatic cholangiocarcinoma (ICC) and identify circulating plasma miRNAs with potential diagnostic and prognostic utility. Methods Using deep-sequencing techniques, miRNA expression between tumor samples and non-neoplastic liver parenchyma were compared. Overexpressed miRNAs were measured in plasma from an independent cohort of patients with cholangiocarcinoma using RT-qPCR and compared with that healthy volunteers. The discriminatory ability of the evaluated plasma miRNAs between patients and controls was evaluated with receiving operating characteristic (ROC) curves. Results Small RNAs from 12 ICC and 11 tumor-free liver samples were evaluated. Unsupervised hierarchical clustering using the miRNA expression data showed clear grouping of ICC vs. non-neoplastic liver parenchyma. We identified 134 down-regulated and 128 upregulated miRNAs. Based on overexpression and high fold-change, miR21, miR200b, miR221, and miR34c were measured in plasma from an independent cohort of patients with ICC (n = 25) and healthy controls (n = 7). Significant overexpression of miR-21 and miR-221 was found in plasma from ICC patients. Furthermore, circulating miR-21 demonstrated a high discriminatory ability between patients with ICC and healthy controls (AUC: 0.94). Conclusion Among the differentially expressed miRNAs in ICC, miR-21 and miR-221 are overexpressed and detectable in the circulation. Plasma expression levels of these miRNAs, particularly miR-21, accurately differentiates patients with ICC from healthy controls and could potentially serve as adjuncts in diagnosis. Prospective validation and comparison with other hepatobiliary malignancies is required to establish their potential role as diagnostic and prognostic biomarkers. PMID:27685844

A Specific miRNA Signature Correlates With Complete Pathological Response to Neoadjuvant Chemoradiotherapy in Locally Advanced Rectal Cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Della Vittoria Scarpati, Giuseppina; Falcetta, Francesca; Carlomagno, Chiara, E-mail: chiara.carlomagno@unina.it

2012-07-15

Purpose: MicroRNAs (miRNAs) are small, noncoding RNA molecules that can be down- or upregulated in colorectal cancer and have been associated to prognosis and response to treatment. We studied miRNA expression in tumor biopsies of patients with rectal cancer to identify a specific 'signature' correlating with pathological complete response (pCR) after neoadjuvant chemoradiotherapy. Methods and Materials: A total of 38 T3-4/N+ rectal cancer patients received capecitabine-oxaliplatin and radiotherapy followed by surgery. Pathologic response was scored according to the Mandard TRG scale. MiRNA expression was analyzed by microarray and confirmed by real-time Reverse Transcription Polymerase Chain Reaction (qRT-PCR) on frozen biopsiesmore » obtained before treatment. The correlation between miRNA expression and TRG, coded as TRG1 (pCR) vs. TRG >1 (no pCR), was assessed by methods specifically designed for this study. Results: Microarray analysis selected 14 miRNAs as being differentially expressed in TRG1 patients, and 13 were confirmed by qRT-PCR: 11 miRNAs (miR-1183, miR-483-5p, miR-622, miR-125a-3p, miR-1224-5p, miR-188-5p, miR-1471, miR-671-5p, miR-1909 Asterisk-Operator , miR-630, miR-765) were significantly upregulated in TRG1 patients, 2 (miR-1274b, miR-720) were downexpressed. MiR-622 and miR-630 had a 100% sensitivity and specificity in selecting TRG1 cases. Conclusions: A set of 13 miRNAs is strongly associated with pCR and may represent a specific predictor of response to chemoradiotherapy in rectal cancer patients.« less

Genome wide predictions of miRNA regulation by transcription factors.

PubMed

Ruffalo, Matthew; Bar-Joseph, Ziv

2016-09-01

Reconstructing regulatory networks from expression and interaction data is a major goal of systems biology. While much work has focused on trying to experimentally and computationally determine the set of transcription-factors (TFs) and microRNAs (miRNAs) that regulate genes in these networks, relatively little work has focused on inferring the regulation of miRNAs by TFs. Such regulation can play an important role in several biological processes including development and disease. The main challenge for predicting such interactions is the very small positive training set currently available. Another challenge is the fact that a large fraction of miRNAs are encoded within genes making it hard to determine the specific way in which they are regulated. To enable genome wide predictions of TF-miRNA interactions, we extended semi-supervised machine-learning approaches to integrate a large set of different types of data including sequence, expression, ChIP-seq and epigenetic data. As we show, the methods we develop achieve good performance on both a labeled test set, and when analyzing general co-expression networks. We next analyze mRNA and miRNA cancer expression data, demonstrating the advantage of using the predicted set of interactions for identifying more coherent and relevant modules, genes, and miRNAs. The complete set of predictions is available on the supporting website and can be used by any method that combines miRNAs, genes, and TFs. Code and full set of predictions are available from the supporting website: http://cs.cmu.edu/~mruffalo/tf-mirna/ zivbj@cs.cmu.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Development of Novel Antisense Oligonucleotides for the Functional Regulation of RNA-Induced Silencing Complex (RISC) by Promoting the Release of microRNA from RISC.

PubMed

Ariyoshi, Jumpei; Momokawa, Daiki; Eimori, Nao; Kobori, Akio; Murakami, Akira; Yamayoshi, Asako

2015-12-16

MicroRNAs (miRNAs) are known to be important post-transcription regulators of gene expression. Aberrant miRNA expression is associated with pathological disease processes, including carcinogenesis. Therefore, miRNAs are considered significant therapeutic targets for cancer therapy. MiRNAs do not act alone, but exhibit their functions by forming RNA-induced silencing complex (RISC). Thus, the regulation of RISC activity is a promising approach for cancer therapy. MiRNA is a core component of RISC and is an essential to RISC for recognizing target mRNA. Thereby, it is expected that development of the method to promote the release of miRNA from RISC would be an effective approach for inhibition of RISC activity. In this study, we synthesized novel peptide-conjugated oligonucleotides (RINDA-as) to promote the release of miRNA from RISC. RINDA-as showed a high rate of miRNA release from RISC and high level of inhibitory effect on RISC activity.

Whole mouse blood microRNA as biomarkers for exposure to γ-rays and 56Fe ions

PubMed Central

Templin, Thomas; Amundson, Sally A.; Brenner, David J.; Smilenov, Lubomir B.

2013-01-01

Purpose Biomarkers of ionising radiation exposure are useful in a variety of scenarios, such as medical diagnostic imaging, occupational exposures, and spaceflight. This study investigates to what extent microRNA (miRNA) expression signatures in mouse peripheral blood can be used as biomarkers for exposures to radiation with low and high linear energy transfers. Materials and methods Mice were irradiated with doses of 0.5, 1.5, or 5.0 Gy γ-rays (dose rate of 0.0136 Gy/s) or with doses of 0.1 or 0.5 Gy 56Fe ions (dose rate of 0.00208 Gy/s). Total RNA was isolated from whole blood at 6 h or 24 h after irradiation. Three animals per irradiation condition were used. Differentially expressed miRNA were determined by means of quantitative real-time polymerase chain reaction. Results miRNA expression signatures were radiation type-specific and dose- and time-dependent. The differentially expressed miRNA were expressed in either one condition (71%) or multiple conditions (29%). Classifiers based on the differentially expressed miRNA predicted radiation type or dose with accuracies between 75% and 100%. Gene-ontology analyses show that miRNA induced by irradiation are involved in the control of several biological processes, such as mRNA transcription regulation, nucleic-acid metabolism, and development. Conclusion miRNA signatures induced by ionising radiation in mouse blood are radiation type- and radiation dose-specific. These findings underline the complexity of the radiation response and the importance of miRNA in it. PMID:21271940

MicroRNAs show a wide diversity of expression profiles in the developing and mature central nervous system

PubMed Central

Kapsimali, Marika; Kloosterman, Wigard P; de Bruijn, Ewart; Rosa, Frederic; Plasterk, Ronald HA; Wilson, Stephen W

2007-01-01

Background MicroRNA (miRNA) encoding genes are abundant in vertebrate genomes but very few have been studied in any detail. Bioinformatic tools allow prediction of miRNA targets and this information coupled with knowledge of miRNA expression profiles facilitates formulation of hypotheses of miRNA function. Although the central nervous system (CNS) is a prominent site of miRNA expression, virtually nothing is known about the spatial and temporal expression profiles of miRNAs in the brain. To provide an overview of the breadth of miRNA expression in the CNS, we performed a comprehensive analysis of the neuroanatomical expression profiles of 38 abundant conserved miRNAs in developing and adult zebrafish brain. Results Our results show miRNAs have a wide variety of different expression profiles in neural cells, including: expression in neuronal precursors and stem cells (for example, miR-92b); expression associated with transition from proliferation to differentiation (for example, miR-124); constitutive expression in mature neurons (miR-124 again); expression in both proliferative cells and their differentiated progeny (for example, miR-9); regionally restricted expression (for example, miR-222 in telencephalon); and cell-type specific expression (for example, miR-218a in motor neurons). Conclusion The data we present facilitate prediction of likely modes of miRNA function in the CNS and many miRNA expression profiles are consistent with the mutual exclusion mode of function in which there is spatial or temporal exclusion of miRNAs and their targets. However, some miRNAs, such as those with cell-type specific expression, are more likely to be co-expressed with their targets. Our data provide an important resource for future functional studies of miRNAs in the CNS. PMID:17711588

Proton radiation-induced miRNA signatures in mouse blood: Characterization and comparison with 56Fe-ion and gamma radiation

PubMed Central

Templin, Thomas; Young, Erik F.; Smilenov, Lubomir B.

2013-01-01

Purpose Previously, we showed that microRNA (miRNA) signatures derived from the peripheral blood of mice are highly specific for both radiation energy (γ-rays or high linear energy transfer [LET] 56Fe ions) and radiation dose. Here, we investigate to what extent miRNA expression signatures derived from mouse blood can be used as biomarkers for exposure to 600 MeV proton radiation. Materials and methods We exposed mice to 600 MeV protons, using doses of 0.5 or 1.0 Gy, isolated total RNA at 6 h or 24 h after irradiation, and used quantitative real-time polymerase chain reaction (PCR) to determine the changes in miRNA expression. Results A total of 26 miRNA were differentially expressed after proton irradiation, in either one (77%) or multiple conditions (23%). Statistical classifiers based on proton, γ, and 56Fe-ion miRNA expression signatures predicted radiation type and proton dose with accuracies of 81% and 88%, respectively. Importantly, gene ontology analysis for proton-irradiated cells shows that genes targeted by radiation-induced miRNA are involved in biological processes and molecular functions similar to those controlled by miRNA in γ ray- and 56Fe-irradiated cells. Conclusions Mouse blood miRNA signatures induced by proton, γ, or 56Fe irradiation are radiation type- and dose-specific. These findings underline the complexity of the miRNA-mediated radiation response. PMID:22551419

TGF-β1 resulting in differential microRNA expression in bovine granulosa cells.

PubMed

Xu, Yefen; Niu, Jiaqiang; Xi, Guangying; Niu, Xuezhi; Wang, Yuheng; Guo, Ming; Yangzong, Qiangba; Yao, Yilong; Sizhu, Suo Lang; Tian, Jianhui

2018-07-15

To explore the expression profile of the cellular miRNAs in bovine ovarian granulosa cells responding to transforming growth factor-β1 (TGF-β1), the effect of TGF-β1 on cell proliferation was firstly investigated by CCK-8 method and the results showed that there was a significant inhibitory effect on bovine granulosa cell proliferation treated with 5/10 ng/mL human recombinant TGF-β1 for 24 h compared to the control (P < 0.05). Then, we performed high-throughput sequencing of two small RNA libraries prepared from cultured bovine granulosa cells stimulated with or without 10 ng/mL human recombinant TGF-β1. A total of 13,257,248 and 138,726,391 clean reads per library were obtained from TGF-β1 and control groups, respectively. There were 498 and 499 bovine-specific exist miRNAs (exist miRNAs), 627 and 570 conserved known miRNAs (known miRNAs), and 593 and 585 predicted novel miRNAs in TGF-β1 and control groups, respectively. A total of 78 miRNAs with significant differential expression, including 39 up-regulated miRNAs and 39 down-regulated miRNAs were identified in the TGF-β1 group compared with the control. Real-time quantitative PCR analyses of bta-miR-106a and bta-miR-1434-5p showed that their up-expressions were interrupted by SB431542, an inhibitor that blocks TGFβ1/Smad signaling, which supported the sequencing data. GO analysis showed involvement of the predicted genes of the differentially expressed miRNAs in a broad spectrum of cell biological processes, cell components, and molecular functions. KEGG pathway analysis of the predicted miRNA targets further indicated that these differentially expressed miRNAs are involved in various signaling pathways, such as Wnt, MAPK, and TGF-β signaling, which might be involved in follicular development. These results provide valuable information on the composition, expression, and function of miRNAs in bovine granulosa cells responding to TGF-β1, and will aid in understanding the molecular mechanisms of TGF-β1 in granulosa cells. Copyright © 2018 Elsevier B.V. All rights reserved.

snoU6 and 5S RNAs are not reliable miRNA reference genes in neuronal differentiation.

PubMed

Lim, Q E; Zhou, L; Ho, Y K; Wan, G; Too, H P

2011-12-29

Accurate profiling of microRNAs (miRNAs) is an essential step for understanding the functional significance of these small RNAs in both physiological and pathological processes. Quantitative real-time PCR (qPCR) has gained acceptance as a robust and reliable transcriptomic method to profile subtle changes in miRNA levels and requires reference genes for accurate normalization of gene expression. 5S and snoU6 RNAs are commonly used as reference genes in microRNA quantification. It is currently unknown if these small RNAs are stably expressed during neuronal differentiation. Panels of miRNAs have been suggested as alternative reference genes to 5S and snoU6 in various physiological contexts. To test the hypothesis that miRNAs may serve as stable references during neuronal differentiation, the expressions of eight miRNAs, 5S and snoU6 RNAs in five differentiating neuronal cell types were analyzed using qPCR. The stabilities of the expressions were evaluated using two complementary statistical approaches (geNorm and Normfinder). Expressions of 5S and snoU6 RNAs were stable under some but not all conditions of neuronal differentiation and thus are not suitable reference genes. In contrast, a combination of three miRNAs (miR-103, miR-106b and miR-26b) allowed accurate expression normalization across different models of neuronal differentiation. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Identification and Characterization of Novel MicroRNAs from Schistosoma japonicum

PubMed Central

Xue, Xiangyang; Sun, Jun; Zhang, Qingfeng; Wang, Zhangxun; Huang, Yufu; Pan, Weiqing

2008-01-01

Background Schistosomiasis japonica remains a major public health problem in China. Its pathogen, Schistosoma japonicum has a complex life cycle and a unique repertoire of genes expressed at different life cycle stages. Exploring schistosome gene regulation will yield the best prospects for new drug targets and vaccine candidates. MicroRNAs (miRNAs) are a highly conserved class of noncoding RNA that control many biological processes by sequence-specific inhibition of gene expression. Although a large number of miRNAs have been identified from plants to mammals, it remains no experimental proof whether schistosome exist miRNAs. Methodology and Results We have identified novel miRNAs from Schistosoma japonicum by cloning and sequencing a small (18–26 nt) RNA cDNA library from the adult worms. Five novel miRNAs were identified from 227 cloned RNA sequences and verified by Northern blot. Alignments of the miRNAs with corresponding family members indicated that four of them belong to a metazoan miRNA family: let-7, miR-71, bantam and miR-125. The fifth potentially new (non conserved) miRNA appears to belong to a previously undescribed family in the genus Schistosome. The novel miRNAs were designated as sja-let-7, sja-miR-71, sja-bantam, sja-miR-125 and sja-miR-new1, respectively. Expression of sja-let-7, sja-miR-71 and sja-bantam were analyzed in six stages of the life cycle, i.e. egg, miracidium, sporocyst, cercaria, schistosomulum, and adult worm, by a modified stem-loop reverse transcribed polymerase chain reaction (RT-PCR) method developed in our laboratory. The expression patterns of these miRNAs were highly stage-specific. In particular, sja-miR-71 and sja-bantam expression reach their peaks in the cercaria stage and then drop quickly to the nadirs in the schistosomulum stage, following penetration of cercaria into a mammalian host. Conclusions Authentic miRNAs were identified for the first time in S. japonicum, including a new schistosome family member. The different expression patterns of the novel miRNAs over the life stages of S. japonicum suggest that they may mediate important roles in Schistosome growth and development. PMID:19107204

Regulation of Bone Formation During Disuse by miRNA

NASA Technical Reports Server (NTRS)

Thomas, Nicholas; Choi, Catherine Y.; Alwood, Joshua S.

2016-01-01

Astronauts lose bone structure during long-duration spaceflight. These changes are due, in part, to insufficient bone formation by the osteoblast cells. Little is known about the role that small (approximately 22 nucleotide), non-coding micro-RNAs (miRNAs) play in the osteoblast response to microgravity. We hypothesize that osteoblast-lineage cells alter their miRNA status during microgravity exposure, contributing to impaired bone formation during weightlessness. To simulate weightlessness, female mice (C57BL/6, Charles River, 10 weeks of age, n = 6) were hindlimb unloaded for 12 days. Age-matched and normally ambulating mice served as controls (n=6). To assess the expression of miRNAs in skeletal tissue, the right and left tibia of the mice were collected ex vivo and cleaned of soft-tissue and marrow. Total RNA was collected from tibial bone and relative abundance was measured for miRNAs of interest using quantitative real time PCR array looking at 372 unique and well-characterized mature miRNAs using the delta-delta Ct method. Transcripts of interest were normalized to an average of 6 reference RNAs. Preliminary results show that hindlimb unloading decreased the expression of 14 miRNAs to less than 1.4-2.9X control levels and increased the expression of 5 miRNAs relative to the control mice greater than 1-2-1.5X (p less than 0.05, respectively). Using the miRSystem we assessed overlapping target genes predicted to be regulated by multiple members of the 19 differentially expressed miRNAs as well as in silico predicted targets of our individual miRNAs. Our miRSystem results indicated that a number of our differentially expressed miRNAs were regulators of genes related to the Wnt-Beta Catenin pathway-a known regulator of bone health-and, interestingly, the estrogen-mediated cell-cycle regulation pathway, which may indicate that simulated weightlessness induced systemic hormonal changes that contributed to bone loss. We plan to follow up these findings by measuring gene expression of miRNA-regulated genes within these two pathways with the aim of furthering our understanding of the function of miRNAs in the skeletal response to spaceflight.

Insilico profiling of microRNAs in Korean ginseng (Panax ginseng Meyer)

PubMed Central

Mathiyalagan, Ramya; Subramaniyam, Sathiyamoorthy; Natarajan, Sathishkumar; Kim, Yeon Ju; Sun, Myung Suk; Kim, Se Young; Kim, Yu-Jin; Yang, Deok Chun

2013-01-01

MicroRNAs (miRNAs) are a class of recently discovered non-coding small RNA molecules, on average approximately 21 nucleotides in length, which underlie numerous important biological roles in gene regulation in various organisms. The miRNA database (release 18) has 18,226 miRNAs, which have been deposited from different species. Although miRNAs have been identified and validated in many plant species, no studies have been reported on discovering miRNAs in Panax ginseng Meyer, which is a traditionally known medicinal plant in oriental medicine, also known as Korean ginseng. It has triterpene ginseng saponins called ginsenosides, which are responsible for its various pharmacological activities. Predicting conserved miRNAs by homology-based analysis with available expressed sequence tag (EST) sequences can be powerful, if the species lacks whole genome sequence information. In this study by using the EST based computational approach, 69 conserved miRNAs belonging to 44 miRNA families were identified in Korean ginseng. The digital gene expression patterns of predicted conserved miRNAs were analyzed by deep sequencing using small RNA sequences of flower buds, leaves, and lateral roots. We have found that many of the identified miRNAs showed tissue specific expressions. Using the insilico method, 346 potential targets were identified for the predicted 69 conserved miRNAs by searching the ginseng EST database, and the predicted targets were mainly involved in secondary metabolic processes, responses to biotic and abiotic stress, and transcription regulator activities, as well as a variety of other metabolic processes. PMID:23717176

Expression differences of circulating microRNAs in metastatic castration resistant prostate cancer and low-risk, localized prostate cancer.

PubMed

Nguyen, Han Christine Ngoc; Xie, Wanling; Yang, Ming; Hsieh, Chen-Lin; Drouin, Sarah; Lee, Gwo-Shu Mary; Kantoff, Philip W

2013-03-01

Recent studies show that microRNAs (miRNAs), small non-coding RNAs that negatively regulate gene expression, may have potential for monitoring cancer status. We investigated circulating miRNAs in prostate cancer that may be associated with the progression of hormone-sensitive primary tumors to metastatic castration resistant prostate cancer (CRPC) after androgen deprivation therapy. Using genome-wide expression profiling by TaqMan Human MicroRNA Arrays (Applied Biosystems) and/or quantitative real-time polymerase chain reaction, we compared the expression levels of miRNAs in serum samples from 28 patients of low-risk localized disease, 30 of high-risk localized disease and 26 of metastatic CRPC. We demonstrated that serum samples from patients of low risk, localized prostate cancer and metastatic CRPC patients exhibit distinct circulating miRNA signatures. MiR-375, miR-378*, and miR-141 were significantly over-expressed in serum from CRPC patients compared with serum from low-risk localized patients, while miR-409-3p was significantly under-expressed. In prostate primary tumor samples, miR-375 and miR-141 also had significantly higher expression levels compared with those in normal prostate tissue. Circulating miRNAs, particularly miR-375, miR-141, miR-378*, and miR-409-3p, are differentially expressed in serum samples from prostate cancer patients. In the search for improved minimally invasive methods to follow cancer pathogenesis, the correlation of disease status with the expression patterns of circulating miRNAs may indicate the potential importance of circulating miRNAs as prognostic markers for prostate cancer progression. Copyright © 2012 Wiley Periodicals, Inc.

Effect of miRNA-203 on cervical cancer cells and its underlying mechanism.

PubMed

Yin, X Z; Zhao, D M; Zhang, G X; Liu, L

2016-09-23

miRNA-203 is involved in the development and progression of various types of cancer. However, its role in cervical cancer remains unclear. The aim of this study was to investigate the effect of miRNA-203 on the proliferation and migration of HeLa cervical cancer cells, as well as survivin expression in these cells. A miRNA-203 primer probe was designed according to a sequence obtained from NCBI. The expression of miRNA-203 in cervical epithelial cells and cervical cancer cells was detected by quantitative reverse transcriptase-polymerase chain reaction. The miRNA-203 expression pattern was compared between these two cell lines. The cervical cancer cells were transfected with miRNA-203 mimic or inhibitor to determine their effects on proliferation and migration. The expression of the miRNA-203 target protein (survivin) was analyzed by western blot. Cervical cancer cells showed reduced miRNA-203 expression compared to cervical epithelial cells. Transfection of miRNA-203 mimic upregulated the expression of miRNA-203, suppressed cell proliferation and migration, and downregulated survivin expression (P < 0.05). However, downregulation of miRNA-203 expression did not affect proliferation, migration, and survivin expression in cervical cancer cells (P > 0.05). In conclusion, upregulation of miRNA-203 in cervical cancer cells inhibits the proliferative and migratory capacities of these cells by downregulating the expression of survivin.

Technical variables in high-throughput miRNA expression profiling: much work remains to be done.

PubMed

Nelson, Peter T; Wang, Wang-Xia; Wilfred, Bernard R; Tang, Guiliang

2008-11-01

MicroRNA (miRNA) gene expression profiling has provided important insights into plant and animal biology. However, there has not been ample published work about pitfalls associated with technical parameters in miRNA gene expression profiling. One source of pertinent information about technical variables in gene expression profiling is the separate and more well-established literature regarding mRNA expression profiling. However, many aspects of miRNA biochemistry are unique. For example, the cellular processing and compartmentation of miRNAs, the differential stability of specific miRNAs, and aspects of global miRNA expression regulation require specific consideration. Additional possible sources of systematic bias in miRNA expression studies include the differential impact of pre-analytical variables, substrate specificity of nucleic acid processing enzymes used in labeling and amplification, and issues regarding new miRNA discovery and annotation. We conclude that greater focus on technical parameters is required to bolster the validity, reliability, and cultural credibility of miRNA gene expression profiling studies.

Identification and Characterization of MicroRNAs in Ovary and Testis of Nile Tilapia (Oreochromis niloticus) by Using Solexa Sequencing Technology

PubMed Central

Zhou, Yi; Yu, Fan; Gao, Yun; Luo, Yongju; Tang, Zhanyang; Guo, Zhongbao; Guo, Enyan; Gan, Xi; Zhang, Ming; Zhang, Yaping

2014-01-01

MicroRNAs (miRNAs) are endogenous non-coding small RNAs which play important roles in the regulation of gene expression by cleaving or inhibiting the translation of target gene transcripts. Thereinto, some specific miRNAs show regulatory activities in gonad development via translational control. In order to further understand the role of miRNA-mediated posttranscriptional regulation in Nile tilapia (Oreochromis niloticus) ovary and testis, two small RNA libraries of Nile tilapia were sequenced by Solexa small RNA deep sequencing methods. A total of 9,731,431 and 8,880,497 raw reads, representing 5,407,800 and 4,396,281 unique sequences were obtained from the sexually mature ovaries and testes, respectively. After comparing the small RNA sequences with the Rfam database, 1,432,210 reads in ovaries and 984,146 reads in testes were matched to the genome sequence of Nile tilapia. Bioinformatic analysis identified 764 mature miRNA, 209 miRNA-5p and 202 miRNA-3p were found in the two libraries, of which 525 known miRNAs are both expressed in the ovary and testis of Nile tilapia. Comparison of expression profiles of the testis, miR-727, miR-129 and miR-29 families were highly expressed in tilapia ovary. Additionally, miR-132, miR-212, miR-33a and miR-135b families, showed significant higher expression in testis compared with that in ovary. Furthermore, the expression patterns of the miRNAs were analyzed in different developmental stages of gonad. The result showed different expression patterns were observed during development of testis and ovary. In addition, the identification and characterization of differentially expressed miRNAs in the ovaries and testis of Nile tilapia provides important information on the role of miRNA in the regulation of the ovarian and testicular development and function. This data will be helpful to facilitate studies on the regulation of miRNAs during teleosts reproduction. PMID:24466258

Regulation of miRNA Processing and miRNA Mediated Gene Repression in Cancer

PubMed Central

Bajan, Sarah; Hutvagner, Gyorgy

2014-01-01

The majority of human protein-coding genes are predicted to be targets of miRNA-mediated post-transcriptional regulation. The widespread influence of miRNAs is illustrated by their essential roles in all biological processes. Regulated miRNA expression is essential for maintaining cellular differentiation; therefore alterations in miRNA expression patterns are associated with several diseases, including various cancers. High-throughput sequencing technologies revealed low level expressing miRNA isoforms, termed isomiRs. IsomiRs may differ in sequence, length, target preference and expression patterns from their parental miRNA and can arise from differences in miRNA biosynthesis, RNA editing, or SNPs inherent to the miRNA gene. The association between isomiR expression and disease progression is largely unknown. Misregulated miRNA expression is thought to contribute to the formation and/or progression of cancer. However, due to the diversity of targeted transcripts, miRNAs can function as both tumor-suppressor genes and oncogenes as defined by cellular context. Despite this, miRNA profiling studies concluded that the differential expression of particular miRNAs in diseased tissue could aid the diagnosis and treatment of some cancers. PMID:25069508

Identification of extracellular miRNA in archived serum samples by next-generation sequencing from RNA extracted using multiple methods.

PubMed

Gautam, Aarti; Kumar, Raina; Dimitrov, George; Hoke, Allison; Hammamieh, Rasha; Jett, Marti

2016-10-01

miRNAs act as important regulators of gene expression by promoting mRNA degradation or by attenuating protein translation. Since miRNAs are stably expressed in bodily fluids, there is growing interest in profiling these miRNAs, as it is minimally invasive and cost-effective as a diagnostic matrix. A technical hurdle in studying miRNA dynamics is the ability to reliably extract miRNA as small sample volumes and low RNA abundance create challenges for extraction and downstream applications. The purpose of this study was to develop a pipeline for the recovery of miRNA using small volumes of archived serum samples. The RNA was extracted employing several widely utilized RNA isolation kits/methods with and without addition of a carrier. The small RNA library preparation was carried out using Illumina TruSeq small RNA kit and sequencing was carried out using Illumina platform. A fraction of five microliters of total RNA was used for library preparation as quantification is below the detection limit. We were able to profile miRNA levels in serum from all the methods tested. We found out that addition of nucleic acid based carrier molecules had higher numbers of processed reads but it did not enhance the mapping of any miRBase annotated sequences. However, some of the extraction procedures offer certain advantages: RNA extracted by TRIzol seemed to align to the miRBase best; extractions using TRIzol with carrier yielded higher miRNA-to-small RNA ratios. Nuclease free glycogen can be carrier of choice for miRNA sequencing. Our findings illustrate that miRNA extraction and quantification is influenced by the choice of methodologies. Addition of nucleic acid- based carrier molecules during extraction procedure is not a good choice when assaying miRNA using sequencing. The careful selection of an extraction method permits the archived serum samples to become valuable resources for high-throughput applications.

Keratinization-associated miR-7 and miR-21 Regulate Tumor Suppressor Reversion-inducing Cysteine-rich Protein with Kazal Motifs (RECK) in Oral Cancer*

PubMed Central

Jung, Hyun Min; Phillips, Brittany L.; Patel, Rushi S.; Cohen, Donald M.; Jakymiw, Andrew; Kong, William W.; Cheng, Jin Q.; Chan, Edward K. L.

2012-01-01

MicroRNAs (miRNAs) are small non-coding RNAs that posttranscriptionally regulate gene expression during many biological processes. Recently, the aberrant expressions of miRNAs have become a major focus in cancer research. The purpose of this study was to identify deregulated miRNAs in oral cancer and further focus on specific miRNAs that were related to patient survival. Here, we report that miRNA expression profiling provided more precise information when oral squamous cell carcinomas were subcategorized on the basis of clinicopathological parameters (tumor primary site, histological subtype, tumor stage, and HPV16 status). An innovative radar chart analysis method was developed to depict subcategories of cancers taking into consideration the expression patterns of multiple miRNAs combined with the clinicopathological parameters. Keratinization of tumors and the high expression of miR-21 were the major factors related to the poor prognosis of patients. Interestingly, a majority of the keratinized tumors expressed high levels of miR-21. Further investigations demonstrated the regulation of the tumor suppressor gene reversion-inducing cysteine-rich protein with kazal motifs (RECK) by two keratinization-associated miRNAs, miR-7 and miR-21. Transfection of miR-7 and miR-21-mimics reduced the expression of RECK through direct miRNA-mediated regulation, and these miRNAs were inversely correlated with RECK in CAL 27 orthotopic xenograft tumors. Furthermore, a similar inverse correlation was demonstrated in CAL 27 cells treated in vitro by different external stimuli such as trypsinization, cell density, and serum concentration. Taken together, our data show that keratinization is associated with poor prognosis of oral cancer patients and keratinization-associated miRNAs mediate deregulation of RECK which may contribute to the aggressiveness of tumors. PMID:22761427

Integrated Analyses of microRNAs Demonstrate Their Widespread Influence on Gene Expression in High-Grade Serous Ovarian Carcinoma

PubMed Central

Levine, Douglas A.; Mankoo, Parminder; Schultz, Nikolaus; Du, Ying; Zhang, Yiqun; Larsson, Erik; Sheridan, Robert; Xiao, Weimin; Spellman, Paul T.; Getz, Gad; Wheeler, David A.; Perou, Charles M.; Gibbs, Richard A.; Sander, Chris; Hayes, D. Neil; Gunaratne, Preethi H.

2012-01-01

Background The Cancer Genome Atlas (TCGA) Network recently comprehensively catalogued the molecular aberrations in 487 high-grade serous ovarian cancers, with much remaining to be elucidated regarding the microRNAs (miRNAs). Here, using TCGA ovarian data, we surveyed the miRNAs, in the context of their predicted gene targets. Methods and Results Integration of miRNA and gene patterns yielded evidence that proximal pairs of miRNAs are processed from polycistronic primary transcripts, and that intronic miRNAs and their host gene mRNAs derive from common transcripts. Patterns of miRNA expression revealed multiple tumor subtypes and a set of 34 miRNAs predictive of overall patient survival. In a global analysis, miRNA:mRNA pairs anti-correlated in expression across tumors showed a higher frequency of in silico predicted target sites in the mRNA 3′-untranslated region (with less frequency observed for coding sequence and 5′-untranslated regions). The miR-29 family and predicted target genes were among the most strongly anti-correlated miRNA:mRNA pairs; over-expression of miR-29a in vitro repressed several anti-correlated genes (including DNMT3A and DNMT3B) and substantially decreased ovarian cancer cell viability. Conclusions This study establishes miRNAs as having a widespread impact on gene expression programs in ovarian cancer, further strengthening our understanding of miRNA biology as it applies to human cancer. As with gene transcripts, miRNAs exhibit high diversity reflecting the genomic heterogeneity within a clinically homogeneous disease population. Putative miRNA:mRNA interactions, as identified using integrative analysis, can be validated. TCGA data are a valuable resource for the identification of novel tumor suppressive miRNAs in ovarian as well as other cancers. PMID:22479643

Deregulation of the miRNAs Expression in Cervical Cancer: Human Papillomavirus Implications

PubMed Central

Gómez-Gómez, Yazmín; Organista-Nava, Jorge; Gariglio, Patricio

2013-01-01

MicroRNAs (miRNAs) are a class of small non coding RNAs of 18–25 nucleotides in length. The temporal or short-lived expression of the miRNAs modulates gene expression post transcriptionally. Studies have revealed that miRNAs deregulation correlates and is involved with the initiation and progression of human tumors. Cervical cancer (CC) displays notably increased or decreased expression of a large number of cellular oncogenic or tumor suppressive miRNAs, respectively. However, understanding the potential role of miRNAs in CC is still limited. In CC, the high-risk human papillomaviruses (HR-HPVs) infection can affect the miRNAs expression through oncoprotein E6 and E7 that contribute to viral pathogenesis, although other viral proteins might also be involved. This deregulation in the miRNAs expression has an important role in the hallmarks of CC. Interestingly, the miRNA expression profile in CC can discriminate between normal and tumor tissue and the extraordinary stability of miRNAs makes it suitable to serve as diagnostic and prognostic biomarkers of cancer. In this review, we will summarize the role of the HR-HPVs in miRNA expression, the role of miRNAs in the hallmarks of CC, and the use of miRNAs as potential prognostic biomarkers in CC. PMID:24490161

Predicting miRNA targets for head and neck squamous cell carcinoma using an ensemble method.

PubMed

Gao, Hong; Jin, Hui; Li, Guijun

2018-01-01

This study aimed to uncover potential microRNA (miRNA) targets in head and neck squamous cell carcinoma (HNSCC) using an ensemble method which combined 3 different methods: Pearson's correlation coefficient (PCC), Lasso and a causal inference method (i.e., intervention calculus when the directed acyclic graph (DAG) is absent [IDA]), based on Borda count election. The Borda count election method was used to integrate the top 100 predicted targets of each miRNA generated by individual methods. Afterwards, to validate the performance ability of our method, we checked the TarBase v6.0, miRecords v2013, miRWalk v2.0 and miRTarBase v4.5 databases to validate predictions for miRNAs. Pathway enrichment analysis of target genes in the top 1,000 miRNA-messenger RNA (mRNA) interactions was conducted to focus on significant KEGG pathways. Finally, we extracted target genes based on occurrence frequency ≥3. Based on an absolute value of PCC >0.7, we found 33 miRNAs and 288 mRNAs for further analysis. We extracted 10 target genes with predicted frequencies not less than 3. The target gene MYO5C possessed the highest frequency, which was predicted by 7 different miRNAs. Significantly, a total of 8 pathways were identified; the pathways of cytokine-cytokine receptor interaction and chemokine signaling pathway were the most significant. We successfully predicted target genes and pathways for HNSCC relying on miRNA expression data, mRNA expression profile, an ensemble method and pathway information. Our results may offer new information for the diagnosis and estimation of the prognosis of HNSCC.

Ursolic Acid Attenuates Diabetic Mesangial Cell Injury through the Up-Regulation of Autophagy via miRNA-21/PTEN/Akt/mTOR Suppression

PubMed Central

Lu, Xinxing; Fan, Qiuling; Xu, Li; Li, Lin; Yue, Yuan; Xu, Yanyan; Su, Yan; Zhang, Dongcheng; Wang, Lining

2015-01-01

Objective To investigate the effect of ursolic acid on autophagy mediated through the miRNA-21-targeted phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway in rat mesangial cells cultured under high glucose (HG) conditions. Methods Rat glomerular mesangial cells were cultured under normal glucose, HG, HG with the PI3K inhibitor LY294002 or HG with ursolic acid conditions. Cell proliferation and hypertrophy were assayed using an MTT assay and the ratio of total protein to cell number, respectively. The miRNA-21 expression was detected using RT-qPCR. The expression of phosphatase and tensin homolog (PTEN)/AKT/mTOR signaling signatures, autophagy-associated protein and collagen I was detected by western blotting and RT-qPCR. Autophagosomes were observed using electron microscopy. Results Compared with mesangial cells cultured under normal glucose conditions, the cells exposed to HG showed up-regulated miRNA-21 expression, down-regulated PTEN protein and mRNA expression, up-regulated p85PI3K, pAkt, pmTOR, p62/SQSTMI, and collagen I expression and down-regulated LC3II expression. Ursolic acid and LY294002 inhibited HG-induced mesangial cell hypertrophy and proliferation, down-regulated p85PI3K, pAkt, pmTOR, p62/SQSTMI, and collagen I expression and up-regulated LC3II expression. However, LY294002 did not affect the expression of miRNA-21 and PTEN. Ursolic acid down-regulated miRNA-21 expression and up-regulated PTEN protein and mRNA expression. Conclusions Ursolic acid inhibits the glucose-induced up-regulation of mesangial cell miRNA-21 expression, up-regulates PTEN expression, inhibits the activation of PI3K/Akt/mTOR signaling pathway, and enhances autophagy to reduce the accumulation of the extracellular matrix and ameliorate cell hypertrophy and proliferation. PMID:25689721

Improving power to detect changes in blood miRNA expression by accounting for sources of variability in experimental designs.

PubMed

Daniels, Sarah I; Sillé, Fenna C M; Goldbaum, Audrey; Yee, Brenda; Key, Ellen F; Zhang, Luoping; Smith, Martyn T; Thomas, Reuben

2014-12-01

Blood miRNAs are a new promising area of disease research, but variability in miRNA measurements may limit detection of true-positive findings. Here, we measured sources of miRNA variability and determine whether repeated measures can improve power to detect fold-change differences between comparison groups. Blood from healthy volunteers (N = 12) was collected at three time points. The miRNAs were extracted by a method predetermined to give the highest miRNA yield. Nine different miRNAs were quantified using different qPCR assays and analyzed using mixed models to identify sources of variability. A larger number of miRNAs from a publicly available blood miRNA microarray dataset with repeated measures were used for a bootstrapping procedure to investigate effects of repeated measures on power to detect fold changes in miRNA expression for a theoretical case-control study. Technical variability in qPCR replicates was identified as a significant source of variability (P < 0.05) for all nine miRNAs tested. Variability was larger in the TaqMan qPCR assays (SD = 0.15-0.61) versus the qScript qPCR assays (SD = 0.08-0.14). Inter- and intraindividual and extraction variability also contributed significantly for two miRNAs. The bootstrapping procedure demonstrated that repeated measures (20%-50% of N) increased detection of a 2-fold change for approximately 10% to 45% more miRNAs. Statistical power to detect small fold changes in blood miRNAs can be improved by accounting for sources of variability using repeated measures and choosing appropriate methods to minimize variability in miRNA quantification. This study demonstrates the importance of including repeated measures in experimental designs for blood miRNA research. See all the articles in this CEBP Focus section, "Biomarkers, Biospecimens, and New Technologies in Molecular Epidemiology." ©2014 American Association for Cancer Research.

PUFA diets alter the microRNA expression profiles in an inflammation rat model

PubMed Central

ZHENG, ZHENG; GE, YINLIN; ZHANG, JINYU; XUE, MEILAN; LI, QUAN; LIN, DONGLIANG; MA, WENHUI

2015-01-01

Omega-3 and -6 polyunsaturated fatty acids (PUFAs) can directly or indirectly regulate immune homeostasis via inflammatory pathways, and components of these pathways are crucial targets of microRNAs (miRNAs). However, no study has examined the changes in the miRNA transcriptome during PUFA-regulated inflammatory processes. Here, we established PUFA diet-induced autoimmune-prone (AP) and autoimmune-averse (AA) rat models, and studied their physical characteristics and immune status. Additionally, miRNA expression patterns in the rat models were compared using microarray assays and bioinformatic methods. A total of 54 miRNAs were differentially expressed in common between the AP and the AA rats, and the changes in rno-miR-19b-3p, -146b-5p and -183-5p expression were validated using stem-loop reverse transcription-quantitative polymerase chain reaction. To better understand the mechanisms underlying PUFA-regulated miRNA changes during inflammation, computational algorithms and biological databases were used to identify the target genes of the three validated miRNAs. Furthermore, Gene Ontology (GO) term annotation and KEGG pathway analyses of the miRNA targets further allowed to explore the potential implication of the miRNAs in inflammatory pathways. The predicted PUFA-regulated inflammatory pathways included the Toll-like receptor (TLR), T cell receptor (TCR), NOD-like receptor (NLR), RIG-I-like receptor (RLR), mitogen-activated protein kinase (MAPK) and the transforming growth factor-β (TGF-β) pathway. This study is the first report, to the best of our knowledge, on in vivo comparative profiling of miRNA transcriptomes in PUFA diet-induced inflammatory rat models using a microarray approach. The results provide a useful resource for future investigation of the role of PUFA-regulated miRNAs in immune homeostasis. PMID:25672643

Analysis of Antisense Expression by Whole Genome Tiling Microarrays and siRNAs Suggests Mis-Annotation of Arabidopsis Orphan Protein-Coding Genes

PubMed Central

Richardson, Casey R.; Luo, Qing-Jun; Gontcharova, Viktoria; Jiang, Ying-Wen; Samanta, Manoj; Youn, Eunseog; Rock, Christopher D.

2010-01-01

Background MicroRNAs (miRNAs) and trans-acting small-interfering RNAs (tasi-RNAs) are small (20–22 nt long) RNAs (smRNAs) generated from hairpin secondary structures or antisense transcripts, respectively, that regulate gene expression by Watson-Crick pairing to a target mRNA and altering expression by mechanisms related to RNA interference. The high sequence homology of plant miRNAs to their targets has been the mainstay of miRNA prediction algorithms, which are limited in their predictive power for other kingdoms because miRNA complementarity is less conserved yet transitive processes (production of antisense smRNAs) are active in eukaryotes. We hypothesize that antisense transcription and associated smRNAs are biomarkers which can be computationally modeled for gene discovery. Principal Findings We explored rice (Oryza sativa) sense and antisense gene expression in publicly available whole genome tiling array transcriptome data and sequenced smRNA libraries (as well as C. elegans) and found evidence of transitivity of MIRNA genes similar to that found in Arabidopsis. Statistical analysis of antisense transcript abundances, presence of antisense ESTs, and association with smRNAs suggests several hundred Arabidopsis ‘orphan’ hypothetical genes are non-coding RNAs. Consistent with this hypothesis, we found novel Arabidopsis homologues of some MIRNA genes on the antisense strand of previously annotated protein-coding genes. A Support Vector Machine (SVM) was applied using thermodynamic energy of binding plus novel expression features of sense/antisense transcription topology and siRNA abundances to build a prediction model of miRNA targets. The SVM when trained on targets could predict the “ancient” (deeply conserved) class of validated Arabidopsis MIRNA genes with an accuracy of 84%, and 76% for “new” rapidly-evolving MIRNA genes. Conclusions Antisense and smRNA expression features and computational methods may identify novel MIRNA genes and other non-coding RNAs in plants and potentially other kingdoms, which can provide insight into antisense transcription, miRNA evolution, and post-transcriptional gene regulation. PMID:20520764

Skeletal Micro-RNA Responses to Simulated Weightlessness

NASA Technical Reports Server (NTRS)

Thomas, Nicholas J.; Choi, Catherine Y.; Alwood, Joshua S.

2016-01-01

Astronauts lose bone structure during long-duration spaceflight. These changes are due, in part, to insufficient bone formation by the osteoblast cells. Little is known about the role that small (approximately 22 nucleotides), non-coding micro-RNAs (miRNAs) play in the osteoblast response to microgravity. We hypothesize that osteoblast-lineage cells alter their miRNA status during microgravity exposure, contributing to impaired bone formation during weightlessness. To simulate weightlessness, female mice (C57BL/6, Charles River, 10 weeks of age, n = 7) were hindlimb unloaded up to 12 days. Age-matched and normally ambulating mice served as controls (n=7). To assess the expression of miRNAs in skeletal tissue, the tibia was collected ex vivo and cleaned of soft-tissue and marrow. Total RNA was collected from tibial bone and relative abundance was measured for miRNAs of interest using quantitative real time PCR array looking at 372 unique and well-characterized mature miRNAs using the delta-delta Ct method. Transcripts of interest were normalized to an average of 6 reference RNAs. Preliminary results show that hindlimb unloading decreased the expression of 14 miRNAs to less than 0.5 times that of the control levels and increased the expression of 5 miRNAs relative to the control mice between 1.2-1.5-fold (p less than 0.05, respectively). Using the miRSystem we assessed overlapping target genes predicted to be regulated by multiple members of the 19 differentially expressed miRNAs as well as in silico predicted targets of our individual miRNAs. Our miRsystem results indicated that a number of our differentially expressed miRNAs were regulators of genes related to the Wnt-Beta Catenin pathway-a known regulator of bone health-and, interestingly, the estrogen-mediated cell-cycle regulation pathway, which may indicate that simulated weightlessness modulated systemic hormonal levels or hormonal transduction that additionally contributed to bone loss. We plan to follow up these findings by measuring gene expression of miRNA-regulated genes within these two pathways with the aim of furthering our understanding of the function of miRNAs in the skeletal response to spaceflight.

Serum miRNAs Signature Plays an Important Role in Keloid Disease.

PubMed

Luan, Y; Liu, Y; Liu, C; Lin, Q; He, F; Dong, X; Xiao, Z

2016-01-01

The molecular mechanism underlying the pathogenesis of keloid is largely unknown. MicroRNA (miRNA) is a class of small regulatory RNA that has emerged as a group of posttranscriptional gene repressors, participating in diverse pathophysiological processes of skin diseases. We investigated the expression profiles of miRNAs in the sera of patients to decipher the complicated factors involved in the development of keloid disease. MiRNA expression profiling in the sera from 9 keloid patients and 7 normal controls were characterized using a miRNA microarray containing established human mature and precursor miRNA sequences. Quantitative real-time PCR was performed to confirm the expression of miRNAs. The putative targets of differentially expressed miRNAs were functionally annotated by bioinformatics. MiRNA microarray analysis identified 37 differentially expressed miRNAs (17 upregulated and 20 downregulated) in keloid patients, compared to the healthy controls. Functional annotations revealed that the targets of those differentially expressed miRNAs were enriched in signaling pathways essential for scar formation and wound healing. The expression profiling of miRNAs is altered in the keloid, providing a clue for the molecular mechanisms underlying its initiation and progression. MiRNAs may partly contribute to the etiology of keloids by affecting the critical signaling pathways relevant to keloid pathogenesis.

Integrated analysis of miRNA and mRNA expression data identifies multiple miRNAs regulatory networks for the tumorigenesis of colorectal cancer.

PubMed

Xu, Peng; Wang, Junhua; Sun, Bo; Xiao, Zhongdang

2018-06-15

Investigating the potential biological function of differential changed genes through integrating multiple omics data including miRNA and mRNA expression profiles, is always hot topic. However, how to evaluate the repression effect on target genes integrating miRNA and mRNA expression profiles are not fully solved. In this study, we provide an analyzing method by integrating both miRNAs and mRNAs expression data simultaneously. Difference analysis was adopted based on the repression score, then significantly repressed mRNAs were screened out by DEGseq. Pathway analysis for the significantly repressed mRNAs shows that multiple pathways such as MAPK signaling pathway, TGF-beta signaling pathway and so on, may correlated to the colorectal cancer(CRC). Focusing on the MAPK signaling pathway, a miRNA-mRNA network that centering the cell fate genes was constructed. Finally, the miRNA-mRNAs that potentially important in the CRC carcinogenesis were screened out and scored by impact index. Copyright © 2018 Elsevier B.V. All rights reserved.

Identification of miRNAs Expression Profile in Gastric Cancer Using Self-Organizing Maps (SOM)

PubMed Central

Gomes, Larissa Luz; Moreira, Fabiano Cordeiro; Hamoy, Igor Guerreiro; Santos, Sidney; Assumpção, Paulo; Santana, Ádamo L.; Ribeiro-dos-Santos, Ândrea

2014-01-01

In this paper, an unsupervised artificial neural network was implemented to identify the patters of specific signatures. The network was based on the differential expression of miRNAs (under or over expression) found in healthy or cancerous gastric tissues. Among the tissues analyzes, the neural network evaluated 514 miRNAs of gastric tissue that exhibited significant differential expression. The result suggested a specific expression signature nine miRNAs (hsa-mir-21, hsa-mir-29a, hsa-mir-29c, hsa-mir-148a, hsa-mir-141, hsa-let-7b, hsa-mir-31, hsa-mir-451, and hsa-mir-192), all with significant values (p-value < 0.01 and fold change > 5) that clustered the samples into two groups: healthy tissue and gastric cancer tissue. The results obtained “in silico” must be validated in a molecular biology laboratory; if confirmed, this method may be used in the future as a risk marker for gastric cancer development. PMID:24966529

Differential Expression of MicroRNA and Predicted Targets in Pulmonary Sarcoidosis

PubMed Central

Crouser, Elliott D.; Julian, Mark W.; Crawford, Melissa; Shao, Guohong; Yu, Lianbo; Planck, Stephen R.; Rosenbaum, James T.; Nana-Sinkam, S. Patrick

2014-01-01

Background Recent studies show that various inflammatory diseases are regulated at the level of RNA translation by small non-coding RNAs, termed microRNAs (miRNAs). We sought to determine whether sarcoidosis tissues harbor a distinct pattern of miRNA expression and then considered their potential molecular targets. Methods and Results Genome-wide microarray analysis of miRNA expression in lung tissue and peripheral blood mononuclear cells (PBMCs) was performed and differentially expressed (DE)-miRNAs were then validated by real-time PCR. A distinct pattern of DE-miRNA expression was identified in both lung tissue and PBMCs of sarcoidosis patients. A subgroup of DE-miRNAs common to lung and lymph node tissues were predicted to target transforming growth factor (TGFβ)-regulated pathways. Likewise, the DE-miRNAs identified in PBMCs of sarcoidosis patients were predicted to target the TGFβ-regulated “wingless and integrase-1” (WNT) pathway. Conclusions This study is the first to profile miRNAs in sarcoidosis tissues and to consider their possible roles in disease pathogenesis. Our results suggest that miRNA regulate TGFβ and related WNT pathways in sarcoidosis tissues, pathways previously incriminated in the pathogenesis of sarcoidosis. PMID:22209793

Differences in microRNA expression during tumor development in the transition and peripheral zones of the prostate

PubMed Central

2013-01-01

Background The prostate is divided into three glandular zones, the peripheral zone (PZ), the transition zone (TZ), and the central zone. Most prostate tumors arise in the peripheral zone (70-75%) and in the transition zone (20-25%) while only 10% arise in the central zone. The aim of this study was to investigate if differences in miRNA expression could be a possible explanation for the difference in propensity of tumors in the zones of the prostate. Methods Patients with prostate cancer were included in the study if they had a tumor with Gleason grade 3 in the PZ, the TZ, or both (n=16). Normal prostate tissue was collected from men undergoing cystoprostatectomy (n=20). The expression of 667 unique miRNAs was investigated using TaqMan low density arrays for miRNAs. Student’s t-test was used in order to identify differentially expressed miRNAs, followed by hierarchical clustering and principal component analysis (PCA) to study the separation of the tissues. The ADtree algorithm was used to identify markers for classification of tissues and a cross-validation procedure was used to test the generality of the identified miRNA-based classifiers. Results The t-tests revealed that the major differences in miRNA expression are found between normal and malignant tissues. Hierarchical clustering and PCA based on differentially expressed miRNAs between normal and malignant tissues showed perfect separation between samples, while the corresponding analyses based on differentially expressed miRNAs between the two zones showed several misplaced samples. A classification and cross-validation procedure confirmed these results and several potential miRNA markers were identified. Conclusions The results of this study indicate that the major differences in the transcription program are those arising during tumor development, rather than during normal tissue development. In addition, tumors arising in the TZ have more unique differentially expressed miRNAs compared to the PZ. The results also indicate that separate miRNA expression signatures for diagnosis might be needed for tumors arising in the different zones. MicroRNA signatures that are specific for PZ and TZ tumors could also lead to more accurate prognoses, since tumors arising in the PZ tend to be more aggressive than tumors arising in the TZ. PMID:23890084

The expression of Argonaute2 and related microRNA biogenesis proteins in normal and hypoxic trophoblasts.

PubMed

Donker, Rogier B; Mouillet, Jean-François; Nelson, D Michael; Sadovsky, Yoel

2007-04-01

Endogenous microRNAs (miRNAs) post-transcriptionally regulate mRNA and protein expression during tissue development and function. Whereas adaptation to environmental insults are tightly regulated in human tissues, the role of miRNAs and miRNA biogenesis proteins in this context is inadequately explored. We sought to analyse the expression of the key RNAi enzyme Argonaute2 (Ago2) and other miRNA biogenesis proteins in human trophoblasts during differentiation and in hypoxic environment. Using an in vitro analysis of primary term human trophoblasts, we identified the expression of the core miRNA biogenesis proteins in human villous trophoblasts, with expression levels unaffected by cellular differentiation. We found that the miRNA biosynthetic pathway was functional and produced miRNAs, with miR-93 up-regulated and miR-424 down-regulated in hypoxic environment. In contrast, hypoxia did not alter the expression of key miRNA machinery proteins. The pivotal miRNA processing enzyme Ago2, along with its interacting protein DP103, were expressed in normal placentas as well as in placentas from pregnancies complicated by placental hypoperfusion that resulted in fetal growth restriction. Ago2 and DP103 co-immunoprecipitated, and did not limit trophoblast response to hypoxic stress. We concluded that the core miRNA machinery proteins are expressed and functional in human trophoblasts. The influence of hypoxia on the expression of a subset of placental miRNA species is unlikely to reflect altered expression of key miRNA biogenesis proteins.

Expression in Whole Blood Samples of miRNA-191 and miRNA-455-3p in Patients with AAA and Their Relationship to Clinical Outcomes after Endovascular Repair.

PubMed

Tenorio, Emanuel Junio Ramos; Braga, Andre Felipe Farias; Tirapelli, Daniela Pretti Da Cunha; Ribeiro, Mauricio Serra; Piccinato, Carlos Eli; Joviliano, Edwaldo Edner

2018-03-05

The purpose of this study was to quantify and evaluate the expression response of miRNA-191 and miRNA-455-3p endovascular repair of abdominal aortic aneurysm (AAA) based in whole blood samples. This report describes a prospective study of a single center of 30 patients with AAA who underwent endovascular repair. Blood samples were collected preoperatively and 6 months postoperatively. The differential expression of the miRNAs was performed by the real-time polymerase chain reaction method, after extraction of the RNA from the blood samples at the 2 moments. In addition, bioinformatic tools were used to determine pathophysiological pathways related to AAA. The miR-191 and miR-455-3p were overexpressed preoperatively. After 6 months postoperatively, miR-191 (median 0.98, IQR 0.5-2.1, P < 0.0001) and miR-455-3p (median 1.4, IQR 0.6-3.1, P = 0.0003) presented a significant reduction in their expressions. There was no correlation between the diameter of the aneurysm and the expression of the miRNAs studied. In addition, analysis of the influence of the various types of devices used for the endovascular treatment of AAA showed no significant differences in the expression of miR-191 and miR-455-3p. Exclusion of the aneurysmal sac after endovascular treatment induces a decrease in the expression of the studied miRNAs in whole blood samples, which suggests a possible use of them as biomarkers of therapeutic success. Copyright © 2018 Elsevier Inc. All rights reserved.

Association of Cigarette Smoking and microRNA Expression in Rectal Cancer: Insight into Tumor Phenotype

PubMed Central

Mullany, Lila E.; Herrick, Jennifer S.; Wolff, Roger K.; Stevens, John R.; Slattery, Martha L.

2016-01-01

Smoking is known to influence messenger RNA (mRNA) expression in colorectal cancer (CRC) cases. As microRNAs (miRNAs) are known repressors of mRNAs, we hypothesize that smoking may influence miRNA expression, thus altering mRNA expression. Our sample consisted of 1447 CRC cases that had normal colorectal mucosa and carcinoma miRNA data and lifestyle data. We examined current smoking, current versus never and former versus never (C/F/N) smoking1, and pack-years smoked with miRNA expression in normal mucosa as well as differential miRNA expression between paired normal and carcinoma tissue for colon and rectal tissue to determine associations between smoking and miRNA expression. We adjusted for multiple comparisons using the Benjamini Hochberg false discovery rate (FDR). Significant associations were seen for rectal differential miRNA expression only. We analyzed miRNAs significantly associated with smoking with CIMP and MSI status, using a polytomous logistic regression. Two hundred and thirty-one miRNAs were differentially expressed with current smoking, 172 with C/F/N, and 206 with pack-years smoked; 111 were associated with all three. Forty-three miRNAs were unique to current smoking, 14 were unique to C/F/N and 57 were unique to pack years smoked. Of the 306 unique miRNAs associated with cigarette smoking, 41 were inversely associated and 200 were directly associated with CIMP high or MSI tumor molecular phenotype for either colon or rectal cancer. Our results suggest that cigarette smoking can alter miRNA expression and, given associations with CIMP high and MSI tumor molecular phenotype, it is possible that smoking influences tumor phenotype through altered miRNA expression. PMID:27780077

Dysregulation of miRNAs in bladder cancer: altered expression with aberrant biogenesis procedure

PubMed Central

Dong, Fan; Xu, Tianyuan; Shen, Yifan; Zhong, Shan; Chen, Shanwen; Ding, Qiang; Shen, Zhoujun

2017-01-01

Aberrant expression profiles of miRNAs are widely observed in the clinical tissue specimens and urine samples as well as the blood samples of bladder cancer patients. These profiles are closely related to the pathological features of bladder cancer, such as the tumour stage/grade, metastasis, recurrence and chemo-sensitivity. MiRNA biogenesis forms the basis of miRNA expression and function, and its dysregulation has been shown to be essential for variations in miRNA expression profiles as well as tumourigenesis and cancer progression. In this review, we summarize the up-to-date and widely reported miRNAs in bladder cancer that display significantly altered expression. We then compare the miRNA expression profiles among three different sample types (tissue, urine and blood) from patients with bladder cancer. Moreover, for the first time, we outline the dysregulated miRNA biogenesis network in bladder cancer from different levels and analyse its possible relationship with aberrant miRNA expression and the pathological characteristics of the disease. PMID:28187437

Prediction of miRNA-mRNA associations in Alzheimer's disease mice using network topology.

PubMed

Noh, Haneul; Park, Charny; Park, Soojun; Lee, Young Seek; Cho, Soo Young; Seo, Hyemyung

2014-08-03

Little is known about the relationship between miRNA and mRNA expression in Alzheimer's disease (AD) at early- or late-symptomatic stages. Sequence-based target prediction algorithms and anti-correlation profiles have been applied to predict miRNA targets using omics data, but this approach often leads to false positive predictions. Here, we applied the joint profiling analysis of mRNA and miRNA expression levels to Tg6799 AD model mice at 4 and 8 months of age using a network topology-based method. We constructed gene regulatory networks and used the PageRank algorithm to predict significant interactions between miRNA and mRNA. In total, 8 cluster modules were predicted by the transcriptome data for co-expression networks of AD pathology. In total, 54 miRNAs were identified as being differentially expressed in AD. Among these, 50 significant miRNA-mRNA interactions were predicted by integrating sequence target prediction, expression analysis, and the PageRank algorithm. We identified a set of miRNA-mRNA interactions that were changed in the hippocampus of Tg6799 AD model mice. We determined the expression levels of several candidate genes and miRNA. For functional validation in primary cultured neurons from Tg6799 mice (MT) and littermate (LM) controls, the overexpression of ARRDC3 enhanced PPP1R3C expression. ARRDC3 overexpression showed the tendency to decrease the expression of miR139-5p and miR3470a in both LM and MT primary cells. Pathological environment created by Aβ treatment increased the gene expression of PPP1R3C and Sfpq but did not significantly alter the expression of miR139-5p or miR3470a. Aβ treatment increased the promoter activity of ARRDC3 gene in LM primary cells but not in MT primary cells. Our results demonstrate AD-specific changes in the miRNA regulatory system as well as the relationship between the expression levels of miRNAs and their targets in the hippocampus of Tg6799 mice. These data help further our understanding of the function and mechanism of various miRNAs and their target genes in the molecular pathology of AD.

Simultaneous visualization of the subfemtomolar expression of microRNA and microRNA target gene using HILO microscopy† †Electronic supplementary information (ESI) available: The LED device for the sample photobleaching, a schematic presentation of HILO microscopy, fluorescence spectra and hybridization curves of the molecular beacons, the linear correlation between the miRNA fluorescence intensity and the miRNA copy number, a validation of the miRNA adsorption and miRNA target gene expression via RT-qPCR, a validation of RT-qPCR using capillary electrophoresis, the reproducibility of RT-qPCR and Poisson distribution of the miRNA pipetting as well as a complete list of the oligonucleotides used in this study. See DOI: 10.1039/c7sc02701j Click here for additional data file.

PubMed Central

Lin, Yi-Zhen; Ou, Da-Liang; Chang, Hsin-Yuan; Lin, Wei-Yu; Hsu, Chiun

2017-01-01

The family of microRNAs (miRNAs) not only plays an important role in gene regulation but is also useful for the diagnosis of diseases. A reliable method with high sensitivity may allow researchers to detect slight fluctuations in ultra-trace amounts of miRNA. In this study, we propose a sensitive imaging method for the direct probing of miR-10b (miR-10b-3p, also called miR-10b*) and its target (HOXD10 mRNA) in fixed cells based on the specific recognition of molecular beacons combined with highly inclined and laminated optical sheet (HILO) fluorescence microscopy. The designed dye-quencher-labelled molecular beacons offer excellent efficiencies of fluorescence resonance energy transfer that allow us to detect miRNA and the target mRNA simultaneously in hepatocellular carcinoma cells using HILO fluorescence microscopy. Not only can the basal trace amount of miRNA be observed in each individual cell, but the obtained images also indicate that this method is useful for monitoring the fluctuations in ultra-trace amounts of miRNA when the cells are transfected with a miRNA precursor or a miRNA inhibitor (anti-miR). Furthermore, a reasonable causal relation between the miR-10b and HOXD10 expression levels was observed in miR-10b* precursor-transfected cells and miR-10b* inhibitor-transfected cells. The trends of the miRNA alterations obtained using HILO microscopy completely matched the RT-qPCR data and showed remarkable reproducibility (the coefficient of variation [CV] = 0.86%) and sensitivity (<1.0 fM). This proposed imaging method appears to be useful for the simultaneous visualisation of ultra-trace amounts of miRNA and target mRNA and excludes the procedures for RNA extraction and amplification. Therefore, the visualisation of miRNA and the target mRNA should facilitate the exploration of the functions of ultra-trace amounts of miRNA in fixed cells in biological studies and may serve as a powerful tool for diagnoses based on circulating cancer cells. PMID:28989695

Identification and profiling of Cyprinus carpio microRNAs during ovary differentiation by deep sequencing.

PubMed

Wang, Fang; Jia, Yongfang; Wang, Po; Yang, Qianwen; Du, QiYan; Chang, ZhongJie

2017-04-28

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that regulate gene expression by targeting specific mRNAs. However, the possible role of miRNAs in the ovary differentiation and development of fish is not well understood. In this study, we examined the expression profiles and differential expression of miRNAs during three key stages of ovarian development and different developmental stages in common carp Cyprinus carpio. A total of 8765 miRNAs were identified, including 2155 conserved miRNAs highly conserved among various species, 145 miRNAs registered in miRBase for common carp, and 6505 novel miRNAs identified in common carp for the first time. Comparison of miRNA expression profiles among the five libraries identified 714 co-expressed and 2382 specific expressed miRNAs. Overall, 150, 628, and 431 specifically expressed miRNAs were identified in primordial gonad, juvenile ovary, and adult ovary, respectively. MiR-6758-3p, miR-3050-5p, and miR-2985-3p were highly expressed in primordial gonad, miR-3544-5p, miR-6877-3p, and miR-9086-5p were highly expressed in juvenile ovary, and miR-154-3p, miR-5307-5p, and miR-3958-3p were highly expressed in adult ovary. Predicted target genes of specific miRNAs in primordial gonad were involved in many reproductive biology signaling pathways, including transforming growth factor-β, Wnt, oocyte meiosis, mitogen-activated protein kinase, Notch, p53, and gonadotropin-releasing hormone pathways. Target-gene prediction revealed upward trends in miRNAs targeting male-bias genes, including dmrt1, atm, gsdf, and sox9, and downward trends in miRNAs targeting female-bias genes including foxl2, smad3, and smad4. Other sex-related genes such as sf1 were also predicted to be miRNA target genes. This comprehensive miRNA transcriptome analysis demonstrated differential expression profiles of miRNAs during ovary development in common carp. These results could facilitate future exploitation of the sex-regulatory roles and mechanisms of miRNAs, especially in primordial gonads, while the specifically expressed miRNAs represent candidates for studying the mechanisms of ovary determination in Yellow River carp.

An improved method for detecting circulating microRNAs with S-Poly(T) Plus real-time PCR

PubMed Central

Niu, Yanqin; Zhang, Limin; Qiu, Huiling; Wu, Yike; Wang, Zhiwei; Zai, Yujia; Liu, Lin; Qu, Junle; Kang, Kang; Gou, Deming

2015-01-01

We herein describe a simple, sensitive and specific method for analysis of circulating microRNAs (miRNA), termed S-Poly(T) Plus real-time PCR assay. This new method is based on our previously developed S-Poly(T) method, in which a unique S-Poly(T) primer is used during reverse-transcription to increase sensitivity and specificity. Further increased sensitivity and simplicity of S-Poly(T) Plus, in comparison with the S-Poly(T) method, were achieved by a single-step, multiple-stage reaction, where RNAs were polyadenylated and reverse-transcribed at the same time. The sensitivity of circulating miRNA detection was further improved by a modified method of total RNA isolation from serum/plasma, S/P miRsol, in which glycogen was used to increase the RNA yield. We validated our methods by quantifying miRNA expression profiles in the sera of the patients with pulmonary arterial hypertension associated with congenital heart disease. In conclusion, we developed a simple, sensitive, and specific method for detecting circulating miRNAs that allows the measurement of 266 miRNAs from 100 μl of serum or plasma. This method presents a promising tool for basic miRNA research and clinical diagnosis of human diseases based on miRNA biomarkers. PMID:26459910

Identification of a Polyomavirus microRNA Highly Expressed in Tumors

PubMed Central

Chen, Chun Jung; Cox, Jennifer E.; Azarm, Kristopher; Wylie, Karen N.; Woolard, Kevin D.; Pesavento, Patricia A.; Sullivan, Christopher S.

2014-01-01

Polyomaviruses (PyVs) are associated with tumors including Merkel cell carcinoma (MCC). Several PyVs encode microRNAs (miRNAs) but to date no abundant PyV miRNAs have been reported in tumors. To better understand the function of the Merkel cell PyV (MCPyV) miRNA, we examined phylogenetically-related viruses for miRNA expression. We show that two primate PyVs and the more distantly-related raccoon PyV (RacPyV) encode miRNAs that share genomic position and partial sequence identity with MCPyV miRNAs. Unlike MCPyV miRNA in MCC, RacPyV miRNA is highly abundant in raccoon tumors. RacPyV miRNA negatively regulates reporters of early viral (T antigen) transcripts, yet robust viral miRNA expression is tolerated in tumors. We also identify raccoon miRNAs expressed in RacPyV-associated neuroglial brain tumors, including several likely oncogenic miRNAs (oncomiRs). This work describes the first PyV miRNA abundantly expressed in tumors and is consistent with a possible role for both host and viral miRNAs in RacPyV-associated tumors. PMID:25514573

Finding quasi-modules of human and viral miRNAs: a case study of human cytomegalovirus (HCMV)

PubMed Central

2012-01-01

Background MicroRNAs (miRNAs) are important regulators of gene expression encoded by a variety of organisms, including viruses. Although the function of most of the viral miRNAs is currently unknown, there is evidence that both viral and host miRNAs contribute to the interactions between viruses and their hosts. miRNAs constitute a complex combinatorial network, where one miRNA may target many genes and one gene may be targeted by multiple miRNAs. In particular, viral and host miRNAs may also have mutual target genes. Based on published evidence linking viral and host miRNAs there are three modes of mutual regulation: competing, cooperating, and compensating modes. Results In this paper we explore the compensating mode of mutual regulation upon Human Cytomegalovirus (HCMV) infection, when host miRNAs are down regulated and viral miRNAs compensate by mimicking their function. To achieve this, we develop a new algorithm which finds groups, called quasi-modules, of viral and host miRNAs and their mutual target genes, and use a new host miRNA expression data for HCMV-infected and uninfected cells. For two of the reported quasi-modules, supporting evidence from biological and medical literature is provided. Conclusions The modules found by our method may advance the understanding of the role of miRNAs in host-viral interactions, and the genes in these modules may serve as candidates for further experimental validation. PMID:23206407

Literature-based condition-specific miRNA-mRNA target prediction.

PubMed

Oh, Minsik; Rhee, Sungmin; Moon, Ji Hwan; Chae, Heejoon; Lee, Sunwon; Kang, Jaewoo; Kim, Sun

2017-01-01

miRNAs are small non-coding RNAs that regulate gene expression by binding to the 3'-UTR of genes. Many recent studies have reported that miRNAs play important biological roles by regulating specific mRNAs or genes. Many sequence-based target prediction algorithms have been developed to predict miRNA targets. However, these methods are not designed for condition-specific target predictions and produce many false positives; thus, expression-based target prediction algorithms have been developed for condition-specific target predictions. A typical strategy to utilize expression data is to leverage the negative control roles of miRNAs on genes. To control false positives, a stringent cutoff value is typically set, but in this case, these methods tend to reject many true target relationships, i.e., false negatives. To overcome these limitations, additional information should be utilized. The literature is probably the best resource that we can utilize. Recent literature mining systems compile millions of articles with experiments designed for specific biological questions, and the systems provide a function to search for specific information. To utilize the literature information, we used a literature mining system, BEST, that automatically extracts information from the literature in PubMed and that allows the user to perform searches of the literature with any English words. By integrating omics data analysis methods and BEST, we developed Context-MMIA, a miRNA-mRNA target prediction method that combines expression data analysis results and the literature information extracted based on the user-specified context. In the pathway enrichment analysis using genes included in the top 200 miRNA-targets, Context-MMIA outperformed the four existing target prediction methods that we tested. In another test on whether prediction methods can re-produce experimentally validated target relationships, Context-MMIA outperformed the four existing target prediction methods. In summary, Context-MMIA allows the user to specify a context of the experimental data to predict miRNA targets, and we believe that Context-MMIA is very useful for predicting condition-specific miRNA targets.

Association of microRNA-200c expression levels with clinicopathological factors and prognosis in endometrioid endometrial cancer.

PubMed

Wilczynski, Milosz; Danielska, Justyna; Domanska-Senderowska, Daria; Dzieniecka, Monika; Szymanska, Bozena; Malinowski, Andrzej

2018-05-01

MicroRNAs (miRNAs) are regulators of gene expression, which play an important role in many critical cellular processes including apoptosis, proliferation and cell differentiation. Aberrant miRNA expression has been reported in a variety of human malignancies. Therefore, miRNAs may be potentially used as cancer biomarkers. miRNA-200c, which is a member of the miRNA-200 family, might play an essential role in tumor progression. The purpose of this study was to evaluate the prognostic and clinical significance of miRNA-200c in women with endometrioid endometrial cancer. Total RNA extraction from 90 archival formalin-fixed paraffin-embedded tissue samples of endometri-oid endometrial cancer and 10 normal endometrium samples was performed. After cDNA synthesis, real-time polymerase chain reaction was conducted and relative expression of miRNA-200c was assessed. Then, miRNA-200c expression levels were evaluated with regard to clinicopathological characteristics. The expression levels of miRNA-200c were significantly increased in endometrioid endometrial cancer samples. Expression of miRNA-200c maintained at significantly higher levels in the early stage endometrioid endometrial cancer compared with more advanced stages. In the Kaplan-Meier analysis, lower levels of miRNA-200c expression were associated with inferior survival. Expression levels of miRNA-200c might be associated with clinicopathological factors and survival in endometrioid endometrial cancer. © 2018 Nordic Federation of Societies of Obstetrics and Gynecology.

MicroRNAs hsa-miR-99b, hsa-miR-330, hsa-miR-126 and hsa-miR-30c: Potential Diagnostic Biomarkers in Natural Killer (NK) Cells of Patients with Chronic Fatigue Syndrome (CFS)/ Myalgic Encephalomyelitis (ME)

PubMed Central

Petty, Robert D.; McCarthy, Neil E.; Le Dieu, Rifca; Kerr, Jonathan R.

2016-01-01

Background Chronic Fatigue Syndrome (CFS/ME) is a complex multisystem disease of unknown aetiology which causes debilitating symptoms in up to 1% of the global population. Although a large cohort of genes have been shown to exhibit altered expression in CFS/ME patients, it is currently unknown whether microRNA (miRNA) molecules which regulate gene translation contribute to disease pathogenesis. We hypothesized that changes in microRNA expression in patient leukocytes contribute to CFS/ME pathology, and may therefore represent useful diagnostic biomarkers that can be detected in the peripheral blood of CFS/ME patients. Methods miRNA expression in peripheral blood mononuclear cells (PBMC) from CFS/ME patients and healthy controls was analysed using the Ambion Bioarray V1. miRNA demonstrating differential expression were validated by qRT-PCR and then replicated in fractionated blood leukocyte subsets from an independent patient cohort. The CFS/ME associated miRNA identified by these experiments were then transfected into primary NK cells and gene expression analyses conducted to identify their gene targets. Results Microarray analysis identified differential expression of 34 miRNA, all of which were up-regulated. Four of the 34 miRNA had confirmed expression changes by qRT-PCR. Fractionating PBMC samples by cell type from an independent patient cohort identified changes in miRNA expression in NK-cells, B-cells and monocytes with the most significant abnormalities occurring in NK cells. Transfecting primary NK cells with hsa-miR-99b or hsa-miR-330-3p, resulted in gene expression changes consistent with NK cell activation but diminished cytotoxicity, suggesting that defective NK cell function contributes to CFS/ME pathology. Conclusion This study demonstrates altered microRNA expression in the peripheral blood mononuclear cells of CFS/ME patients, which are potential diagnostic biomarkers. The greatest degree of miRNA deregulation was identified in NK cells with targets consistent with cellular activation and altered effector function. PMID:26967895

MicroRNA-146b-5p Identified in Porcine Liver Donation Model is Associated with Early Allograft Dysfunction in Human Liver Transplantation

PubMed Central

Li, Cheukfai; Zhao, Qiang; Zhang, Wei; Chen, Maogen; Ju, Weiqiang; Wu, Linwei; Han, Ming; Ma, Yi; Zhu, Xiaofeng; Wang, Dongping; Guo, Zhiyong; He, Xiaoshun

2017-01-01

Background Poor transplant outcome was observed in donation after brain death followed by circulatory death (DBCD), since the donor organs suffered both cytokine storm of brain death and warm ischemia injury. MicroRNAs (miRNAs) have emerged as promising disease biomarkers, so we sought to establish a miRNA signature of porcine DBCD and verify the findings in human liver transplantation. Material/Methods MiRNA expression was determined with miRNA sequencing in 3 types of the porcine model of organ donation, including donation after brain death (DBD) group, donation after circulatory death (DCD) group, and DBCD group. Bioinformatics analysis was performed to reveal the potential regulatory behavior of target miRNA. Human liver graft biopsy samples after reperfusion detected by fluorescence in situ hybridization were used to verify the expression of target miRNA. Results We compared miRNA expression profiles of the 3 donation types. The porcine liver graft miR-146b was significantly increased and selected in the DBCD group versus in the DBD and DCD groups. The donor liver expression of human miR-146b-5p, which is homologous to porcine miR-146b, was further examined in 42 cases of human liver transplantations. High expression of miR-146b-5p successfully predicted the post-transplant early allograft dysfunction (EAD) with the area under the ROC curve (AUC) 0.759 (P=0.004). Conclusions Our results revealed the miRNA signature of DBCD liver grafts for the first time. The miR-146b-5p may have important clinical implications for monitoring liver graft function and predicating transplant outcomes. PMID:29227984

Titanium and Zirconium Levels Are Associated with Changes in MicroRNAs Expression: Results from a Human Cross-Sectional Study on Obese Population

PubMed Central

Dioni, Laura; Angelici, Laura; Vigna, Luisella; Farronato, Giampietro; Pesatori, Angela Cecilia; Bollati, Valentina

2016-01-01

Objectives In this study on 90 individuals we aimed at evaluating the microRNAs (miRNAs) expression profile associated with personal levels of Titanium (Ti) and Zirconium (Zr) traced in hair samples. Ti and Zr materials are broadly used for dental implants but the biological reactions triggered by a long term presence of these materials in the oral cavity still need to be assessed. MiRNAs are mechanisms that need to be investigated as they play a fundamental role in the control of gene expression following external stimuli and contribute to a wide range of pathophysiological processes. Methods Using the TaqMan® Low-Density Array, we assessed the expression levels of 377 human miRNAs in peripheral blood of 90 subjects. Hair samples were analyzed for Ti and Zr content using Inductively Coupled Plasma-Mass Spectrometry. We performed multivariable regression analysis to investigate the effects of Ti and Zr exposure on miRNA expression levels. We used the Ingenuity Pathway Analysis (IPA) software to explore the functional role of the investigated miRNAs and the related target genes. Results Seven miRNAs (miR-99b, miR-142-5p, miR-152, miR-193a-5p, miR-323-3p, miR-335, miR-494) resulted specifically associated with Zr levels. The functional target analysis showed that miRNAs are involved in mechanisms such as inflammation, skeletal and connective tissue disorders. Conclusions Our data suggest that Zr is more bioactive than Ti and show that miRNAs are relevant molecular mechanisms sensitive to Zr exposure. PMID:27611787

microRNA-342, microRNA-191 and microRNA-510 are differentially expressed in T regulatory cells of type 1 diabetic patients.

PubMed

Hezova, Renata; Slaby, Ondrej; Faltejskova, Petra; Mikulkova, Zuzana; Buresova, Ivana; Raja, K R Muthu; Hodek, Jan; Ovesna, Jaroslava; Michalek, Jaroslav

2010-01-01

Regulatory T cells (Tregs) are critical regulators of autoimmune diseases, including type 1 diabetes mellitus. It is hypothesised that Tregs' function can be influenced by changes in the expression of specific microRNAs (miRNAs). Thus, we performed miRNAs profiling in a population of Tregs separated from peripheral blood of five type 1 diabetic patients and six healthy donors. For more detailed molecular characterisation of Tregs, we additionally compared miRNAs expression profiles of Tregs and conventional T cells. Tregs were isolated according to CD3+, CD4+, CD25(hi)+ and CD127- by flow cytometry, and miRNA expression profiling was performed using TaqMan Array Human MicroRNA Panel-1 (384-well low density array). In Tregs of diabetic patients we found significantly increased expression of miRNA-510 (p=0.05) and decreased expression of both miRNA-342 (p<0.0001) and miRNA-191 (p=0.0079). When comparing Tregs and T cells, we revealed that Tregs had significant higher expression of miRNA-146a and lower expression of eight specific miRNAs (20b, 31, 99a, 100, 125b, 151, 335, and 365). To our knowledge, this is the first study demonstrating changes in miRNA expression profiles occurring in Tregs of T1D patients and a miRNAs signature of adult Tregs.

Comparisons of serum miRNA expression profiles in patients with diabetic retinopathy and type 2 diabetes mellitus.

PubMed

Ma, Jianping; Wang, Jufang; Liu, Yanfen; Wang, Changyi; Duan, Donghui; Lu, Nanjia; Wang, Kaiyue; Zhang, Lu; Gu, Kaibo; Chen, Sihan; Zhang, Tao; You, Dingyun; Han, Liyuan

2017-02-01

The aim of this study was to compare the expression levels of serum miRNAs in diabetic retinopathy and type 2 diabetes mellitus. Serum miRNA expression profiles from diabetic retinopathy cases (type 2 diabetes mellitus patients with diabetic retinopathy) and type 2 diabetes mellitus controls (type 2 diabetes mellitus patients without diabetic retinopathy) were examined by miRNA-specific microarray analysis. Quantitative real-time polymerase chain reaction was used to validate the significantly differentially expressed serum miRNAs from the microarray analysis of 45 diabetic retinopathy cases and 45 age-, sex-, body mass index- and duration-of-diabetes-matched type 2 diabetes mellitus controls. The relative changes in serum miRNA expression levels were analyzed using the 2-ΔΔCt method. A total of 5 diabetic retinopathy cases and 5 type 2 diabetes mellitus controls were included in the miRNA-specific microarray analysis. The serum levels of miR-3939 and miR-1910-3p differed significantly between the two groups in the screening stage; however, quantitative real-time polymerase chain reaction did not reveal significant differences in miRNA expression for 45 diabetic retinopathy cases and their matched type 2 diabetes mellitus controls. Our findings indicate that miR-3939 and miR-1910-3p may not play important roles in the development of diabetic retinopathy; however, studies with a larger sample size are needed to confirm our findings.

Normalization matters: tracking the best strategy for sperm miRNA quantification.

PubMed

Corral-Vazquez, Celia; Blanco, Joan; Salas-Huetos, Albert; Vidal, Francesca; Anton, Ester

2017-01-01

What is the most reliable normalization strategy for sperm microRNA (miRNA) quantitative Reverse Transcription Polymerase Chain Reactions (qRT-PCR) using singleplex assays? The use of the average expression of hsa-miR-100-5p and hsa-miR-30a-5p as sperm miRNA qRT-PCR data normalizer is suggested as an optimal strategy. Mean-centering methods are the most reliable normalization strategies for miRNA high-throughput expression analyses. Nevertheless, specific trustworthy reference controls must be established in singleplex sperm miRNA qRT-PCRs. Cycle threshold (Ct) values from previously published sperm miRNA expression profiles were normalized using four approaches: (i) Mean-Centering Restricted (MCR) method (taken as the reference strategy); (ii) expression of the small nuclear RNA RNU6B; (iii) expression of four miRNAs selected by the Concordance Correlation Restricted (CCR) algorithm: hsa-miR-100-5p, hsa-miR-146b-5p, hsa-miR-92a-3p and hsa-miR-30a-5p; (iv) the combination of two of these miRNAs that achieved the highest proximity to MCR. Expression profile data from 736 sperm miRNAs were taken from previously published studies performed in fertile donors (n = 10) and infertile patients (n = 38). For each tested normalizer molecule, expression ubiquity and uniformity across the different samples and populations were assessed as indispensable requirements for being considered as valid candidates. The reliability of the different normalizing strategies was compared to MCR based on the set of differentially expressed miRNAs (DE-miRNAs) detected between populations, the corresponding predicted targets and the associated enriched biological processes. All tested normalizers were found to be ubiquitous and non-differentially expressed between populations. RNU6B was the least uniformly expressed candidate across samples. Data normalization through RNU6B led to dramatically misguided results when compared to MCR outputs, with a null prediction of target genes and enriched biological processes. Hsa-miR-146b-5p and hsa-miR-92a-3p were more uniformly expressed than RNU6B, but their results still showed scant proximity to the reference method. The highest resemblance to MCR was achieved by hsa-miR-100-5p and hsa-miR-30a-5p. Normalization against the combination of both miRNAs reached the best proximity rank regarding the detected DE-miRNAs (Area Under the Curve = 0.8). This combination also exhibited the best performance in terms of the target genes predicted (72.3% of True Positives) and their corresponding enriched biological processes (70.4% of True Positives). Not applicable. This study is focused on sperm miRNA qRT-PCR analysis. The use of the selected normalizers in other cell types or tissues would still require confirmation. The search for new fertility biomarkers based on sperm miRNA expression using high-throughput assays is one of the upcoming challenges in the field of reproductive genetics. In this context, validation of the results using singleplex assays would be mandatory. The normalizer strategy suggested in this study would provide a universal option in this area, allowing for normalization of the validated data without causing meaningful variations of the results. Instead, qRT-PCR data normalization by RNU6B should be discarded in sperm-miRNA expression studies. This work was supported by the 2014/SGR00524 project (Agència de Gestió d'Ajuts Universitaris i de Recerca, Generalitat de Catalunya, Spain) and UAB CF-180034 grant (Universitat Autònoma de Barcelona). Celia Corral-Vazquez is a recipient of a Personal Investigador en Formació grant UAB/PIF2015 (Universitat Autònoma de Barcelona). The authors report no conflict of interest. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A Novel Persistence Associated EBV miRNA Expression Profile Is Disrupted in Neoplasia

PubMed Central

Qiu, Jin; Cosmopoulos, Katherine; Pegtel, Michiel; Hopmans, Erik; Murray, Paul; Middeldorp, Jaap; Shapiro, Michael; Thorley-Lawson, David A.

2011-01-01

We have performed the first extensive profiling of Epstein-Barr virus (EBV) miRNAs on in vivo derived normal and neoplastic infected tissues. We describe a unique pattern of viral miRNA expression by normal infected cells in vivo expressing restricted viral latency programs (germinal center: Latency II and memory B: Latency I/0). This includes the complete absence of 15 of the 34 miRNAs profiled. These consist of 12 BART miRNAs (including approximately half of Cluster 2) and 3 of the 4 BHRF1 miRNAs. All but 2 of these absent miRNAs become expressed during EBV driven growth (Latency III). Furthermore, EBV driven growth is accompanied by a 5–10 fold down regulation in the level of the BART miRNAs expressed in germinal center and memory B cells. Therefore, Latency III also expresses a unique pattern of viral miRNAs. We refer to the miRNAs that are specifically expressed in EBV driven growth as the Latency III associated miRNAs. In EBV associated tumors that employ Latency I or II (Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma and gastric carcinoma), the Latency III associated BART but not BHRF1 miRNAs are up regulated. Thus BART miRNA expression is deregulated in the EBV associated tumors. This is the first demonstration that Latency III specific genes (the Latency III associated BARTs) can be expressed in these tumors. The EBV associated tumors demonstrate very similar patterns of miRNA expression yet were readily distinguished when the expression data were analyzed either by heat-map/clustering or principal component analysis. Systematic analysis revealed that the information distinguishing the tumor types was redundant and distributed across all the miRNAs. This resembles “secret sharing” algorithms where information can be distributed among a large number of recipients in such a way that any combination of a small number of recipients is able to understand the message. Biologically, this may be a consequence of functional redundancy between the miRNAs. PMID:21901094

miRNA-556-3p promotes human bladder cancer proliferation, migration and invasion by negatively regulating DAB2IP expression.

PubMed

Feng, Chen; Sun, Ping; Hu, Jing; Feng, Hua; Li, Mingqiu; Liu, Guibo; Pan, Yanming; Feng, Ying; Xu, Yongliang; Feng, Kejian; Feng, Yukuan

2017-06-01

MicroRNAs (miRNAs) play critical roles in tumorigenesis and metastasis by negatively regulating gene expression through complementary binding to the 3'-untranslated region of target mRNAs. The role of miRNAs in expression of the tumor suppressor DAB2IP in bladder cancer (BC) remains unknown. The aim of the present study was to identify miRNAs targeting DAB2IP and determine their expression and function in BC. We predicted candidate miRNAs targeting DAB2IP using TargetScan software. Dual-luciferase reporter assays confirmed that miRNA-556-3p directly regulated DAB2IP expression. Quantitative RT-PCR and RNase protection assays showed that endogenous miRNA-556-3p expression was significantly upregulated in clinical samples of BC patients and BC cell lines and western blot analysis indicated that DAB2IP expression in BC tissues and BC cell lines was concurrently downregulated. Gain or loss of function studies showed that upregulation of miRNA-556-3p promoted proliferation, invasion, migration and colony formation of BC cells, whereas downregulation resulted in opposite effects. Importantly, restoration of DAB2IP expression rescued the effects induced by miRNA-556-3p. Overexpression of miRNA-556-3p in BC cells not only decreased DAB2IP expression, but also markedly increased Ras GTPase activity and ERK1/2 phosphorylation level. These findings suggest that DAB2IP is a direct target of miRNA-556-3p, and endogenous miRNA-556-3p expression shows inverse correlation with simultaneous DAB2IP expression in BC tissues and cells. miRNA-556-3p functions as a tumor promoter in tumorigenesis and metastasis of BC by targeting DAB2IP. Moreover, miRNA-556-3p-mediated DAB2IP suppression plays an oncogenic role by partial activation of the Ras-ERK pathway.

Identification of Mouse Serum miRNA Endogenous References by Global Gene Expression Profiles

PubMed Central

Mi, Qing-Sheng; Weiland, Matthew; Qi, Rui-Qun; Gao, Xing-Hua; Poisson, Laila M.; Zhou, Li

2012-01-01

MicroRNAs (miRNAs) are recently discovered small non-coding RNAs and can serve as serum biomarkers for disease diagnosis and prognoses. Lack of reliable serum miRNA endogenous references for normalization in miRNA gene expression makes single miRNA assays inaccurate. Using TaqMan® real-time PCR miRNA arrays with a global gene expression normalization strategy, we have analyzed serum miRNA expression profiles of 20 female mice of NOD/ShiLtJ (n = 8), NOR/LtJ (n = 6), and C57BL/6J (n = 6) at different ages and disease conditions. We identified five miRNAs, miR-146a, miR-16, miR-195, miR-30e and miR-744, to be stably expressed in all strains, which could serve as mouse serum miRNA endogenous references for single assay experiments. PMID:22348064

An integrated expression atlas of miRNAs and their promoters in human and mouse

PubMed Central

de Rie, Derek; Abugessaisa, Imad; Alam, Tanvir; Arner, Erik; Arner, Peter; Ashoor, Haitham; Åström, Gaby; Babina, Magda; Bertin, Nicolas; Burroughs, A. Maxwell; Carlisle, Ailsa J.; Daub, Carsten O.; Detmar, Michael; Deviatiiarov, Ruslan; Fort, Alexandre; Gebhard, Claudia; Goldowitz, Daniel; Guhl, Sven; Ha, Thomas J.; Harshbarger, Jayson; Hasegawa, Akira; Hashimoto, Kosuke; Herlyn, Meenhard; Heutink, Peter; Hitchens, Kelly J.; Hon, Chung Chau; Huang, Edward; Ishizu, Yuri; Kai, Chieko; Kasukawa, Takeya; Klinken, Peter; Lassmann, Timo; Lecellier, Charles-Henri; Lee, Weonju; Lizio, Marina; Makeev, Vsevolod; Mathelier, Anthony; Medvedeva, Yulia A.; Mejhert, Niklas; Mungall, Christopher J.; Noma, Shohei; Ohshima, Mitsuhiro; Okada-Hatakeyama, Mariko; Persson, Helena; Rizzu, Patrizia; Roudnicky, Filip; Sætrom, Pål; Sato, Hiroki; Severin, Jessica; Shin, Jay W.; Swoboda, Rolf K.; Tarui, Hiroshi; Toyoda, Hiroo; Vitting-Seerup, Kristoffer; Winteringham, Louise; Yamaguchi, Yoko; Yasuzawa, Kayoko; Yoneda, Misako; Yumoto, Noriko; Zabierowski, Susan; Zhang, Peter G.; Wells, Christine A.; Summers, Kim M.; Kawaji, Hideya; Sandelin, Albin; Rehli, Michael; Hayashizaki, Yoshihide; Carninci, Piero; Forrest, Alistair R. R.; de Hoon, Michiel J. L.

2018-01-01

MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libraries, with matching Cap Analysis Gene Expression (CAGE) data, from 396 human and 47 mouse RNA samples. Promoters were identified for 1,357 human and 804 mouse miRNAs and showed strong sequence conservation between species. We also found that primary and mature miRNA expression levels were correlated, allowing us to use the primary miRNA measurements as a proxy for mature miRNA levels in a total of 1,829 human and 1,029 mouse CAGE libraries. We thus provide a broad atlas of miRNA expression and promoters in primary mammalian cells, establishing a foundation for detailed analysis of miRNA expression patterns and transcriptional control regions. PMID:28829439

Microrna expression signatures predict patient progression and disease outcome in pediatric embryonal central nervous system neoplasms.

PubMed

Braoudaki, Maria; Lambrou, George I; Giannikou, Krinio; Milionis, Vasilis; Stefanaki, Kalliopi; Birks, Diane K; Prodromou, Neophytos; Kolialexi, Aggeliki; Kattamis, Antonis; Spiliopoulou, Chara A; Tzortzatou-Stathopoulou, Fotini; Kanavakis, Emmanouel

2014-12-31

Although, substantial experimental evidence related to diagnosis and treatment of pediatric central nervous system (CNS) neoplasms have been demonstrated, the understanding of the etiology and pathogenesis of the disease remains scarce. Recent microRNA (miRNA)-based research reveals the involvement of miRNAs in various aspects of CNS development and proposes that they might compose key molecules underlying oncogenesis. The current study evaluated miRNA differential expression detected between pediatric embryonal brain tumors and normal controls to characterize candidate biomarkers related to diagnosis, prognosis and therapy. Overall, 19 embryonal brain tumors; 15 Medulloblastomas (MBs) and 4 Atypical Teratoid/Rabdoid Tumors (AT/RTs) were studied. As controls, 13 samples were used; The First-Choice Human Brain Reference RNA and 12 samples from deceased children who underwent autopsy and were not present with any brain malignancy. RNA extraction was carried out using the Trizol method, whilst miRNA extraction was performed with the mirVANA miRNA isolation kit. The experimental approach included miRNA microarrays covering 1211 miRNAs. Quantitative Real-Time Polymerase Chain Reaction was performed to validate the expression profiles of miR-34a and miR-601 in all 32 samples initially screened with miRNA microarrays and in an additional independent cohort of 30 patients (21MBs and 9 AT/RTs). Moreover, meta-analyses was performed in total 27 embryonal tumor samples; 19 MBs, 8 ATRTs and 121 control samples. Twelve germinomas were also used as an independent validation cohort. All deregulated miRNAs were correlated to patients' clinical characteristics and pathological measures. In several cases, there was a positive correlation between individual miRNA expression levels and laboratory or clinical characteristics. Based on that, miR-601 could serve as a putative tumor suppressor gene, whilst miR-34a as an oncogene. In general, miR-34a demonstrated oncogenic roles in all pediatric embryonal CNS neoplasms studied. Deeper understanding of the aberrant miRNA expression in pediatric embryonal brain tumors might aid in the development of tumor-specific miRNA signatures, which could potentially afford promising biomarkers related to diagnosis, prognosis and patient targeted therapy.

Over-expression of the miRNA cluster at chromosome 14q32 in the alcoholic brain correlates with suppression of predicted target mRNA required for oligodendrocyte proliferation.

PubMed

Manzardo, A M; Gunewardena, S; Butler, M G

2013-09-10

We examined miRNA expression from RNA isolated from the frontal cortex (Broadman area 9) of 9 alcoholics (6 males, 3 females, mean age 48 years) and 9 matched controls using both the Affymetrix GeneChip miRNA 2.0 and Human Exon 1.0 ST Arrays to further characterize genetic influences in alcoholism and the effects of alcohol consumption on predicted target mRNA expression. A total of 12 human miRNAs were significantly up-regulated in alcohol dependent subjects (fold change≥1.5, false discovery rate (FDR)≤0.3; p<0.05) compared with controls including a cluster of 4 miRNAs (e.g., miR-377, miR-379) from the maternally expressed 14q32 chromosome region. The status of the up-regulated miRNAs was supported using the high-throughput method of exon microarrays showing decreased predicted mRNA gene target expression as anticipated from the same RNA aliquot. Predicted mRNA targets were involved in cellular adhesion (e.g., THBS2), tissue differentiation (e.g., CHN2), neuronal migration (e.g., NDE1), myelination (e.g., UGT8, CNP) and oligodendrocyte proliferation (e.g., ENPP2, SEMA4D1). Our data support an association of alcoholism with up-regulation of a cluster of miRNAs located in the genomic imprinted domain on chromosome 14q32 with their predicted gene targets involved with oligodendrocyte growth, differentiation and signaling. Copyright © 2013 Elsevier B.V. All rights reserved.

Analysis of miRNA expression profile based on SVM algorithm

NASA Astrophysics Data System (ADS)

Ting-ting, Dai; Chang-ji, Shan; Yan-shou, Dong; Yi-duo, Bian

2018-05-01

Based on mirna expression spectrum data set, a new data mining algorithm - tSVM - KNN (t statistic with support vector machine - k nearest neighbor) is proposed. the idea of the algorithm is: firstly, the feature selection of the data set is carried out by the unified measurement method; Secondly, SVM - KNN algorithm, which combines support vector machine (SVM) and k - nearest neighbor (k - nearest neighbor) is used as classifier. Simulation results show that SVM - KNN algorithm has better classification ability than SVM and KNN alone. Tsvm - KNN algorithm only needs 5 mirnas to obtain 96.08 % classification accuracy in terms of the number of mirna " tags" and recognition accuracy. compared with similar algorithms, tsvm - KNN algorithm has obvious advantages.

microRNA Expression Profiling: Technologies, Insights, and Prospects.

PubMed

Roden, Christine; Mastriano, Stephen; Wang, Nayi; Lu, Jun

2015-01-01

Since the early days of microRNA (miRNA) research, miRNA expression profiling technologies have provided important tools toward both better understanding of the biological functions of miRNAs and using miRNA expression as potential diagnostics. Multiple technologies, such as microarrays, next-generation sequencing, bead-based detection system, single-molecule measurements, and quantitative RT-PCR, have enabled accurate quantification of miRNAs and the subsequent derivation of key insights into diverse biological processes. As a class of ~22 nt long small noncoding RNAs, miRNAs present unique challenges in expression profiling that require careful experimental design and data analyses. We will particularly discuss how normalization and the presence of miRNA isoforms can impact data interpretation. We will present one example in which the consideration in data normalization has provided insights that helped to establish the global miRNA expression as a tumor suppressor. Finally, we discuss two future prospects of using miRNA profiling technologies to understand single cell variability and derive new rules for the functions of miRNA isoforms.

Connecting rules from paired miRNA and mRNA expression data sets of HCV patients to detect both inverse and positive regulatory relationships

PubMed Central

2015-01-01

Background Intensive research based on the inverse expression relationship has been undertaken to discover the miRNA-mRNA regulatory modules involved in the infection of Hepatitis C virus (HCV), the leading cause of chronic liver diseases. However, biological studies in other fields have found that inverse expression relationship is not the only regulatory relationship between miRNAs and their targets, and some miRNAs can positively regulate a mRNA by binding at the 5' UTR of the mRNA. Results This work focuses on the detection of both inverse and positive regulatory relationships from a paired miRNA and mRNA expression data set of HCV patients through a 'change-to-change' method which can derive connected discriminatory rules. Our study uncovered many novel miRNA-mRNA regulatory modules. In particular, it was revealed that GFRA2 is positively regulated by miR-557, miR-765 and miR-17-3p that probably bind at different locations of the 5' UTR of this mRNA. The expression relationship between GFRA2 and any of these three miRNAs has not been studied before, although separate research for this gene and these miRNAs have all drawn conclusions linked to hepatocellular carcinoma. This suggests that the binding of mRNA GFRA2 with miR-557, miR-765, or miR-17-3p, or their combinations, is worthy of further investigation by experimentation. We also report another mRNA QKI which has a strong inverse expression relationship with miR-129 and miR-493-3p which may bind at the 3' UTR of QKI with a perfect sequence match. Furthermore, the interaction between hsa-miR-129-5p (previous ID: hsa-miR-129) and QKI is supported with CLIP-Seq data from starBase. Our method can be easily extended for the expression data analysis of other diseases. Conclusion Our rule discovery method is useful for integrating binding information and expression profile for identifying HCV miRNA-mRNA regulatory modules and can be applied to the study of the expression profiles of other complex human diseases. PMID:25707620

Xenopus microRNA genes are predominantly located within introns and are differentially expressed in adult frog tissues via post-transcriptional regulation

PubMed Central

Tang, Guo-Qing; Maxwell, E. Stuart

2008-01-01

The amphibian Xenopus provides a model organism for investigating microRNA expression during vertebrate embryogenesis and development. Searching available Xenopus genome databases using known human pre-miRNAs as query sequences, more than 300 genes encoding 142 Xenopus tropicalis miRNAs were identified. Analysis of Xenopus tropicalis miRNA genes revealed a predominate positioning within introns of protein-coding and nonprotein-coding RNA Pol II-transcribed genes. MiRNA genes were also located in pre-mRNA exons and positioned intergenically between known protein-coding genes. Many miRNA species were found in multiple locations and in more than one genomic context. MiRNA genes were also clustered throughout the genome, indicating the potential for the cotranscription and coordinate expression of miRNAs located in a given cluster. Northern blot analysis confirmed the expression of many identified miRNAs in both X. tropicalis and X. laevis. Comparison of X. tropicalis and X. laevis blots revealed comparable expression profiles, although several miRNAs exhibited species-specific expression in different tissues. More detailed analysis revealed that for some miRNAs, the tissue-specific expression profile of the pri-miRNA precursor was distinctly different from that of the mature miRNA profile. Differential miRNA precursor processing in both the nucleus and cytoplasm was implicated in the observed tissue-specific differences. These observations indicated that post-transcriptional processing plays an important role in regulating miRNA expression in the amphibian Xenopus. PMID:18032731

Solexa Sequencing of Novel and Differentially Expressed MicroRNAs in Testicular and Ovarian Tissues in Holstein Cattle

PubMed Central

Huang, Jinming; Ju, Zhihua; Li, Qiuling; Hou, Qinlei; Wang, Changfa; Li, Jianbin; Li, Rongling; Wang, Lingling; Sun, Tao; Hang, Suqin; Gao, Yundong; Hou, Minghai; Zhong, Jifeng

2011-01-01

The posttranscriptional gene regulation mediated by microRNA plays an important role in the development and function of male and female reproductive organs and germ cells in mammals, including cattle. In the present study, we identified novel and differentially expressed miRNAs in the testis and ovary in Holstein cattle by combining the Solexa sequencing with bioinformatics. In total 100 and 104 novel pre-miRNAs were identified in testicular and ovarian tissues, encoding 122 and 136 mature miRNAs, respectively. Of these, 6 miRNAs appear to be bovine-specific. A total of 246 known miRNAs were co-expressed in the testicular and ovarian tissues. Of the known miRNAs, twenty-one testis-specific and nine ovary-specific (1-23 reads) were found. Approximately 30.5% of the known bovine miRNAs in this study were found to have >2-fold differential expression within the two respective reproductive organ systems. The putative miRNA target genes of miRNAs were involved in pathways associated with reproductive physiology. Both known and novel tissue-specific miRNAs are expressed by Real-time quantitative PCR analysis in dairy cattle. This study expands the number of miRNAs known to be expressed in cattle. The patterns of miRNAs expression differed significantly between the bovine testicular and ovarian tissues, which provide important information on sex differences in miRNA expression. Diverse miRNAs may play an important regulatory role in the development of the reproductive organs in Holstein cattle. PMID:21912509

Simultaneous visualization of the subfemtomolar expression of microRNA and microRNA target gene using HILO microscopy.

PubMed

Lin, Yi-Zhen; Ou, Da-Liang; Chang, Hsin-Yuan; Lin, Wei-Yu; Hsu, Chiun; Chang, Po-Ling

2017-09-01

The family of microRNAs (miRNAs) not only plays an important role in gene regulation but is also useful for the diagnosis of diseases. A reliable method with high sensitivity may allow researchers to detect slight fluctuations in ultra-trace amounts of miRNA. In this study, we propose a sensitive imaging method for the direct probing of miR-10b (miR-10b-3p, also called miR-10b*) and its target ( HOXD10 mRNA) in fixed cells based on the specific recognition of molecular beacons combined with highly inclined and laminated optical sheet (HILO) fluorescence microscopy. The designed dye-quencher-labelled molecular beacons offer excellent efficiencies of fluorescence resonance energy transfer that allow us to detect miRNA and the target mRNA simultaneously in hepatocellular carcinoma cells using HILO fluorescence microscopy. Not only can the basal trace amount of miRNA be observed in each individual cell, but the obtained images also indicate that this method is useful for monitoring the fluctuations in ultra-trace amounts of miRNA when the cells are transfected with a miRNA precursor or a miRNA inhibitor (anti-miR). Furthermore, a reasonable causal relation between the miR-10b and HOXD10 expression levels was observed in miR-10b* precursor-transfected cells and miR-10b* inhibitor-transfected cells. The trends of the miRNA alterations obtained using HILO microscopy completely matched the RT-qPCR data and showed remarkable reproducibility (the coefficient of variation [CV] = 0.86%) and sensitivity (<1.0 fM). This proposed imaging method appears to be useful for the simultaneous visualisation of ultra-trace amounts of miRNA and target mRNA and excludes the procedures for RNA extraction and amplification. Therefore, the visualisation of miRNA and the target mRNA should facilitate the exploration of the functions of ultra-trace amounts of miRNA in fixed cells in biological studies and may serve as a powerful tool for diagnoses based on circulating cancer cells.

Expression Profiling Analysis Reveals Key MicroRNA–mRNA Interactions in Early Retinal Degeneration in Retinitis Pigmentosa

PubMed Central

Anasagasti, Ander; Ezquerra-Inchausti, Maitane; Barandika, Olatz; Muñoz-Culla, Maider; Caffarel, María M.; Otaegui, David; López de Munain, Adolfo

2018-01-01

Purpose The aim of this study was to identify differentially expressed microRNAs (miRNAs) that might play an important role in the etiology of retinal degeneration in a genetic mouse model of retinitis pigmentosa (rd10 mice) at initial stages of the disease. Methods miRNAs–mRNA interaction networks were generated for analysis of biological pathways involved in retinal degeneration. Results Of more than 1900 miRNAs analyzed, we selected 19 miRNAs on the basis of (1) a significant differential expression in rd10 retinas compared with control samples and (2) an inverse expression relationship with predicted mRNA targets involved in biological pathways relevant to retinal biology and/or degeneration. Seven of the selected miRNAs have been associated with retinal dystrophies, whereas, to our knowledge, nine have not been previously linked to any disease. Conclusions This study contributes to our understanding of the etiology and progression of retinal degeneration. PMID:29847644

Cloning and analysis of fetal ovary microRNAs in cattle.

PubMed

Tripurani, Swamy K; Xiao, Caide; Salem, Mohamed; Yao, Jianbo

2010-07-01

Ovarian folliculogenesis and early embryogenesis are complex processes, which require tightly regulated expression and interaction of a multitude of genes. Small endogenous RNA molecules, termed microRNAs (miRNAs), are involved in the regulation of gene expression during folliculogenesis and early embryonic development. To identify miRNAs in bovine oocytes/ovaries, a bovine fetal ovary miRNA library was constructed. Sequence analysis of random clones from the library identified 679 miRNA sequences, which represent 58 distinct bovine miRNAs. Of these distinct miRNAs, 42 are known bovine miRNAs present in the miRBase database and the remaining 16 miRNAs include 15 new bovine miRNAs that are homologous to miRNAs identified in other species, and one novel miRNA, which does not match any miRNAs in the database. The precursor sequences for 14 of the new 15 miRNAs as well as the novel miRNA were identified from the bovine genome database and their hairpin structures were predicted. Expression analysis of the 58 miRNAs in fetal ovaries in comparison to somatic tissue pools identified 8 miRNAs predominantly expressed in fetal ovaries. Further analysis of the eight miRNAs in germinal vesicle (GV) stage oocytes identified two miRNAs (bta-mir424 and bta-mir-10b), that are highly abundant in GV oocytes. Both miRNAs show similar expression patterns during oocyte maturation and preimplantation development of bovine embryos, being abundant in GV and MII stage oocytes, as well as in early stage embryos (until 16-cell stage). The amount of the novel miRNA is relatively small in oocytes and early cleavage embryos but greater in blastocysts, suggesting a role of this miRNA in blastocyst cell differentiation. Copyright 2010 Elsevier B.V. All rights reserved.

Pre-microRNA and Mature microRNA in Human Mitochondria

PubMed Central

Barrey, Eric; Saint-Auret, Gaelle; Bonnamy, Blandine; Damas, Dominique; Boyer, Orane; Gidrol, Xavier

2011-01-01

Background Because of the central functions of the mitochondria in providing metabolic energy and initiating apoptosis on one hand and the role that microRNA (miRNA) play in gene expression, we hypothesized that some miRNA could be present in the mitochondria for post-transcriptomic regulation by RNA interference. We intend to identify miRNA localized in the mitochondria isolated from human skeletal primary muscular cells. Methodology/Principal Findings To investigate the potential origin of mitochondrial miRNA, we in-silico searched for microRNA candidates in the mtDNA. Twenty five human pre-miRNA and 33 miRNA aligments (E-value<0.1) were found in the reference mitochondrial sequence and some of the best candidates were chosen for a co-localization test. In situ hybridization of pre-mir-302a, pre-let-7b and mir-365, using specific labelled locked nucleic acids and confocal microscopy, demonstrated that these miRNA were localized in mitochondria of human myoblasts. Total RNA was extracted from enriched mitochondria isolated by an immunomagnetic method from a culture of human myotubes. The detection of 742 human miRNA (miRBase) were monitored by RT-qPCR at three increasing mtRNA inputs. Forty six miRNA were significantly expressed (2nd derivative method Cp>35) for the smallest RNA input concentration and 204 miRNA for the maximum RNA input concentration. In silico analysis predicted 80 putative miRNA target sites in the mitochondrial genome (E-value<0.05). Conclusions/Significance The present study experimentally demonstrated for the first time the presence of pre-miRNA and miRNA in the human mitochondria isolated from skeletal muscular cells. A set of miRNA were significantly detected in mitochondria fraction. The origin of these pre-miRNA and miRNA should be further investigate to determine if they are imported from the cytosol and/or if they are partially processed in the mitochondria. PMID:21637849

miRNA-15a, miRNA-15b, and miRNA-499 are Reduced in Erythrocytes of Pre-Diabetic African-American Adults.

PubMed

Fluitt, Maurice B; Kumari, Namita; Nunlee-Bland, Gail; Nekhai, Sergei; Gambhir, Kanwal K

2016-12-01

The use of circulatory miRNAs as biomarkers and therapeutic targets for T2DM is an explosive area of study. However, no study has investigated circulatory miRNA expression exclusively in African-American adults. The aim of this study was to identify the expression of nine selected miRNAs in erythrocytes of pre-diabetic and type 2 diabetic African-American adults. Patients were recruited from the Howard University Hospital Diabetes Treatment Center following an 8 to 10 hour overnight fast. Expression of the nine selected miRNAs (miRNA-499, miRNA-146, miRNA-126, miRNA-223, miRNA-15a, miRNA-15b, miRNA-224, miRNA-326, and miRNA-375) was evaluated using quantitative real time PCR. miRNA-15a, miRNA-15b, and miRNA-499 were significantly reduced in erythrocytes of pre-diabetic African-American adults. In the T2DM group, we found significant correlations between miRNA-15a and BMI (r=0.59, p=0.04), miRNA-15a and weight (r=0.52, p=0.01), and miRNA-15b and diastolic blood pressure (r=-0.52, p=0.02). In the pre-diabetic group, we found significant correlations between miRNA-15b and weight (r=0.90, p=0.02) and miRNA-499 and HbA1c (r=-0.89, p=0.01). To our knowledge, this is the first study investigating miRNA expression in erythrocytes of non-diabetic high-risk obese--pre-diabetic and type 2 diabetic African-American adults. The findings of this study are consistent with previous reports of reduced expression of miRNA-15a, miRNA-15b, and miRNA-499 in human plasma or serum and in animal models. The current findings support the use of circulating miRNA-15a, miRNA-15b, and miRNA-499 as potential biomarkers for T2DM in African-American adults.

Differential miRNA expression in B cells is associated with inter-individual differences in humoral immune response to measles vaccination

PubMed Central

Haralambieva, Iana H.; Kennedy, Richard B.; Simon, Whitney L.; Goergen, Krista M.; Grill, Diane E.; Ovsyannikova, Inna G.

2018-01-01

Background MicroRNAs are important mediators of post-transcriptional regulation of gene expression through RNA degradation and translational repression, and are emerging biomarkers of immune system activation/response after vaccination. Methods We performed Next Generation Sequencing (mRNA-Seq) of intracellular miRNAs in measles virus-stimulated B and CD4+ T cells from high and low antibody responders to measles vaccine. Negative binomial generalized estimating equation (GEE) models were used for miRNA assessment and the DIANA tool was used for gene/target prediction and pathway enrichment analysis. Results We identified a set of B cell-specific miRNAs (e.g., miR-151a-5p, miR-223, miR-29, miR-15a-5p, miR-199a-3p, miR-103a, and miR-15a/16 cluster) and biological processes/pathways, including regulation of adherens junction proteins, Fc-receptor signaling pathway, phosphatidylinositol-mediated signaling pathway, growth factor signaling pathway/pathways, transcriptional regulation, apoptosis and virus-related processes, significantly associated with neutralizing antibody titers after measles vaccination. No CD4+ T cell-specific miRNA expression differences between high and low antibody responders were found. Conclusion Our study demonstrates that miRNA expression directly or indirectly influences humoral immunity to measles vaccination and suggests that B cell-specific miRNAs may serve as useful predictive biomarkers of vaccine humoral immune response. PMID:29381765

Identification and consequences of miRNA-target interactions--beyond repression of gene expression.

PubMed

Hausser, Jean; Zavolan, Mihaela

2014-09-01

Comparative genomics analyses and high-throughput experimental studies indicate that a microRNA (miRNA) binds to hundreds of sites across the transcriptome. Although the knockout of components of the miRNA biogenesis pathway has profound phenotypic consequences, most predicted miRNA targets undergo small changes at the mRNA and protein levels when the expression of the miRNA is perturbed. Alternatively, miRNAs can establish thresholds in and increase the coherence of the expression of their target genes, as well as reduce the cell-to-cell variability in target gene expression. Here, we review the recent progress in identifying miRNA targets and the emerging paradigms of how miRNAs shape the dynamics of target gene expression.

Integrative analysis of multi-omics data for identifying multi-markers for diagnosing pancreatic cancer

PubMed Central

2015-01-01

Background microRNA (miRNA) expression plays an influential role in cancer classification and malignancy, and miRNAs are feasible as alternative diagnostic markers for pancreatic cancer, a highly aggressive neoplasm with silent early symptoms, high metastatic potential, and resistance to conventional therapies. Methods In this study, we evaluated the benefits of multi-omics data analysis by integrating miRNA and mRNA expression data in pancreatic cancer. Using support vector machine (SVM) modelling and leave-one-out cross validation (LOOCV), we evaluated the diagnostic performance of single- or multi-markers based on miRNA and mRNA expression profiles from 104 PDAC tissues and 17 benign pancreatic tissues. For selecting even more reliable and robust markers, we performed validation by independent datasets from the Gene Expression Omnibus (GEO) and the Cancer Genome Atlas (TCGA) data depositories. For validation, miRNA activity was estimated by miRNA-target gene interaction and mRNA expression datasets in pancreatic cancer. Results Using a comprehensive identification approach, we successfully identified 705 multi-markers having powerful diagnostic performance for PDAC. In addition, these marker candidates annotated with cancer pathways using gene ontology analysis. Conclusions Our prediction models have strong potential for the diagnosis of pancreatic cancer. PMID:26328610

Comprehensive analysis of the functional microRNA–mRNA regulatory network identifies miRNA signatures associated with glioma malignant progression

PubMed Central

Li, Yongsheng; Xu, Juan; Chen, Hong; Bai, Jing; Li, Shengli; Zhao, Zheng; Shao, Tingting; Jiang, Tao; Ren, Huan; Kang, Chunsheng; Li, Xia

2013-01-01

Glioma is the most common and fatal primary brain tumour with poor prognosis; however, the functional roles of miRNAs in glioma malignant progression are insufficiently understood. Here, we used an integrated approach to identify miRNA functional targets during glioma malignant progression by combining the paired expression profiles of miRNAs and mRNAs across 160 Chinese glioma patients, and further constructed the functional miRNA–mRNA regulatory network. As a result, most tumour-suppressive miRNAs in glioma progression were newly discovered, whose functions were widely involved in gliomagenesis. Moreover, three miRNA signatures, with different combinations of hub miRNAs (regulations≥30) were constructed, which could independently predict the survival of patients with all gliomas, high-grade glioma and glioblastoma. Our network-based method increased the ability to identify the prognostic biomarkers, when compared with the traditional method and random conditions. Hsa-miR-524-5p and hsa-miR-628-5p, shared by these three signatures, acted as protective factors and their expression decreased gradually during glioma progression. Functional analysis of these miRNA signatures highlighted their critical roles in cell cycle and cell proliferation in glioblastoma malignant progression, especially hsa-miR-524-5p and hsa-miR-628-5p exhibited dominant regulatory activities. Therefore, network-based biomarkers are expected to be more effective and provide deep insights into the molecular mechanism of glioma malignant progression. PMID:24194606

Evolutionary conserved microRNAs are ubiquitously expressed compared to tick-specific miRNAs in the cattle tick Rhipicephalus (Boophilus) microplus

PubMed Central

2011-01-01

Background MicroRNAs (miRNAs) are small non-coding RNAs that act as regulators of gene expression in eukaryotes modulating a large diversity of biological processes. The discovery of miRNAs has provided new opportunities to understand the biology of a number of species. The cattle tick, Rhipicephalus (Boophilus) microplus, causes significant economic losses in cattle production worldwide and this drives us to further understand their biology so that effective control measures can be developed. To be able to provide new insights into the biology of cattle ticks and to expand the repertoire of tick miRNAs we utilized Illumina technology to sequence the small RNA transcriptomes derived from various life stages and selected organs of R. microplus. Results To discover and profile cattle tick miRNAs we employed two complementary approaches, one aiming to find evolutionary conserved miRNAs and another focused on the discovery of novel cattle-tick specific miRNAs. We found 51 evolutionary conserved R. microplus miRNA loci, with 36 of these previously found in the tick Ixodes scapularis. The majority of the R. microplus miRNAs are perfectly conserved throughout evolution with 11, 5 and 15 of these conserved since the Nephrozoan (640 MYA), Protostomian (620MYA) and Arthropoda (540 MYA) ancestor, respectively. We then employed a de novo computational screening for novel tick miRNAs using the draft genome of I. scapularis and genomic contigs of R. microplus as templates. This identified 36 novel R. microplus miRNA loci of which 12 were conserved in I. scapularis. Overall we found 87 R. microplus miRNA loci, of these 15 showed the expression of both miRNA and miRNA* sequences. R. microplus miRNAs showed a variety of expression profiles, with the evolutionary-conserved miRNAs mainly expressed in all life stages at various levels, while the expression of novel tick-specific miRNAs was mostly limited to particular life stages and/or tick organs. Conclusions Anciently acquired miRNAs in the R. microplus lineage not only tend to accumulate the least amount of nucleotide substitutions as compared to those recently acquired miRNAs, but also show ubiquitous expression profiles through out tick life stages and organs contrasting with the restricted expression profiles of novel tick-specific miRNAs. PMID:21699734

MicroRNA221-3p modulates Ets-1 expression in synovial fibroblasts from patients with osteoarthritis of temporomandibular joint.

PubMed

Xu, J; Liu, Y; Deng, M; Li, J; Cai, H; Meng, Q; Fang, W; Long, X; Ke, J

2016-11-01

This study aimed to screen differential expression of microRNAs (miRNAs), and investigate function of the specifically selected miRNA in synovial fibroblasts from patients suffering osteoarthritis of temporomandibular joint (TMJOA). MiRNA microarray was used to select differentially expressed miRNAs between TMJOA and normal synovial fibroblasts. The expression of screened miRNA221-3p was quantified using real-time PCR, and its specific target gene was predicted by bioinformatics. After transfection of miRNA221-3p mimics or inhibitor into synovial fibroblasts, the expression of v-Ets avian erythroblastosis virus E26 oncogene homolog 1 (Ets-1) was detected by immunohistochemistry, real-time PCR and Western blot, respectively. Dual luciferase activity was performed to identify the direct regulation of miRNA221-3p on Ets-1. Interlukin-1β (IL-1β) mimics an inflammatory situation. In TMJOA synovial fibroblasts, eight miRNAs were up-regulated and six miRNAs were down-regulated. MiRNA221-3p was the most down-expressed. A sequence in the 3'-untranslated (3'-UTR) of Ets-1 complementary to the seed sequence of miRNA221-3p. Elevated expression of Ets-1 associated with attenuation of miRNA221-3p. Over-expression of miRNA221-3p suppressed the activity of a reporter construct containing the 3'-UTR of Ets-1 transcript and inhibited the expression of Ets-1 as well as its downstream molecules, matrix metalloproteinase 1 (MMP1) and MMP9 in TMJOA synovial fibroblasts. IL-1β suppressed the expression of miRNA221-3p in both a dose-dependent and time-dependent manner. The reduction of miRNA221-3p in synovial fibroblasts, attributed from abundance of IL-1β in inflamed circumstance, induces Ets-1 up-regulation and then, initiates MMP1 and MMP9 secretion, thereby leading to continuously pathological development in TMJOA. Copyright © 2016 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Identification of novel, highly expressed retroviral microRNAs in cells infected by bovine foamy virus.

PubMed

Whisnant, Adam W; Kehl, Timo; Bao, Qiuying; Materniak, Magdalena; Kuzmak, Jacek; Löchelt, Martin; Cullen, Bryan R

2014-05-01

While numerous viral microRNAs (miRNAs) expressed by DNA viruses, especially herpesvirus family members, have been reported, there have been very few reports of miRNAs derived from RNA viruses. Here we describe three miRNAs expressed by bovine foamy virus (BFV), a member of the spumavirus subfamily of retroviruses, in both BFV-infected cultured cells and BFV-infected cattle. All three viral miRNAs are initially expressed in the form of an ∼ 122-nucleotide (nt) pri-miRNA, encoded within the BFV long terminal repeat U3 region, that is subsequently cleaved to generate two pre-miRNAs that are then processed to yield three distinct, biologically active miRNAs. The BFV pri-miRNA is transcribed by RNA polymerase III, and the three resultant mature miRNAs were found to contribute a remarkable ∼ 70% of all miRNAs expressed in BFV-infected cells. These data document the second example of a retrovirus that is able to express viral miRNAs by using embedded proviral RNA polymerase III promoters. Foamy viruses are a ubiquitous family of nonpathogenic retroviruses that have potential as gene therapy vectors in humans. Here we demonstrate that bovine foamy virus (BFV) expresses high levels of three viral microRNAs (miRNAs) in BFV-infected cells in culture and also in infected cattle. The BFV miRNAs are unusual in that they are initially transcribed by RNA polymerase III as a single, ∼ 122-nt pri-miRNA that is subsequently processed to release three fully functional miRNAs. The observation that BFV, a foamy virus, is able to express viral miRNAs in infected cells adds to emerging evidence that miRNA expression is a common, albeit clearly not universal, property of retroviruses and suggests that these miRNAs may exert a significant effect on viral replication in vivo.

Identification of Novel, Highly Expressed Retroviral MicroRNAs in Cells Infected by Bovine Foamy Virus

PubMed Central

Whisnant, Adam W.; Kehl, Timo; Bao, Qiuying; Materniak, Magdalena; Kuzmak, Jacek; Löchelt, Martin

2014-01-01

ABSTRACT While numerous viral microRNAs (miRNAs) expressed by DNA viruses, especially herpesvirus family members, have been reported, there have been very few reports of miRNAs derived from RNA viruses. Here we describe three miRNAs expressed by bovine foamy virus (BFV), a member of the spumavirus subfamily of retroviruses, in both BFV-infected cultured cells and BFV-infected cattle. All three viral miRNAs are initially expressed in the form of an ∼122-nucleotide (nt) pri-miRNA, encoded within the BFV long terminal repeat U3 region, that is subsequently cleaved to generate two pre-miRNAs that are then processed to yield three distinct, biologically active miRNAs. The BFV pri-miRNA is transcribed by RNA polymerase III, and the three resultant mature miRNAs were found to contribute a remarkable ∼70% of all miRNAs expressed in BFV-infected cells. These data document the second example of a retrovirus that is able to express viral miRNAs by using embedded proviral RNA polymerase III promoters. IMPORTANCE Foamy viruses are a ubiquitous family of nonpathogenic retroviruses that have potential as gene therapy vectors in humans. Here we demonstrate that bovine foamy virus (BFV) expresses high levels of three viral microRNAs (miRNAs) in BFV-infected cells in culture and also in infected cattle. The BFV miRNAs are unusual in that they are initially transcribed by RNA polymerase III as a single, ∼122-nt pri-miRNA that is subsequently processed to release three fully functional miRNAs. The observation that BFV, a foamy virus, is able to express viral miRNAs in infected cells adds to emerging evidence that miRNA expression is a common, albeit clearly not universal, property of retroviruses and suggests that these miRNAs may exert a significant effect on viral replication in vivo. PMID:24522910

Novel prediction of anticancer drug chemosensitivity in cancer cell lines: evidence of moderation by microRNA expressions.

PubMed

Yang, Daniel S

2014-01-01

The objectives of this study are (1) to develop a novel "moderation" model of drug chemosensitivity and (2) to investigate if miRNA expression moderates the relationship between gene expression and drug chemosensitivity, specifically for HSP90 inhibitors applied to human cancer cell lines. A moderation model integrating the interaction between miRNA and gene expressions was developed to examine if miRNA expression affects the strength of the relationship between gene expression and chemosensitivity. Comprehensive datasets on miRNA expressions, gene expressions, and drug chemosensitivities were obtained from National Cancer Institute's NCI-60 cell lines including nine different cancer types. A workflow including steps of selecting genes, miRNAs, and compounds, correlating gene expression with chemosensitivity, and performing multivariate analysis was utilized to test the proposed model. The proposed moderation model identified 12 significantly-moderating miRNAs: miR-15b*, miR-16-2*, miR-9, miR-126*, miR-129*, miR-138, miR-519e*, miR-624*, miR-26b, miR-30e*, miR-32, and miR-196a, as well as two genes ERCC2 and SF3B1 which affect chemosensitivities of Tanespimycin and Alvespimycin - both HSP90 inhibitors. A bootstrap resampling of 2,500 times validates the significance of all 12 identified miRNAs. The results confirm that certain miRNA and gene expressions interact to produce an effect on drug response. The lack of correlation between miRNA and gene expression themselves suggests that miRNA transmits its effect through translation inhibition/control rather than mRNA degradation. The results suggest that miRNAs could serve not only as prognostic biomarkers for cancer treatment outcome but also as interventional agents to modulate desired chemosensitivity.

Characterization and identification of differentially expressed microRNAs during the process of the peribiliary fibrosis induced by Clonorchis sinensis.

PubMed

Yan, Chao; Shen, Li-Ping; Ma, Rui; Li, Bo; Li, Xiang-Yang; Hua, Hui; Zhang, Bo; Yu, Qian; Wang, Yu-Gang; Tang, Ren-Xian; Zheng, Kui-Yang

2016-09-01

Clonorchis sinensis (C. sinensis) infection can lead to biliary fibrosis. MicroRNAs (miRNAs) play important roles in regulation of genes expression in the liver diseases. However, the differential expression of miRNAs that probably regulates the portal fibrogenesis caused by C. sinensis has not yet been investigated. Hepatic miRNAs expression profiles from C. sinensis-infected mice at different time-points were analyzed by miRNA microarray and validated by quantitative real-time PCR (qRT-PCR). 349 miRNAs were differentially expressed in the liver of the C. sinensis-infected mice at 2, 8 or 16weeks post infection (p.i.), compared with those at 0week p.i., and there were 143 down-regulated and 206 up-regulated miRNAs among them. These all dysregulated miRNAs were potentially involved in the pathological processes of clonorchiasis by regulation of cancer-related signaling pathway, TGF-β signaling pathway, MAPK signaling pathway, Toll-like receptor signaling pathway, PI3K /AKT signaling pathway, etc. 169 of these dysregulated miRNAs were predicted to be involved in the TGF/Smads signaling pathway which plays an important role in the biliary fibrosis caused by C. sinensis. Additionally, miRNA-32, miRNA-34a, miRNA-125b and miRNA-497 were negatively correlated with Smad7 expression, indicating these miRNAs may specifically down-regulate Smad7 expression and participate in regulation of biliary fibrosis caused by C. sinensis. The results of the present study for the first time demonstrated that miRNAs were differentially expressed in the liver of mice infected by C. sinensis, and these miRNAs may play important roles in regulation of peribiliary fibrosis caused by C. sinensis, which may provide possible therapeutic targets for clonorchiasis. Copyright © 2016 Elsevier B.V. All rights reserved.

Diversity, expression and mRNA targeting abilities of Argonaute-targeting miRNAs among selected vascular plants.

PubMed

Jagtap, Soham; Shivaprasad, Padubidri V

2014-12-02

Micro (mi)RNAs are important regulators of plant development. Across plant lineages, Dicer-like 1 (DCL1) proteins process long ds-like structures to produce micro (mi) RNA duplexes in a stepwise manner. These miRNAs are incorporated into Argonaute (AGO) proteins and influence expression of RNAs that have sequence complementarity with miRNAs. Expression levels of AGOs are greatly regulated by plants in order to minimize unwarranted perturbations using miRNAs to target mRNAs coding for AGOs. AGOs may also have high promoter specificity-sometimes expression of AGO can be limited to just a few cells in a plant. Viral pathogens utilize various means to counter antiviral roles of AGOs including hijacking the host encoded miRNAs to target AGOs. Two host encoded miRNAs namely miR168 and miR403 that target AGOs have been described in the model plant Arabidopsis and such a mechanism is thought to be well conserved across plants because AGO sequences are well conserved. We show that the interaction between AGO mRNAs and miRNAs is species-specific due to the diversity in sequences of two miRNAs that target AGOs, sequence diversity among corresponding target regions in AGO mRNAs and variable expression levels of these miRNAs among vascular plants. We used miRNA sequences from 68 plant species representing 31 plant families for this analysis. Sequences of miR168 and miR403 are not conserved among plant lineages, but surprisingly they differ drastically in their sequence diversity and expression levels even among closely related plants. Variation in miR168 expression among plants correlates well with secondary structures/length of loop sequences of their precursors. Our data indicates a complex AGO targeting interaction among plant lineages due to miRNA sequence diversity and sequences of miRNA targeting regions among AGO mRNAs, thus leading to the assumption that the perturbations by viruses that use host miRNAs to target antiviral AGOs can only be species-specific. We also show that rapid evolution and likely loss of expression of miR168 isoforms in tobacco is related to the insertion of MITE-like transposons between miRNA and miRNA* sequences, a possible mechanism showing how miRNAs are lost in few plant lineages even though other close relatives have abundantly expressing miRNAs.

Isolation and identification of gene-specific microRNAs.

PubMed

Lin, Shi-Lung; Chang, Donald C; Ying, Shao-Yao

2006-01-01

Prediction of microRNA (miRNA) candidates using computer programming has identified hundreds and hundreds of genomic hairpin sequences, of which, the functions remain to be determined. Because direct transfection of hairpin-like miRNA precursors (pre)-miRNAs in mammalian cells is not always sufficient to trigger effective RNA-induced gene-silencing complex (RISC) assembly, a key step for RNA interference (RNAi)-related gene silencing, we developed an intronic miRNA-expressing system to overcome this problem, and successfully increased the efficiency and effectiveness of miRNA-associated RNAi induction in vitro and in vivo. By insertion of a hairpin-like pre-miRNA structure into the intron region of a gene, this intronic miRNA biogenesis system has been found to depend on a coupled interaction of nascent precursor messenger RNA transcription and intron excision within a specific nuclear region proximal to genomic perichromatin fibrils. The intronic miRNA was transcribed by RNA type II polymerases, coexpressed with a primary gene transcript, and excised out of its encoding gene transcript by intracellular RNA splicing and processing mechanisms. Currently, some ribonuclease III endonucleases have been found to be involved in the processing of spliced introns and probably facilitating the intronic miRNA maturation. Using this miRNA-expressing system, we have shown for the first time that the intron-derived miRNAs were able to induce strong RNAi effects in not only human and mouse cells but also zebrafish, chicken embryos, and adult mice. Based on the strand complementarity between the designed miRNA and its target gene sequence, we have also developed a miRNA isolation protocol to purify and identify the mature miRNAs generated by the intronic miRNA-expressing system. Several intronic miRNA identities and structures are currently confirmed to be active in vitro and in vivo. According to this proof- of-principle method, we now have the knowledge to design pre-miRNA inserts that are more efficient and effective for the intronic miRNA-expressing system.

MicroRNA expression in melanocytic nevi: the usefulness of formalin-fixed, paraffin-embedded material for miRNA microarray profiling.

PubMed

Glud, Martin; Klausen, Mikkel; Gniadecki, Robert; Rossing, Maria; Hastrup, Nina; Nielsen, Finn C; Drzewiecki, Krzysztof T

2009-05-01

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate cellular differentiation, proliferation, and apoptosis. MiRNAs are expressed in a developmentally regulated and tissue-specific manner. Aberrant expression may contribute to pathological processes such as cancer, and miRNA may therefore serve as biomarkers that may be useful in a clinical environment for diagnosis of various diseases. Most miRNA profiling studies have used fresh tissue samples. However, in some types of cancer, including malignant melanoma, fresh material is difficult to obtain from primary tumors, and most surgical specimens are formalin fixed and paraffin embedded (FFPE). To explore whether FFPE material would be suitable for miRNA profiling in melanocytic lesions, we compared miRNA expression patterns in FFPE versus fresh frozen samples, obtained from 15 human melanocytic nevi. Out of microarray data, we identified 84 miRNAs that were expressed in both types of samples and represented an miRNA profile of melanocytic nevi. Our results showed a high correlation in miRNA expression (Spearman r-value of 0.80) between paired FFPE and fresh frozen material. The data were further validated by quantitative RT-PCR. In conclusion, FFPE specimens of melanocytic lesions are suitable as a source for miRNA microarray profiling.

Cancer survival classification using integrated data sets and intermediate information.

PubMed

Kim, Shinuk; Park, Taesung; Kon, Mark

2014-09-01

Although numerous studies related to cancer survival have been published, increasing the prediction accuracy of survival classes still remains a challenge. Integration of different data sets, such as microRNA (miRNA) and mRNA, might increase the accuracy of survival class prediction. Therefore, we suggested a machine learning (ML) approach to integrate different data sets, and developed a novel method based on feature selection with Cox proportional hazard regression model (FSCOX) to improve the prediction of cancer survival time. FSCOX provides us with intermediate survival information, which is usually discarded when separating survival into 2 groups (short- and long-term), and allows us to perform survival analysis. We used an ML-based protocol for feature selection, integrating information from miRNA and mRNA expression profiles at the feature level. To predict survival phenotypes, we used the following classifiers, first, existing ML methods, support vector machine (SVM) and random forest (RF), second, a new median-based classifier using FSCOX (FSCOX_median), and third, an SVM classifier using FSCOX (FSCOX_SVM). We compared these methods using 3 types of cancer tissue data sets: (i) miRNA expression, (ii) mRNA expression, and (iii) combined miRNA and mRNA expression. The latter data set included features selected either from the combined miRNA/mRNA profile or independently from miRNAs and mRNAs profiles (IFS). In the ovarian data set, the accuracy of survival classification using the combined miRNA/mRNA profiles with IFS was 75% using RF, 86.36% using SVM, 84.09% using FSCOX_median, and 88.64% using FSCOX_SVM with a balanced 22 short-term and 22 long-term survivor data set. These accuracies are higher than those using miRNA alone (70.45%, RF; 75%, SVM; 75%, FSCOX_median; and 75%, FSCOX_SVM) or mRNA alone (65.91%, RF; 63.64%, SVM; 72.73%, FSCOX_median; and 70.45%, FSCOX_SVM). Similarly in the glioblastoma multiforme data, the accuracy of miRNA/mRNA using IFS was 75.51% (RF), 87.76% (SVM) 85.71% (FSCOX_median), 85.71% (FSCOX_SVM). These results are higher than the results of using miRNA expression and mRNA expression alone. In addition we predict 16 hsa-miR-23b and hsa-miR-27b target genes in ovarian cancer data sets, obtained by SVM-based feature selection through integration of sequence information and gene expression profiles. Among the approaches used, the integrated miRNA and mRNA data set yielded better results than the individual data sets. The best performance was achieved using the FSCOX_SVM method with independent feature selection, which uses intermediate survival information between short-term and long-term survival time and the combination of the 2 different data sets. The results obtained using the combined data set suggest that there are some strong interactions between miRNA and mRNA features that are not detectable in the individual analyses. Copyright © 2014 Elsevier B.V. All rights reserved.

Haloperidol induces pharmacoepigenetic response by modulating miRNA expression, global DNA methylation and expression profiles of methylation maintenance genes and genes involved in neurotransmission in neuronal cells

PubMed Central

Swathy, Babu

2017-01-01

Introduction Haloperidol has been extensively used in various psychiatric conditions. It has also been reported to induce severe side effects. We aimed to evaluate whether haloperidol can influence host methylome, and if so what are the possible mechanisms for it in neuronal cells. Impact on host methylome and miRNAs can have wide spread alterations in gene expression, which might possibly help in understanding how haloperidol may impact treatment response or induce side effects. Methods SK-N-SH, a neuroblasoma cell line was treated with haloperidol at 10μm concentration for 24 hours and global DNA methylation was evaluated. Methylation at global level is maintained by methylation maintenance machinery and certain miRNAs. Therefore, the expression of methylation maintenance genes and their putative miRNA expression profiles were assessed. These global methylation alterations could result in gene expression changes. Therefore genes expressions for neurotransmitter receptors, regulators, ion channels and transporters were determined. Subsequently, we were also keen to identify a strong candidate miRNA based on biological and in-silico approach which can reflect on the pharmacoepigenetic trait of haloperidol and can also target the altered neuroscience panel of genes used in the study. Results Haloperidol induced increase in global DNA methylation which was found to be associated with corresponding increase in expression of various epigenetic modifiers that include DNMT1, DNMT3A, DNMT3B and MBD2. The expression of miR-29b that is known to putatively regulate the global methylation by modulating the expression of epigenetic modifiers was observed to be down regulated by haloperidol. In addition to miR-29b, miR-22 was also found to be downregulated by haloperidol treatment. Both these miRNA are known to putatively target several genes associated with various epigenetic modifiers, pharmacogenes and neurotransmission. Interestingly some of these putative target genes involved in neurotransmission were observed to be upregulated while CHRM2 gene expression was down regulated. Conclusions Haloperidol can influence methylation traits thereby inducing a pharmacoepigenomic response, which seems to be regulated by DNMTs and their putative miRNA expression. Increased methylation seems to influence CHRM2 gene expression while microRNA could influence neurotransmission, pharmacogene expression and methylation events. Altered expression of various therapeutically relevant genes and miRNA expression, could account for their role in therapeutic response or side effects. PMID:28886082

The Silkworm (Bombyx mori) microRNAs and Their Expressions in Multiple Developmental Stages

PubMed Central

Luo, Qibin; Cai, Yimei; Lin, Wen-chang; Chen, Huan; Yang, Yue; Hu, Songnian; Yu, Jun

2008-01-01

Background MicroRNAs (miRNAs) play crucial roles in various physiological processes through post-transcriptional regulation of gene expressions and are involved in development, metabolism, and many other important molecular mechanisms and cellular processes. The Bombyx mori genome sequence provides opportunities for a thorough survey for miRNAs as well as comparative analyses with other sequenced insect species. Methodology/Principal Findings We identified 114 non-redundant conserved miRNAs and 148 novel putative miRNAs from the B. mori genome with an elaborate computational protocol. We also sequenced 6,720 clones from 14 developmental stage-specific small RNA libraries in which we identified 35 unique miRNAs containing 21 conserved miRNAs (including 17 predicted miRNAs) and 14 novel miRNAs (including 11 predicted novel miRNAs). Among the 114 conserved miRNAs, we found six pairs of clusters evolutionarily conserved cross insect lineages. Our observations on length heterogeneity at 5′ and/or 3′ ends of nine miRNAs between cloned and predicted sequences, and three mature forms deriving from the same arm of putative pre-miRNAs suggest a mechanism by which miRNAs gain new functions. Analyzing development-related miRNAs expression at 14 developmental stages based on clone-sampling and stem-loop RT PCR, we discovered an unusual abundance of 33 sequences representing 12 different miRNAs and sharply fluctuated expression of miRNAs at larva-molting stage. The potential functions of several stage-biased miRNAs were also analyzed in combination with predicted target genes and silkworm's phenotypic traits; our results indicated that miRNAs may play key regulatory roles in specific developmental stages in the silkworm, such as ecdysis. Conclusions/Significance Taking a combined approach, we identified 118 conserved miRNAs and 151 novel miRNA candidates from the B. mori genome sequence. Our expression analyses by sampling miRNAs and real-time PCR over multiple developmental stages allowed us to pinpoint molting stages as hotspots of miRNA expression both in sorts and quantities. Based on the analysis of target genes, we hypothesized that miRNAs regulate development through a particular emphasis on complex stages rather than general regulatory mechanisms. PMID:18714353

MicroRNA profile of silk gland reveals different silk yields of three silkworm strains.

PubMed

Qin, Sheng; Danso, Blessing; Zhang, Jing; Li, Juan; Liu, Na; Sun, Xia; Hou, Chengxiang; Luo, Heng; Chen, Keping; Zhang, Guozheng; Li, Muwang

2018-05-05

Silk proteins are synthesized and secreted by the silk gland. The differential gene expression in it leads to different silk yield among various silkworm strains. As crucial factors, microRNAs (miRNAs) regulate protein synthesis at post-transcriptional level in silk gland. MiRNAs expression level in the silk gland of three silkworm strains (Jingsong, Lan10 and Dazao) was analyzed and 33 differentially expressed miRNAs (DEMs) were discovered between JingSong (JS) and Lan10 (L10), 60 DEMs between JS and Dazao, 54 DEMs between L10 and Dazao respectively. The DEMs target genes were predicted combing with two different methods and their functions were annotated according to gene ontology. Our previous studies showed that a batch of genes related to silk yield were identified in JS and L10 strains by comparative transcriptome and quantitative trait loci (QTL) method. Thirteen DEMs whose target genes are related to protein biosynthesis processes were screened by combining with these researches. Twelve DEMs potentially regulate nineteen genes which exist in our QTL results. Six common DEMs potentially regulate the genes in both of previous results. Finally, five DEMs were selected to verify their expression levels between JS and L10 by qRT-PCR, which showed similar difference as the results of small RNA-sequencing. MiRNAs in the silk gland may directly affect silk protein biosynthesis in different silkworm strains. In current work, we identified a batch of DEMs which potentially regulate the genes related to silk yield. Further functionally study of these miRNAs will contribute to improve varieties and boost the silk yield. Our research provides a basis for studying these miRNAs and their functions in silk production. Copyright © 2018 Elsevier B.V. All rights reserved.

miRNA and mRNA Expression Profiles Reveal Insight into Chitosan-Mediated Regulation of Plant Growth.

PubMed

Zhang, Xiaoqian; Li, Kecheng; Xing, Ronge; Liu, Song; Chen, Xiaolin; Yang, Haoyue; Li, Pengcheng

2018-04-18

Chitosan has been numerously studied as a plant growth regulator and stress tolerance inducer. To investigate the roles of chitosan as bioregulator on plant and unravel its possible metabolic responses mechanisms, we simultaneously investigated mRNAs and microRNAs (miRNAs) expression profiles of wheat seedlings in response to chitosan heptamer. We found 400 chitosan-responsive differentially expressed genes, including 268 up-regulated and 132 down-regulated mRNAs, many of which were related to photosynthesis, primary carbon and nitrogen metabolism, defense responses, and transcription factors. Moreover, miRNAs also participate in chitosan-mediated regulation on plant growth. We identified 87 known and 21 novel miRNAs, among which 56 miRNAs were induced or repressed by chitosan heptamer, such as miRNA156, miRNA159a, miRNA164, miRNA171a, miRNA319, and miRNA1127. The integrative analysis of miRNA and mRNA expression profiles in this case provides fundamental information for further investigation of regulation mechanisms of chitosan on plant growth and will facilitate its application in agriculture.

miRNAs Expressions and Interaction with Biological Systems in Patients with Alzheimer`s Disease. Using miRNAs as a Diagnosis and Prognosis Biomarker.

PubMed

Negoita, Silvius I; Sandesc, Dorel; Rogobete, Alexandru F; Dutu, Madalina; Bedreag, Ovidiu H; Papurica, Marius; Ercisli, Muhammed F; Popovici, Sonia E; Dumache, Raluca; Sandesc, Mihai; Dinu, Anca; Sas, Adriana M; Serban, Denis; Corneci, Dan

2017-09-01

A high percentage of patients develop Alzheimer`s disease (AD). The main signs are loss of memory and cognitive functions which have a significant impact on lifestyle. Numerous studies have been conducted to identify new biomarkers for early diagnosis of patients with AD. An ideal biomarker is represented by the expression of miRNAs. In this paper, we want to summarize expressions miRNAs in AD. We also want to present the pathophysiological and genetic interactions of miRNAs with protein systems in these patients. For the study, we examined available studies in scientific databases, such as PubMed and Scopus. The studies were searched using the keywords "miRNAs expression", "Alzheimer`s disease", "genetic polymorphisms", and "genetic biomarkers". For the assessment and monitoring of patients with AD, the expression of miRNAs can be used successfully due to increased specificity and selectivity. Moreover, the expression of miRNAs can provide important answers regarding possible genetic interactions and genetic therapeutic regimens. For the evaluation and non-invasive monitoring of patients with Alzheimer`s disease the expression of miRNAs can be successfully used.

Identification and characterization of novel and differentially expressed microRNAs in peripheral blood from healthy and mastitis Holstein cattle by deep sequencing.

PubMed

Li, Zhixiong; Wang, Hongliang; Chen, Ling; Wang, Lijun; Liu, Xiaolin; Ru, Caixia; Song, Ailong

2014-02-01

MicroRNA (miRNA) mediates post-transcriptional gene regulation and plays an important role in regulating the development of immune cells and in modulating innate and adaptive immune responses in mammals, including cattle. In the present study, we identified novel and differentially expressed miRNAs in peripheral blood from healthy and mastitis Holstein cattle by Solexa sequencing and bioinformatics. In total, 608 precursor hairpins (pre-miRNAs) encoding for 753 mature miRNAs were detected. Statistically, 173 unique miRNAs (of 753, 22.98%) were identified that had significant differential expression between healthy and mastitis Holstein cattle (P < 0.001). Most differentially expressed miRNAs (118 of 173, 68.21%) belonged to the chemokine signaling pathway involved in the immune responses. This study expands the number of miRNAs known to be expressed in cattle. The patterns of miRNAs expression differed significantly between the peripheral blood from healthy and mastitis Holstein cattle, which provide important information on mastitis in miRNAs expression. Diverse miRNAs may play an important role in the treatment of mastitis in Holstein cattle. © 2013 Stichting International Foundation for Animal Genetics.

Narcolepsy patients' blood-based miRNA expression profiling: miRNA expression differences with Pandemrix vaccination.

PubMed

Mosakhani, N; Sarhadi, V; Panula, P; Partinen, M; Knuutila, S

2017-11-01

Narcolepsy is a neurological sleep disorder characterized by excessive daytime sleepiness and nighttime sleep disturbance. Among children and adolescents vaccinated with Pandemrix vaccine in Finland and Sweden, the number of narcolepsy cases increased. Our aim was to identify miRNAs involved in narcolepsy and their association with Pandemrix vaccination. We performed global miRNA proofing by miRNA microarrays followed by RT-PCR verification on 20 narcolepsy patients (Pandemrix-associated and Pandemrix-non-associated) and 17 controls (vaccinated and non-vaccinated). Between all narcolepsy patients and controls, 11 miRNAs were differentially expressed; 17 miRNAs showed significantly differential expression between Pandemrix-non-associated narcolepsy patients and non-vaccinated healthy controls. MiR-188-5p and miR-4499 were over-expressed in narcolepsy patients vs healthy controls. Two miRNAs, miR-1470 and miR-4455, were under-expressed in Pandemrix-associated narcolepsy patients vs Pandemrix-non-associated narcolepsy patients. We identified miRNA expression patterns in narcolepsy patients that linked them to mRNA targets known to be involved in brain-related pathways or brain disorders. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The role of miRNAs in human papilloma virus (HPV)-associated cancers: bridging between HPV-related head and neck cancer and cervical cancer

PubMed Central

Lajer, C B; Garnæs, E; Friis-Hansen, L; Norrild, B; Therkildsen, M H; Glud, M; Rossing, M; Lajer, H; Svane, D; Skotte, L; Specht, L; Buchwald, C; Nielsen, F C

2012-01-01

Background: Although the role of human papilloma virus (HPV) in cervical squamous cell carcinoma (CSCC) is well established, the role in head and neck SCC (HNSCC) is less clear. MicroRNAs (miRNAs) have a role in the cancer development, and HPV status may affect the miRNA expression pattern in HNSCC. To explore the influence of HPV in HNSCC, we made a comparative miRNA profile of HPV-positive (HPV+) and HPV-negative (HPV−) HNSCC against CSCC. Methods: Fresh frozen and laser microdissected-paraffin-embedded samples obtained from patients with HPV+/HPV− HNSCC, CSCC and controls were used for microarray analysis. Differentially expressed miRNAs in the HPV+ and HPV− HNSCC samples were compared with the differentially expressed miRNAs in the CSCC samples. Results: Human papilloma virus positive (+) HNSCC had a distinct miRNA profile compared with HPV− HNSCC. Significantly more similarity was seen between HPV+ HNSCC and CSCC than HPV− and CSCC. A set of HPV core miRNAs were identified. Of these especially the miR-15a/miR-16/miR195/miR-497 family, miR-143/miR-145 and the miR-106-363 cluster appear to be important within the known HPV pathogenesis. Conclusion: This study adds new knowledge to the known pathogenic pathways of HPV and substantiates the oncogenic role of HPV in subsets of HNSCCs. PMID:22472886

Evolutionary relationships between miRNA genes and their activity.

PubMed

Zhu, Yan; Skogerbø, Geir; Ning, Qianqian; Wang, Zhen; Li, Biqing; Yang, Shuang; Sun, Hong; Li, Yixue

2012-12-22

The emergence of vertebrates is characterized by a strong increase in miRNA families. MicroRNAs interact broadly with many transcripts, and the evolution of such a system is intriguing. However, evolutionary questions concerning the origin of miRNA genes and their subsequent evolution remain unexplained. In order to systematically understand the evolutionary relationship between miRNAs gene and their function, we classified human known miRNAs into eight groups based on their evolutionary ages estimated by maximum parsimony method. New miRNA genes with new functional sequences accumulated more dynamically in vertebrates than that observed in Drosophila. Different levels of evolutionary selection were observed over miRNA gene sequences with different time of origin. Most genic miRNAs differ from their host genes in time of origin, there is no particular relationship between the age of a miRNA and the age of its host genes, genic miRNAs are mostly younger than the corresponding host genes. MicroRNAs originated over different time-scales are often predicted/verified to target the same or overlapping sets of genes, opening the possibility of substantial functional redundancy among miRNAs of different ages. Higher degree of tissue specificity and lower expression level was found in young miRNAs. Our data showed that compared with protein coding genes, miRNA genes are more dynamic in terms of emergence and decay. Evolution patterns are quite different between miRNAs of different ages. MicroRNAs activity is under tight control with well-regulated expression increased and targeting decreased over time. Our work calls attention to the study of miRNA activity with a consideration of their origin time.

The miRNAome dynamics during developmental and metabolic reprogramming of tomato root infected with potato cyst nematode.

PubMed

Koter, Marek D; Święcicka, Magdalena; Matuszkiewicz, Mateusz; Pacak, Andrzej; Derebecka, Natalia; Filipecki, Marcin

2018-03-01

Cyst-forming plant-parasitic nematodes are pests threatening many crops. By means of their secretions cyst nematodes induce the developmental and metabolic reprogramming of host cells that lead to the formation of a syncytium, which is the sole food source for growing nematodes. The in depth micro RNA (miRNA) dynamics in the syncytia induced by Globodera rostochiensis in tomato roots was studied. The miRNAomes were obtained from syncytia covering the early and intermediate developmental stages, and were the subject of differential expression analysis. The expression of 1235 miRNAs was monitored. The fold change (log 2 FC) ranged from -7.36 to 8.38, indicating that this transcriptome fraction was very variable. Moreover, we showed that the DE (differentially expressed) miRNAs do not fully overlap between the selected time points, suggesting infection stage specific regulation by miRNA. The correctness of RNA-seq expression profiling was confirmed by qRT-PCR (quantitative Real Time Polymerase Chain Reaction) for seven miRNA species. Down- and up-regulated miRNA species, including their isomiRs, were further used to identify their potential targets. Among them there are a large number of transcription factors linked to different aspects of plant development belonging to gene families, such as APETALA2 (AP2), SQUAMOSA (MADS-box), MYB, GRAS, and AUXIN RESPONSE FACTOR (ARF). The substantial portion of potential target genes belong to the NB-LRR and RLK (RECEPTOR-LIKE KINASE) families, indicating the involvement of miRNA mediated regulation in defense responses. We also collected the evidence for target cleavage in the case of 29 miRNAs using one of three alternative methods: 5' RACE (5' Rapid Amplification of cDNA Ends), a search of tasiRNA within our datasets, and the meta-analysis of tomato degradomes in the GEO (Gene Expression Omnibus) database. Eight target transcripts showed a negative correlation with their respective miRNAs at two or three time points. These results indicate a large regulatory potential for miRNAs in tuning the development and defense responses. Copyright © 2017 Elsevier B.V. All rights reserved.

Altered retinal microRNA expression profiles in early diabetic retinopathy: an in silico analysis.

PubMed

Xiong, Fen; Du, Xinhua; Hu, Jianyan; Li, Tingting; Du, Shanshan; Wu, Qiang

2014-07-01

MicroRNAs (miRNAs) - as negative regulators of target genes - are associated with various human diseases, but their precise role(s) in diabetic retinopathy (DR) remains to be elucidated. The aim of this study was to elucidate the involvement of miRNAs in early DR using in silico analysis to explore their gene expression patterns. We used the streptozotocin (STZ)-induced diabetic rat to investigate the roles of miRNAs in early DR. Retinal miRNA expression profiles from diabetic versus healthy control rats were examined by miRNA array analysis. Based on several bioinformatic systems, specifically, gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, we identified signatures of the potential pathological processes, gene functions, and signaling pathways that are influenced by dysregulated miRNAs. We used quantitative real-time polymerase chain reaction (qRT-PCR) to validate six (i.e. those with significant changes in expression levels) of the 17 miRNAs that were detected in the miRNA array. We also describe the significant role of the miRNA-gene network, which is based on the interactions between miRNAs and target genes. GO analysis of the 17 miRNAs detected in the miRNA array analysis revealed the most prevalent miRNAs to be those related to biological processes, olfactory bulb development and axonogenesis. These miRNAs also exert significant influence on additional pathways, including the mitogen-activated protein and calcium signaling pathways. Six of the seventeen miRNAs were chosen for qRT-PCR validation. With the exception of a slight difference in miRNA-350, our results are in close agreement with the differential expressions detected by array analysis. This study, which describes miRNA expression during the early developmental phases of DR, revealed extensive miRNA interactions. Based on both their target genes and signaling pathways, we suggest that miRNAs perform critical regulatory functions during the early stages of DR evolution.

Multicolor microRNA FISH effectively differentiates tumor types

PubMed Central

Renwick, Neil; Cekan, Pavol; Masry, Paul A.; McGeary, Sean E.; Miller, Jason B.; Hafner, Markus; Li, Zhen; Mihailovic, Aleksandra; Morozov, Pavel; Brown, Miguel; Gogakos, Tasos; Mobin, Mehrpouya B.; Snorrason, Einar L.; Feilotter, Harriet E.; Zhang, Xiao; Perlis, Clifford S.; Wu, Hong; Suárez-Fariñas, Mayte; Feng, Huichen; Shuda, Masahiro; Moore, Patrick S.; Tron, Victor A.; Chang, Yuan; Tuschl, Thomas

2013-01-01

MicroRNAs (miRNAs) are excellent tumor biomarkers because of their cell-type specificity and abundance. However, many miRNA detection methods, such as real-time PCR, obliterate valuable visuospatial information in tissue samples. To enable miRNA visualization in formalin-fixed paraffin-embedded (FFPE) tissues, we developed multicolor miRNA FISH. As a proof of concept, we used this method to differentiate two skin tumors, basal cell carcinoma (BCC) and Merkel cell carcinoma (MCC), with overlapping histologic features but distinct cellular origins. Using sequencing-based miRNA profiling and discriminant analysis, we identified the tumor-specific miRNAs miR-205 and miR-375 in BCC and MCC, respectively. We addressed three major shortcomings in miRNA FISH, identifying optimal conditions for miRNA fixation and ribosomal RNA (rRNA) retention using model compounds and high-pressure liquid chromatography (HPLC) analyses, enhancing signal amplification and detection by increasing probe-hapten linker lengths, and improving probe specificity using shortened probes with minimal rRNA sequence complementarity. We validated our method on 4 BCC and 12 MCC tumors. Amplified miR-205 and miR-375 signals were normalized against directly detectable reference rRNA signals. Tumors were classified using predefined cutoff values, and all were correctly identified in blinded analysis. Our study establishes a reliable miRNA FISH technique for parallel visualization of differentially expressed miRNAs in FFPE tumor tissues. PMID:23728175

miR-ID: A novel, circularization-based platform for detection of microRNAs

PubMed Central

Kumar, Pavan; Johnston, Brian H.; Kazakov, Sergei A.

2011-01-01

MicroRNAs (miRNAs) are important regulators of gene expression and have great potential as biomarkers, prognostic indicators, and therapeutic targets. Determining the expression patterns of these molecules is essential for elucidating their biogenesis, regulation, relation to disease, and response to therapy. Although PCR-based assays are commonly used for expression profiling of miRNAs, the small size, sequence heterogeneity, and (in some cases) end modifications of miRNAs constrain the performance of existing PCR methods. Here we introduce miR-ID, a novel method that avoids these constraints while providing superior sensitivity and sequence specificity at a lower cost. It also has the unique ability to differentiate unmodified small RNAs from those carrying 2′-OMe groups at their 3′-ends while detecting both forms. miR-ID is comprised of the following steps: (1) circularization of the miRNA by a ligase; (2) reverse transcription of the circularized miRNA (RTC), producing tandem repeats of a DNA sequence complementary to the miRNA; and (3) qPCR amplification of segments of this multimeric cDNA using 5′-overlapping primers and a nonspecific dye such as SYBR Green. No chemically modified probes (e.g., TaqMan) or primers (e.g., LNA) are required. The circular RNA and multimeric cDNA templates provide unmatched flexibility in the positioning of primers, which may include straddling the boundaries between these repetitive miRNA sequences. miR-ID is based on new findings that are themselves of general interest, including reverse transcription of small RNA circles and the use of 5′-overlapping primers for detection of repetitive sequences by qPCR. PMID:21169480

Genome-Wide Identification and Comparative Analysis of Conserved and Novel MicroRNAs in Grafted Watermelon by High-Throughput Sequencing

PubMed Central

Liu, Na; Yang, Jinghua; Guo, Shaogui; Xu, Yong; Zhang, Mingfang

2013-01-01

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs involved in the post-transcriptional gene regulation and play a critical role in plant growth, development and stresses response. However less is known about miRNAs involvement in grafting behaviors, especially with the watermelon (Citrullus lanatus L.) crop, which is one of the most important agricultural crops worldwide. Grafting method is commonly used in watermelon production in attempts to improve its adaptation to abiotic and biotic stresses, in particular to the soil-borne fusarium wilt disease. In this study, Solexa sequencing has been used to discover small RNA populations and compare miRNAs on genome-wide scale in watermelon grafting system. A total of 11,458,476, 11,614,094 and 9,339,089 raw reads representing 2,957,751, 2,880,328 and 2,964,990 unique sequences were obtained from the scions of self-grafted watermelon and watermelon grafted on-to bottle gourd and squash at two true-leaf stage, respectively. 39 known miRNAs belonging to 30 miRNA families and 80 novel miRNAs were identified in our small RNA dataset. Compared with self-grafted watermelon, 20 (5 known miRNA families and 15 novel miRNAs) and 47 (17 known miRNA families and 30 novel miRNAs) miRNAs were expressed significantly different in watermelon grafted on to bottle gourd and squash, respectively. MiRNAs expressed differentially when watermelon was grafted onto different rootstocks, suggesting that miRNAs might play an important role in diverse biological and metabolic processes in watermelon and grafting may possibly by changing miRNAs expressions to regulate plant growth and development as well as adaptation to stresses. The small RNA transcriptomes obtained in this study provided insights into molecular aspects of miRNA-mediated regulation in grafted watermelon. Obviously, this result would provide a basis for further unravelling the mechanism on how miRNAs information is exchanged between scion and rootstock in grafted watermelon, and its relevance to diverse biological processes and environmental adaptation. PMID:23468976

Identification of Appropriate Reference Genes for Normalization of miRNA Expression in Grafted Watermelon Plants under Different Nutrient Stresses

PubMed Central

Wu, Weifang; Deng, Qin; Shi, Pibiao; Yang, Jinghua; Hu, Zhongyuan; Zhang, Mingfang

2016-01-01

Watermelon (Citrullus lanatus) is a globally important crop belonging to the family Cucurbitaceae. The grafting technique is commonly used to improve its tolerance to stress, as well as to enhance its nutrient uptake and utilization. It is believed that miRNA is most likely involved in its nutrient-starvation response as a graft-transportable signal. The quantitative real-time reverse transcriptase polymerase chain reaction is the preferred method for miRNA functional analysis, in which reliable reference genes for normalization are crucial to ensure the accuracy. The purpose of this study was to select appropriate reference genes in scion (watermelon) and rootstocks (squash and bottle gourd) of grafted watermelon plants under normal growth conditions and nutrient stresses (nitrogen and phosphorus starvation). Under nutrient starvation, geNorm identified miR167c and miR167f as two most stable genes in both watermelon leaves and squash roots. miR166b was recommended by both geNorm and NormFinder as the best reference in bottle gourd roots under nutrient limitation. Expression of a new Cucurbitaceae miRNA, miR85, was used to validate the reliability of candidate reference genes under nutrient starvation. Moreover, by comparing several target genes expression in qRT-PCR analysis with those in RNA-seq data, miR166b and miR167c were proved to be the most suitable reference genes to normalize miRNA expression under normal growth condition in scion and rootstock tissues, respectively. This study represents the first comprehensive survey of the stability of miRNA reference genes in Cucurbitaceae and provides valuable information for investigating more accurate miRNA expression involving grafted watermelon plants. PMID:27749935

Identification of Appropriate Reference Genes for Normalization of miRNA Expression in Grafted Watermelon Plants under Different Nutrient Stresses.

PubMed

Wu, Weifang; Deng, Qin; Shi, Pibiao; Yang, Jinghua; Hu, Zhongyuan; Zhang, Mingfang

2016-01-01

Watermelon (Citrullus lanatus) is a globally important crop belonging to the family Cucurbitaceae. The grafting technique is commonly used to improve its tolerance to stress, as well as to enhance its nutrient uptake and utilization. It is believed that miRNA is most likely involved in its nutrient-starvation response as a graft-transportable signal. The quantitative real-time reverse transcriptase polymerase chain reaction is the preferred method for miRNA functional analysis, in which reliable reference genes for normalization are crucial to ensure the accuracy. The purpose of this study was to select appropriate reference genes in scion (watermelon) and rootstocks (squash and bottle gourd) of grafted watermelon plants under normal growth conditions and nutrient stresses (nitrogen and phosphorus starvation). Under nutrient starvation, geNorm identified miR167c and miR167f as two most stable genes in both watermelon leaves and squash roots. miR166b was recommended by both geNorm and NormFinder as the best reference in bottle gourd roots under nutrient limitation. Expression of a new Cucurbitaceae miRNA, miR85, was used to validate the reliability of candidate reference genes under nutrient starvation. Moreover, by comparing several target genes expression in qRT-PCR analysis with those in RNA-seq data, miR166b and miR167c were proved to be the most suitable reference genes to normalize miRNA expression under normal growth condition in scion and rootstock tissues, respectively. This study represents the first comprehensive survey of the stability of miRNA reference genes in Cucurbitaceae and provides valuable information for investigating more accurate miRNA expression involving grafted watermelon plants.

Profound Effect of Profiling Platform and Normalization Strategy on Detection of Differentially Expressed MicroRNAs – A Comparative Study

PubMed Central

Meyer, Swanhild U.; Kaiser, Sebastian; Wagner, Carola; Thirion, Christian; Pfaffl, Michael W.

2012-01-01

Background Adequate normalization minimizes the effects of systematic technical variations and is a prerequisite for getting meaningful biological changes. However, there is inconsistency about miRNA normalization performances and recommendations. Thus, we investigated the impact of seven different normalization methods (reference gene index, global geometric mean, quantile, invariant selection, loess, loessM, and generalized procrustes analysis) on intra- and inter-platform performance of two distinct and commonly used miRNA profiling platforms. Methodology/Principal Findings We included data from miRNA profiling analyses derived from a hybridization-based platform (Agilent Technologies) and an RT-qPCR platform (Applied Biosystems). Furthermore, we validated a subset of miRNAs by individual RT-qPCR assays. Our analyses incorporated data from the effect of differentiation and tumor necrosis factor alpha treatment on primary human skeletal muscle cells and a murine skeletal muscle cell line. Distinct normalization methods differed in their impact on (i) standard deviations, (ii) the area under the receiver operating characteristic (ROC) curve, (iii) the similarity of differential expression. Loess, loessM, and quantile analysis were most effective in minimizing standard deviations on the Agilent and TLDA platform. Moreover, loess, loessM, invariant selection and generalized procrustes analysis increased the area under the ROC curve, a measure for the statistical performance of a test. The Jaccard index revealed that inter-platform concordance of differential expression tended to be increased by loess, loessM, quantile, and GPA normalization of AGL and TLDA data as well as RGI normalization of TLDA data. Conclusions/Significance We recommend the application of loess, or loessM, and GPA normalization for miRNA Agilent arrays and qPCR cards as these normalization approaches showed to (i) effectively reduce standard deviations, (ii) increase sensitivity and accuracy of differential miRNA expression detection as well as (iii) increase inter-platform concordance. Results showed the successful adoption of loessM and generalized procrustes analysis to one-color miRNA profiling experiments. PMID:22723911

Using artificial microRNA sponges to achieve microRNA loss-of-function in cancer cells.

PubMed

Tay, Felix Chang; Lim, Jia Kai; Zhu, Haibao; Hin, Lau Cia; Wang, Shu

2015-01-01

Widely observed dysregulation of microRNAs (miRNAs) in human cancer has led to substantial speculation regarding possible functions of these short, non-coding RNAs in cancer development and manipulation of miRNA expression to treat cancer. To achieve miRNA loss-of-function, miRNA sponge technology has been developed to use plasmid or viral vectors for intracellular expression of tandemly arrayed, bulged miRNA binding sites complementary to a miRNA target to saturate its ability to regulate natural mRNAs. A strong viral promoter can be used in miRNA sponge vectors to generate high-level expression of the competitive inhibitor transcripts for either transient or long-term inhibition of miRNA function. Taking the advantage of sharing a common seed sequence by members of a miRNA family, this technology is especially useful in knocking down the expression of a family of miRNAs, providing a powerful means for simultaneous inhibition of multiple miRNAs of interest with a single inhibitor. Knockdown of overexpressed oncogenic miRNAs with the technology can be a rational therapeutic strategy for cancer, whereas inhibition of tumor-suppressive miRNAs by the sponges will be useful in deciphering functions of miRNAs in oncogenesis. Herein, we discuss the design of miRNA sponge expression vectors and the use of the vectors to gain better understanding of miRNA's roles in cancer biology and as an alternative tool for anticancer gene therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

Strong conservation of inbred mouse strain microRNA loci but broad variation in brain microRNAs due to RNA editing and isomiR expression.

PubMed

Trontti, Kalevi; Väänänen, Juho; Sipilä, Tessa; Greco, Dario; Hovatta, Iiris

2018-05-01

Diversity in the structure and expression of microRNAs, important regulators of gene expression, arises from SNPs, duplications followed by divergence, production of isomiRs, and RNA editing. Inbred mouse strains and crosses using them are important reference populations for genetic mapping, and as models of human disease. We determined the nature and extent of interstrain miRNA variation by (i) identifying miRNA SNPs in whole-genome sequence data from 36 strains, and (ii) examining miRNA editing and expression in hippocampus (Hpc) and frontal cortex (FCx) of six strains, to facilitate the study of miRNAs in neurobehavioral phenotypes. miRNA loci were strongly conserved among the 36 strains, but even the highly conserved seed region contained 16 SNPs. In contrast, we identified RNA editing in 58.9% of miRNAs, including 11 consistent editing events in the seed region. We confirmed the functional significance of three conserved edits in the miR-379/410 cluster, demonstrating that edited miRNAs gained novel target mRNAs not recognized by the unedited miRNAs. We found significant interstrain differences in miRNA and isomiR expression: Of 779 miRNAs expressed in Hpc and 719 in FCx, 262 were differentially expressed (190 in Hpc, 126 in FCx, 54 in both). We also identified 32 novel miRNA candidates using miRNA prediction tools. Our studies provide the first comprehensive analysis of SNP, isomiR, and RNA editing variation in miRNA loci across inbred mouse strains, and a detailed catalog of expressed miRNAs in Hpc and FCx in six commonly used strains. These findings will facilitate the molecular analysis of neurological and behavioral phenotypes in this model organism. © 2018 Trontti et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Ancient human miRNAs are more likely to have broad functions and disease associations than young miRNAs.

PubMed

Patel, Vir D; Capra, John A

2017-08-31

microRNAs (miRNAs) are essential to the regulation of gene expression in eukaryotes, and improper expression of miRNAs contributes to hundreds of diseases. Despite the essential functions of miRNAs, the evolutionary dynamics of how they are integrated into existing gene regulatory and functional networks is not well understood. Knowledge of the origin and evolutionary history a gene has proven informative about its functions and disease associations; we hypothesize that incorporating the evolutionary origins of miRNAs into analyses will help resolve differences in their functional dynamics and how they influence disease. We computed the phylogenetic age of miRNAs across 146 species and quantified the relationship between human miRNA age and several functional attributes. Older miRNAs are significantly more likely to be associated with disease than younger miRNAs, and the number of associated diseases increases with age. As has been observed for genes, the miRNAs associated with different diseases have different age profiles. For example, human miRNAs implicated in cancer are enriched for origins near the dawn of animal multicellularity. Consistent with the increasing contribution of miRNAs to disease with age, older miRNAs target more genes than younger miRNAs, and older miRNAs are expressed in significantly more tissues. Furthermore, miRNAs of all ages exhibit a strong preference to target older genes; 93% of validated miRNA gene targets were in existence at the origin of the targeting miRNA. Finally, we find that human miRNAs in evolutionarily related families are more similar in their targets and expression profiles than unrelated miRNAs. Considering the evolutionary origin and history of a miRNA provides useful context for the analysis of its function. Consistent with recent work in Drosophila, our results support a model in which miRNAs increase their expression and functional regulatory interactions over evolutionary time, and thus older miRNAs have increased potential to cause disease. We anticipate that these patterns hold across mammalian species; however, comprehensively evaluating them will require refining miRNA annotations across species and collecting functional data in non-human systems.

Identification and Expression Analyses of miRNAs from Two Contrasting Flower Color Cultivars of Canna by Deep Sequencing.

PubMed

Roy, Sribash; Tripathi, Abhinandan Mani; Yadav, Amrita; Mishra, Parneeta; Nautiyal, Chandra Shekhar

2016-01-01

miRNAs are endogenous small RNA (sRNA) that play critical roles in plant development processes. Canna is an ornamental plant belonging to family Cannaceae. Here, we report for the first time the identification and differential expression of miRNAs in two contrasting flower color cultivars of Canna, Tropical sunrise and Red president. A total of 313 known miRNAs belonging to 78 miRNA families were identified from both the cultivars. Thirty one miRNAs (17 miRNA families) were specific to Tropical sunrise and 43 miRNAs (10 miRNA families) were specific to Red president. Thirty two and 18 putative new miRNAs were identified from Tropical sunrise and Red president, respectively. One hundred and nine miRNAs were differentially expressed in the two cultivars targeting 1343 genes. Among these, 16 miRNAs families targeting 60 genes were involved in flower development related traits and five miRNA families targeting five genes were involved in phenyl propanoid and pigment metabolic processes. We further validated the expression analysis of a few miRNA and their target genes by qRT-PCR. Transcription factors were the major miRNA targets identified. Target validation of a few randomly selected miRNAs by RLM-RACE was performed but was successful with only miR162. These findings will help in understanding flower development processes, particularly the color development in Canna.

miRNA-148a regulates the expression of the estrogen receptor through DNMT1-mediated DNA methylation in breast cancer cells

PubMed Central

Xu, Yurui; Chao, Lin; Wang, Jianyu; Sun, Yonghong

2017-01-01

Breast cancer remains the most prevalent cancer among women worldwide. The expression of estrogen receptor-α (ER-α) is an important marker for prognosis. ER-α status may be positive or negative in breast cancer cells, although the cause of negative or positive status is not yet fully characterized. In the present study, the expression of ER-α and miRNA-148a was assessed in two breast cancer cell lines, HCC1937 and MCF7. An association between ER-α and miRNA-148a expression was identified. It was then demonstrated that DNA methyltransferase 1 (DNMT1) is a target of miRNA-148a, which may suppress the expression of ER-α via DNA methylation. Finally, an miRNA-148a mimic or inhibitor was transfected into MCF7 cells; the miRNA-148a mimic increased ER-α expression whereas the miRNA-148a inhibitor decreased ER-α expression. In conclusion, it was identified that miRNA-148a regulates ER-α expression through DNMT1-mediated DNA methylation in breast cancer cells. This may represent a potential miRNA-based strategy to modulate the expression of ER-α and provide a novel perspective for investigating the role of miRNAs in treating breast cancer. PMID:29085474

Impact of microRNAs on regulatory networks and pathways in human colorectal carcinogenesis and development of metastasis

PubMed Central

2013-01-01

Background Qualitative alterations or abnormal expression of microRNAs (miRNAs) in colon cancer have mainly been demonstrated in primary tumors. Poorly overlapping sets of oncomiRs, tumor suppressor miRNAs and metastamiRs have been linked with distinct stages in the progression of colorectal cancer. To identify changes in both miRNA and gene expression levels among normal colon mucosa, primary tumor and liver metastasis samples, and to classify miRNAs into functional networks, in this work miRNA and gene expression profiles in 158 samples from 46 patients were analysed. Results Most changes in miRNA and gene expression levels had already manifested in the primary tumors while these levels were almost stably maintained in the subsequent primary tumor-to-metastasis transition. In addition, comparing normal tissue, tumor and metastasis, we did not observe general impairment or any rise in miRNA biogenesis. While only few mRNAs were found to be differentially expressed between primary colorectal carcinoma and liver metastases, miRNA expression profiles can classify primary tumors and metastases well, including differential expression of miR-10b, miR-210 and miR-708. Of 82 miRNAs that were modulated during tumor progression, 22 were involved in EMT. qRT-PCR confirmed the down-regulation of miR-150 and miR-10b in both primary tumor and metastasis compared to normal mucosa and of miR-146a in metastases compared to primary tumor. The upregulation of miR-201 in metastasis compared both with normal and primary tumour was also confirmed. A preliminary survival analysis considering differentially expressed miRNAs suggested a possible link between miR-10b expression in metastasis and patient survival. By integrating miRNA and target gene expression data, we identified a combination of interconnected miRNAs, which are organized into sub-networks, including several regulatory relationships with differentially expressed genes. Key regulatory interactions were validated experimentally. Specific mixed circuits involving miRNAs and transcription factors were identified and deserve further investigation. The suppressor activity of miR-182 on ENTPD5 gene was identified for the first time and confirmed in an independent set of samples. Conclusions Using a large dataset of CRC miRNA and gene expression profiles, we describe the interplay of miRNA groups in regulating gene expression, which in turn affects modulated pathways that are important for tumor development. PMID:23987127

MicroRNA profiles following metformin treatment in a mouse model of non-alcoholic steatohepatitis

PubMed Central

KATSURA, AKIKO; MORISHITA, ASAHIRO; IWAMA, HISAKAZU; TANI, JOJI; SAKAMOTO, TEPPEI; TATSUTA, MIWA; TOYOTA, YUKA; FUJITA, KOJI; KATO, KIYOHITO; MAEDA, EMIKO; NOMURA, TAKAKO; MIYOSHI, HISAAKI; YONEYAMA, HIROHITO; HIMOTO, TAKASHI; FUJIWARA, SHINTARO; KOBARA, HIDEKI; MORI, HIROHITO; NIKI, TOSHIRO; ONO, MASAFUMI; HIRASHIMA, MITSUOMI; MASAKI, TSUTOMU

2015-01-01

Non-alcoholic steatohepatitis (NASH) is one of the most common causes of chronic liver disease and is considered to be a causative factor of cryptogenic cirrhosis and hepatocellular carcinoma. microRNAs (miRNAs) are small non-coding RNAs that negatively regulate messenger RNA (mRNA). Recently, it was demonstrated that the aberrant expression of certain miRNAs plays a pivotal role in liver disease. The aim of the present study was to evaluate changes in miRNA profiles associated with metformin treatment in a NASH model. Eight-week-old male mice were fed a methionine- and choline-deficient (MCD) diet alone or with 0.08% metformin for 15 weeks. Metformin significantly downregulated the level of plasma transaminases and attenuated hepatic steatosis and liver fibrosis. The expression of miRNA-376a, miRNA-127, miRNA-34a, miRNA-300 and miRNA-342-3p was enhanced among the 71 upregulated miRNAs, and the expression of miRNA-122, miRNA-194, miRNA-101b and miRNA-705 was decreased among 60 downregulated miRNAs in the liver of MCD-fed mice when compared with control mice. Of note, miRNA profiles were altered following treatment with metformin in MCD-fed mice. miRNA-376a, miRNA-127, miRNA-34a, miRNA-300 and miRNA-342-3p were down-regulated, but miRNA-122, miRNA-194, miRNA-101b and miRNA-705 were significantly upregulated in MCD-fed mice treated with metformin. miRNA profiles were altered in MCD-fed mice and metformin attenuated this effect on miRNA expression. Therefore, miRNA profiles are a potential tool that may be utilized to clarify the mechanism behind the metformin-induced improvement of hepatic steatosis and liver fibrosis. Furthermore, identification of targetable miRNAs may be used as a novel therapy in human NASH. PMID:25672270

Visual Display of 5p-arm and 3p-arm miRNA Expression with a Mobile Application.

PubMed

Pan, Chao-Yu; Kuo, Wei-Ting; Chiu, Chien-Yuan; Lin, Wen-Chang

2017-01-01

MicroRNAs (miRNAs) play important roles in human cancers. In previous studies, we have demonstrated that both 5p-arm and 3p-arm of mature miRNAs could be expressed from the same precursor and we further interrogated the 5p-arm and 3p-arm miRNA expression with a comprehensive arm feature annotation list. To assist biologists to visualize the differential 5p-arm and 3p-arm miRNA expression patterns, we utilized a user-friendly mobile App to display. The Cancer Genome Atlas (TCGA) miRNA-Seq expression information. We have collected over 4,500 miRNA-Seq datasets from 15 TCGA cancer types and further processed them with the 5p-arm and 3p-arm annotation analysis pipeline. In order to be displayed with the RNA-Seq Viewer App, annotated 5p-arm and 3p-arm miRNA expression information and miRNA gene loci information were converted into SQLite tables. In this distinct application, for any given miRNA gene, 5p-arm miRNA is illustrated on the top of chromosome ideogram and 3p-arm miRNA is illustrated on the bottom of chromosome ideogram. Users can then easily interrogate the differentially 5p-arm/3p-arm expressed miRNAs with their mobile devices. This study demonstrates the feasibility and utility of RNA-Seq Viewer App in addition to mRNA-Seq data visualization.

Differential expression analysis of Paralichthys olivaceus microRNAs in adult ovary and testis by deep sequencing.

PubMed

Gu, Yifeng; Zhang, Lei; Chen, Xiaowu

2014-08-01

MicroRNAs (miRNAs) play an important role in gonadal development and differentiation in fish. However, understanding of the mechanism of this process is hindered by our poor knowledge of miRNA expression patterns in fish gonads. In this study, miRNA libraries derived from adult gonads of Paralichthys olivaceus were generated by using next-generation sequencing (NGS) technology. Bioinformatics analysis was performed to distinguish mature miRNA sequences from two classes of small RNAs represented in the sequencing data. A total of 141 mature miRNAs were identified, in which 21 miRNAs were found in P. olivaceus for the first time. Variance and preference of miRNAs expression were concluded from the deep sequencing reads. Some miRNAs, such as pol-miR-143, pol-miR-26a and pol-let-7a were found with quite high expression levels in both gonads, while some exhibited a clear sex-biased expression in different gonad. Approximate 20.0% and 13.1% of the isolated miRNAs were preferentially expressed in the testis (FC<0.5) or ovary (FC>2), respectively. The identification and the preliminary analysis of the sex-biased expression of miRNAs in P. olivaceus gonads in our work by using NGS will provide us a basic catalog of miRNAs to facilitate future improvement and exploitation of sexual regulatory mechanisms in P. olivaceus. Copyright © 2014. Published by Elsevier Inc.

Detection and comparison of microRNA expression in the serum of Doberman Pinschers with dilated cardiomyopathy and healthy controls

PubMed Central

2013-01-01

Background Dilated cardiomyopathy (DCM) is the most common heart disease in Doberman Pinschers. MicroRNAs (miRNAs) are short non-coding RNAs playing important roles in gene regulation. Different miRNA expression patterns have been described for DCM in humans and might represent potential diagnostic markers. There are no studies investigating miRNA expression profiles in canine DCM. The aims of this study were to screen the miRNA expression profile of canine serum using miRNA microarray and to compare expression patterns of a group of Doberman Pinschers with DCM and healthy controls. Results Eight Doberman Pinschers were examined by echocardiography and 24-hour-ECG and classified as healthy (n = 4) or suffering from DCM (n = 4). Total RNA was extracted from serum and hybridized on a custom-designed 8x60k miRNA microarray (Agilent) containing probes for 1368 individual miRNAs. Although total RNA concentrations were very low in serum samples, 404 different miRNAs were detectable with sufficient signal intensity on miRNA microarray. 22 miRNAs were differentially expressed in the two groups (p < 0.05 and fold change (FC) > 1.5), but did not reach statistical significance after multiple testing correction (false discovery rate adjusted p > 0.05). Five miRNAs were selected for further analysis using quantitative Real-Time RT-PCR (qPCR) assays. No significant differences were found using specific miRNA qPCR assays (p > 0.05). Conclusions Numerous miRNAs can be detected in canine serum. Between healthy and DCM dogs, miRNA expression changes could be detected, but the results did not reach statistical significance most probably due to the small group size. miRNAs are potential new circulating biomarkers in veterinary medicine and should be investigated in larger patient groups and additional canine diseases. PMID:23327631

Detection and comparison of microRNA expression in the serum of Doberman Pinschers with dilated cardiomyopathy and healthy controls.

PubMed

Steudemann, Carola; Bauersachs, Stefan; Weber, Karin; Wess, Gerhard

2013-01-17

Dilated cardiomyopathy (DCM) is the most common heart disease in Doberman Pinschers. MicroRNAs (miRNAs) are short non-coding RNAs playing important roles in gene regulation. Different miRNA expression patterns have been described for DCM in humans and might represent potential diagnostic markers. There are no studies investigating miRNA expression profiles in canine DCM. The aims of this study were to screen the miRNA expression profile of canine serum using miRNA microarray and to compare expression patterns of a group of Doberman Pinschers with DCM and healthy controls. Eight Doberman Pinschers were examined by echocardiography and 24-hour-ECG and classified as healthy (n=4) or suffering from DCM (n=4). Total RNA was extracted from serum and hybridized on a custom-designed 8x60k miRNA microarray (Agilent) containing probes for 1368 individual miRNAs. Although total RNA concentrations were very low in serum samples, 404 different miRNAs were detectable with sufficient signal intensity on miRNA microarray. 22 miRNAs were differentially expressed in the two groups (p<0.05 and fold change (FC)>1.5), but did not reach statistical significance after multiple testing correction (false discovery rate adjusted p>0.05). Five miRNAs were selected for further analysis using quantitative Real-Time RT-PCR (qPCR) assays. No significant differences were found using specific miRNA qPCR assays (p>0.05). Numerous miRNAs can be detected in canine serum. Between healthy and DCM dogs, miRNA expression changes could be detected, but the results did not reach statistical significance most probably due to the small group size. miRNAs are potential new circulating biomarkers in veterinary medicine and should be investigated in larger patient groups and additional canine diseases.

Notch ligand Delta-like 1 as a novel molecular target in childhood neuroblastoma.

PubMed

Bettinsoli, P; Ferrari-Toninelli, G; Bonini, S A; Prandelli, C; Memo, M

2017-05-19

Neuroblastoma is the most common extracranial solid malignancy in childhood, responsible for 15% of all pediatric cancer deaths. It is an heterogeneous disease that does not always respond to classical therapy; so the identification of new and specific molecular targets to improve existing therapy is needed. We have previously demonstrated the involvement of the Notch pathway in the onset and progression of neuroblastoma. In this study we further investigated the role of Notch signaling and identified Delta-like 1 (DLL1) as a novel molecular target in neuroblastoma cells with a high degree of MYCN amplification, which is a major oncogenic driver in neuroblastoma. The possibility to act on DLL1 expression levels by using microRNAs (miRNAs) was assessed. DLL1 mRNA and protein expression levels were measured in three different neuroblastoma cell lines using quantitative real-time PCR and Western Blot analysis, respectively. Activation of the Notch pathway as a result of increased levels of DLL1 was analyzed by Immunofluorescence and Western Blot methods. In silico tools revealed the possibility to act on DLL1 expression levels with miRNAs, in particular with the miRNA-34 family. Neuroblastoma cells were transfected with miRNA-34 family members, and the effect of miRNAs transfection on DLL1 mRNA expression levels, on cell differentiation, proliferation and apoptosis was measured. In this study, the DLL1 ligand was identified as the Notch pathway component highly expressed in neuroblastoma cells with MYCN amplification. In silico analysis demonstrated that DLL1 is one of the targets of miRNA-34 family members that maps on chromosome regions that are frequently deregulated or deleted in neuroblastoma. We studied the possibility to use miRNAs to target DLL1. Among all miRNA-34 family members, miRNA-34b is able to significantly downregulate DLL1 mRNA expression levels, to arrest cell proliferation and to induce neuronal differentiation in malignant neuroblastoma cells. Targeted therapies have emerged as new strategies for cancer treatment. This study identified the Notch ligand DLL1 as a novel and attractive molecular target in childhood neuroblastoma and its results could help to devise a targeted therapy using miRNAs.

Identification and Expression Analysis of microRNAs at the Grain Filling Stage in Rice(Oryza sativa L.)via Deep Sequencing

PubMed Central

Yi, Rong; Zhu, Zhixuan; Hu, Jihong; Qian, Qian; Dai, Jincheng; Ding, Yi

2013-01-01

MicroRNAs (miRNAs) have been shown to play crucial roles in the regulation of plant development. In this study, high-throughput RNA-sequencing technology was used to identify novel miRNAs, and to reveal miRNAs expression patterns at different developmental stages during rice (Oryza sativa L.) grain filling. A total of 434 known miRNAs (380, 402, 390 and 392 at 5, 7, 12 and 17 days after fertilization, respectively.) were obtained from rice grain. The expression profiles of these identified miRNAs were analyzed and the results showed that 161 known miRNAs were differentially expressed during grain development, a high proportion of which were up-regulated from 5 to 7 days after fertilization. In addition, sixty novel miRNAs were identified, and five of these were further validated experimentally. Additional analysis showed that the predicted targets of the differentially expressed miRNAs may participate in signal transduction, carbohydrate and nitrogen metabolism, the response to stimuli and epigenetic regulation. In this study, differences were revealed in the composition and expression profiles of miRNAs among individual developmental stages during the rice grain filling process, and miRNA editing events were also observed, analyzed and validated during this process. The results provide novel insight into the dynamic profiles of miRNAs in developing rice grain and contribute to the understanding of the regulatory roles of miRNAs in grain filling. PMID:23469249

Reference genes for real-time PCR quantification of messenger RNAs and microRNAs in mouse model of obesity.

PubMed

Matoušková, Petra; Bártíková, Hana; Boušová, Iva; Hanušová, Veronika; Szotáková, Barbora; Skálová, Lenka

2014-01-01

Obesity and metabolic syndrome is increasing health problem worldwide. Among other ways, nutritional intervention using phytochemicals is important method for treatment and prevention of this disease. Recent studies have shown that certain phytochemicals could alter the expression of specific genes and microRNAs (miRNAs) that play a fundamental role in the pathogenesis of obesity. For study of the obesity and its treatment, monosodium glutamate (MSG)-injected mice with developed central obesity, insulin resistance and liver lipid accumulation are frequently used animal models. To understand the mechanism of phytochemicals action in obese animals, the study of selected genes expression together with miRNA quantification is extremely important. For this purpose, real-time quantitative PCR is a sensitive and reproducible method, but it depends on proper normalization entirely. The aim of present study was to identify the appropriate reference genes for mRNA and miRNA quantification in MSG mice treated with green tea catechins, potential anti-obesity phytochemicals. Two sets of reference genes were tested: first set contained seven commonly used genes for normalization of messenger RNA, the second set of candidate reference genes included ten small RNAs for normalization of miRNA. The expression stability of these reference genes were tested upon treatment of mice with catechins using geNorm, NormFinder and BestKeeper algorithms. Selected normalizers for mRNA quantification were tested and validated on expression of quinone oxidoreductase, biotransformation enzyme known to be modified by catechins. The effect of selected normalizers for miRNA quantification was tested on two obesity- and diabetes- related miRNAs, miR-221 and miR-29b, respectively. Finally, the combinations of B2M/18S/HPRT1 and miR-16/sno234 were validated as optimal reference genes for mRNA and miRNA quantification in liver and 18S/RPlP0/HPRT1 and sno234/miR-186 in small intestine of MSG mice. These reference genes will be used for mRNA and miRNA normalization in further study of green tea catechins action in obese mice.

Effective Anti-miRNA Oligonucleotides Show High Releasing Rate of MicroRNA from RNA-Induced Silencing Complex.

PubMed

Ariyoshi, Jumpei; Matsuyama, Yohei; Kobori, Akio; Murakami, Akira; Sugiyama, Hiroshi; Yamayoshi, Asako

2017-10-01

MicroRNAs (miRNAs) regulate gene expression by forming RNA-induced silencing complexes (RISCs) and have been considered as promising therapeutic targets. MiRNA is an essential component of RISC for the modulation of gene expression. Therefore, the release of miRNA from RISC is considered as an effective method for the inhibition of miRNA functions. In our previous study, we reported that anti-miRNA oligonucleotides (AMOs), which are composed of the 2'-O-methyl (2'-OMe) RNA, could induce the release of miRNA from RISC. However, the mechanisms underlying the miRNA-releasing effects of chemically modified AMOs, which are conventionally used as anti-cancer drugs, are still unclear. In this study, we investigated the relationship between the miRNA releasing rate from RISC and the inhibitory effect on RISC activity (IC 50 ) using conventional chemically modified AMOs. We demonstrated that the miRNA-releasing effects of AMOs are directly proportional to the IC 50 values, and AMOs, which have an ability to promote the release of miRNA from RISC, can effectively inhibit RISC activity in living cells.

Cloning and expression of porcine Dicer and the impact of developmental stage and culture conditions on MicroRNA expression in porcine embryos.

PubMed

Stowe, Heather M; Curry, Erin; Calcatera, Samantha M; Krisher, Rebecca L; Paczkowski, Melissa; Pratt, Scott L

2012-06-15

MicroRNA (miRNA) is a class of small, single-stranded ribonucleic acids that regulate gene expression post-transcriptionally and are involved in somatic cell, germ cell, and embryonic development. As the enzyme responsible for producing mature miRNA, Dicer is crucial to miRNA production. Characterization of Dicer and its expression at the nucleotide level, as well as the identification of miRNA expression in reproductive tissues, have yet to be reported for the domestic pig (Sus scrofa), a species important for disease modeling, biomedical research, and food production. In this study we determined the primary cDNA sequence of porcine Dicer (pDicer), confirmed its expression in porcine oocytes and early stage embryos, and evaluated the expression of specific miRNA during early embryonic development and between in vivo (IVO) and in vitro (IVF) produced embryos. Total cellular RNA (tcRNA) was isolated and subjected to end point RT-PCR, subcloning, and sequencing. The pDicer coding sequence was found to be highly conserved, and phylogenetic analysis showed that pDicer is more highly conserved to human Dicer (hDicer) than the mouse homolog. Expression of pDicer mRNA was detected in oocytes and in IVO produced blastocyst embryos. Two RT-PCR procedures were conducted to identify and quantitate miRNA expressed in metaphase II oocytes (MII) and embryos. RT-PCR array was conducted using primers designed for human miRNA, and 86 putative porcine miRNA in MII and early embryos were detected. Fewer miRNAs were detected in 8-cell (8C) embryos compared to MII and blastocysts (B) (P=0.026 and P<0.0001, respectively). Twenty-one miRNA (of 88 examined) were differentially expressed between MII and 8C, 8C and B, or MII and B. Transcripts targeted by the differentially expressed miRNA were enriched in gene ontology (GO) categories associated with cellular development and differentiation. Further, we evaluated the effects of IVF culture on the expression of specific miRNA at the blastocyst stage. Quantitative RT-PCR was conducted on blastocyst tcRNA isolated from individual IVO and IVF produced embryos for miR-18a, -21, and -24. Only the expression level of miR-24 differed due to culture conditions, with lower levels detected in the IVO embryos. These data show that pDicer and miRNA are present in porcine oocytes and embryos. In addition, specific miRNA levels are altered due to stage of embryonic development and, in the case of miR-24, due to culture conditions, making this miRNA a candidate for screening of embryo quality. Additional studies characterizing Dicer and miRNA expression during early embryonic development from IVO and IVF sources are required to further examine and evaluate the use of miRNA as a marker for embryo quality. Copyright © 2012 Elsevier B.V. All rights reserved.

Decreased expression of microRNA-29 family in leiomyoma contributes to increased major fibrillar collagen production.

PubMed

Marsh, Erica E; Steinberg, Marissa L; Parker, J Brandon; Wu, Ju; Chakravarti, Debabrata; Bulun, Serdar E

2016-09-01

To determine the expression and function of the microRNA-29 family (miRNA-29a, miRNA-29b, miRNA-29c) in human leiomyoma and myometrium. Basic science experimental design. Academic medical center. Women undergoing surgery for symptomatic uterine fibroids. Overexpression and knockdown of miRNA-29a, miRNA-29b, and miRNA-29c in primary leiomyoma and myometrial cells. [1] Expression of the miRNA-29 family members in vivo in leiomyoma versus myometrium; [2] Major fibrillar collagen (I, II, III) expression in leiomyoma and myometrial cells with manipulation of miRNA-29 species. Members of the miRNA-29 family (29a, 29b, 29c) are all down-regulated in leiomyoma versus myometrium in vivo. The expression of the miRNA-29 family can be successfully modulated in primary leiomyoma and myometrial cells. Overexpression of the miRNA-29 family in leiomyoma cells results in down-regulation of the major fibrillar collagens. Down-regulation of the miRNA-29 species in myometrium results in an increase in collagen type III deposition. The miRNA-29 family is consistently down-regulated in leiomyoma compared to matched myometrial tissue. This down-regulation contributes to the increased collagen seen in leiomyomas versus myometrium. When miRNA-29 members are overexpressed in leiomyoma cells, protein levels of all of the major fibrillar collagens decrease. The miRNA-29 members are potential therapeutic targets in this highly prevalent condition. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Characterisation of microRNAs from apple (Malus domestica 'Royal Gala') vascular tissue and phloem sap.

PubMed

Varkonyi-Gasic, Erika; Gould, Nick; Sandanayaka, Manoharie; Sutherland, Paul; MacDiarmid, Robin M

2010-08-04

Plant microRNAs (miRNAs) are a class of small, non-coding RNAs that play an important role in development and environmental responses. Hundreds of plant miRNAs have been identified to date, mainly from the model species for which there are available genome sequences. The current challenge is to characterise miRNAs from plant species with agricultural and horticultural importance, to aid our understanding of important regulatory mechanisms in crop species and enable improvement of crops and rootstocks. Based on the knowledge that many miRNAs occur in large gene families and are highly conserved among distantly related species, we analysed expression of twenty-one miRNA sequences in different tissues of apple (Malus x domestica 'Royal Gala'). We identified eighteen sequences that are expressed in at least one of the tissues tested. Some, but not all, miRNAs expressed in apple tissues including the phloem tissue were also detected in the phloem sap sample derived from the stylets of woolly apple aphids. Most of the miRNAs detected in apple phloem sap were also abundant in the phloem sap of herbaceous species. Potential targets for apple miRNAs were identified that encode putative proteins shown to be targets of corresponding miRNAs in a number of plant species. Expression patterns of potential targets were analysed and correlated with expression of corresponding miRNAs. This study validated tissue-specific expression of apple miRNAs that target genes responsible for plant growth, development, and stress response. A subset of characterised miRNAs was also present in the apple phloem translocation stream. A comparative analysis of phloem miRNAs in herbaceous species and woody perennials will aid our understanding of non-cell autonomous roles of miRNAs in plants.

Characterisation of microRNAs from apple (Malus domestica 'Royal Gala') vascular tissue and phloem sap

PubMed Central

2010-01-01

Background Plant microRNAs (miRNAs) are a class of small, non-coding RNAs that play an important role in development and environmental responses. Hundreds of plant miRNAs have been identified to date, mainly from the model species for which there are available genome sequences. The current challenge is to characterise miRNAs from plant species with agricultural and horticultural importance, to aid our understanding of important regulatory mechanisms in crop species and enable improvement of crops and rootstocks. Results Based on the knowledge that many miRNAs occur in large gene families and are highly conserved among distantly related species, we analysed expression of twenty-one miRNA sequences in different tissues of apple (Malus x domestica 'Royal Gala'). We identified eighteen sequences that are expressed in at least one of the tissues tested. Some, but not all, miRNAs expressed in apple tissues including the phloem tissue were also detected in the phloem sap sample derived from the stylets of woolly apple aphids. Most of the miRNAs detected in apple phloem sap were also abundant in the phloem sap of herbaceous species. Potential targets for apple miRNAs were identified that encode putative proteins shown to be targets of corresponding miRNAs in a number of plant species. Expression patterns of potential targets were analysed and correlated with expression of corresponding miRNAs. Conclusions This study validated tissue-specific expression of apple miRNAs that target genes responsible for plant growth, development, and stress response. A subset of characterised miRNAs was also present in the apple phloem translocation stream. A comparative analysis of phloem miRNAs in herbaceous species and woody perennials will aid our understanding of non-cell autonomous roles of miRNAs in plants. PMID:20682080

Identification of MicroRNA as Sepsis Biomarker Based on miRNAs Regulatory Network Analysis

PubMed Central

Huang, Jie; Sun, Zhandong; Yan, Wenying; Zhu, Yujie; Lin, Yuxin; Chen, Jiajai; Shen, Bairong

2014-01-01

Sepsis is regarded as arising from an unusual systemic response to infection but the physiopathology of sepsis remains elusive. At present, sepsis is still a fatal condition with delayed diagnosis and a poor outcome. Many biomarkers have been reported in clinical application for patients with sepsis, and claimed to improve the diagnosis and treatment. Because of the difficulty in the interpreting of clinical features of sepsis, some biomarkers do not show high sensitivity and specificity. MicroRNAs (miRNAs) are small noncoding RNAs which pair the sites in mRNAs to regulate gene expression in eukaryotes. They play a key role in inflammatory response, and have been validated to be potential sepsis biomarker recently. In the present work, we apply a miRNA regulatory network based method to identify novel microRNA biomarkers associated with the early diagnosis of sepsis. By analyzing the miRNA expression profiles and the miRNA regulatory network, we obtained novel miRNAs associated with sepsis. Pathways analysis, disease ontology analysis, and protein-protein interaction network (PIN) analysis, as well as ROC curve, were exploited to testify the reliability of the predicted miRNAs. We finally identified 8 novel miRNAs which have the potential to be sepsis biomarkers. PMID:24809055

Dacarbazine inhibits proliferation of melanoma FEMX-1 cells by up-regulating expression of miRNA-200.

PubMed

Chen, Y-N

2017-03-01

Melanoma is a highly aggressive tumour, and treatment efficacy depends on the stage of the tumour. Early stage cutaneous melanoma is efficiently treated by surgical excision. In contrast, late-stage melanoma requires chemotherapy with dacarbazine (DTIC). Unfortunately, advanced melanoma can often be resistant to DTIC. The mechanisms of anti-melanoma effects of DTIC are still poorly understood, which hinders development of more potent therapies. In this study, we examined the effects of DTIC on growth inhibition of FEMX-1 melanoma cell line, expression of apoptosis-related proteins, and expression of micro (mi)RNA-200 (miRNA-200a, miRNA-200b, miRNA-200c, and miRNA-141). DTIC was used at 50 (low dose) or 100 (high dose) mg/ml. Cell growth inhibition was documented by MTT assay. Cell apoptosis was quantified by propidium iodide staining and caspase 3-8 activity assay. Expression of apoptosis-related proteins Bim, Bak, BAX, and Bad were documented by Western blot analysis, while expression of miRNA-200 by PCR. DTIC dose-dependently inhibited growth of FEMX-1 melanoma cell line, induced cell apoptosis, modulated the levels of apoptosis-related proteins, and up-regulated expression of miRNA-200 family members. DTIC inhibits the growth of melanoma cells by up-regulating expression of miRNA-200.

MicroRNA expression profiling in alveolar macrophages of indigenous Chinese Tongcheng pigs infected with PRRSV in vivo.

PubMed

Zhou, Xiang; Michal, Jennifer J; Jiang, Zhihua; Liu, Bang

2017-11-01

Porcine respiratory and reproductive syndrome (PRRS), caused by PRRS virus (PRRSV), is one of the most serious infectious diseases in the swine industry worldwide. Indigenous Chinese Tongcheng (TC) pigs reportedly show strong resistance to PRRSV infection. The miRNA expression profiles of porcine alveolar macrophages (PAMs) of control TC pigs and those infected with PRRSV in vivo were analyzed by high-throughput sequencing to explore changes induced by infection. A total of 182 known miRNAs including 101 miRNA-5p and 81 miRNA-3p were identified with 23 up-regulated differentially expressed miRNAs (DEmiRNAs) and 25 down-regulated DEmiRNAs. Gene Ontology analysis showed that predicted target genes for the DEmiRNAs were enriched in immune response, transcription regulation, and cell death. The integrative analysis of mRNA and miRNA expression revealed that down-regulated methylation-related genes (DNMT1 and DNMT3b) were targeted by five up-regulated DEmiRNAs. Furthermore, 35 pairs of miRNAs (70 miRNAs) were co-expressed after PRRSV infection and six pairs were co-expressed differently. Our results describe miRNA expression profiles of TC pigs in response to PRRSV infection and lay a strong foundation for developing novel therapies to control PRRS in pigs.

Effect of luteal-phase support on endometrial microRNA expression following controlled ovarian stimulation

PubMed Central

2012-01-01

Background Studies suggested that microRNAs influence cellular activities in the uterus including cell differentiation and embryo implantation. In assisted reproduction cycles, luteal phase support, given to improve endometrial characteristics and to facilitate the implantation process, has been a standard practice. The effect of different types of luteal phase support using steroid hormones in relation to endometrial miRNA profiles during the peri-implantation period has not seen described. This study was designed to evaluate the expression of miRNAs during the luteal phase following controlled ovarian stimulation for IVF and the influence of different luteal phase support protocols on miRNA profiles. Methods The study was approved by the Johns Hopkins Hospital Institutional Review Board. Endometrial biopsies were obtained on the day of oocyte retrieval from 9 oocyte donors (group I). An additional endometrial biopsy was obtained 3–5 days later (Group II) after the donors were randomized into three groups. Group IIa had no luteal-phase support, group IIb had luteal support with micronized progesterone (P), and Group IIc had luteal support with progesterone plus 17-beta-estradiol (P + E). Total RNA was isolated and microarray analysis was performed using an Illumina miRNA expression panel. Results A total of 526 miRNAs were identified. Out of those, 216 miRNAs were differentially regulated (p < 0.05) between the comparison groups. As compared to the day of retrieval, 19, 11 and 6 miRNAs were differentially regulated more than 2 fold in the groups of no support, in the P support only, and in the P + E support respectively, 3–5 days after retrieval. During the peri-implantation period (3–5 days after retrieval) the expression of 33 and 6 miRNAs increased, while the expression of 3 and 0 miRNAs decreased, in the P alone and in the P + E group respectively as compared to the no steroid supplementation group. Conclusion Luteal support following COS has a profound influence on miRNA profiles. Up or down regulation of miRNAs after P or P + E support suggest a role(s) of luteal support in the peri-implantation uterus in IVF cycles through the regulation of associated target genes. PMID:22950660

Distribution and differential expression of microRNAs in the intestinal mucosal layer of necrotic enteritis induced Fayoumi chickens

PubMed Central

Rengaraj, Deivendran; Truong, Anh Duc; Ban, Jihye; Lillehoj, Hyun S.; Hong, Yeong Ho

2017-01-01

Objective Despite an increasing number of investigations into the pathophysiology of necrotic enteritis (NE) disease, etiology of NE-associated diseases, and gene expression profiling of NE-affected tissues, the microRNA (miRNA) profiles of NE-affected poultry have been poorly studied. The aim of this study was to induce NE disease in the genetically disparate Fayoumi chicken lines, and to perform non-coding RNA sequencing in the intestinal mucosal layer. Methods NE disease was induced in the Fayoumi chicken lines (M5.1 and M15.2), and non-coding RNA sequencing was performed in the intestinal mucosal layer of both NE-affected and uninfected chickens to examine the differential expression of miRNAs. Next, quantitative real-time polymerase chain reaction (real-time qPCR) was performed to further examine four miRNAs that showed the highest fold differences. Finally, bioinformatics analyses were performed to examine the four miRNAs target genes involvement in the signaling pathways, and to examine their interaction. Results According to non-coding RNA sequencing, total 50 upregulated miRNAs and 26 downregulated miRNAs were detected in the NE-induced M5.1 chickens. While 32 upregulated miRNAs and 11 downregulated miRNAs were detected in the NE-induced M15.2 chickens. Results of real-time qPCR analysis on the four miRNAs (gga-miR-9-5p, gga-miR-20b-5p, gga-miR-196-5p, and gga-let-7d) were mostly correlated with the results of RNAseq. Overall, gga-miR-20b-5p was significantly downregulated in the NE-induced M5.1 chickens and this was associated with the upregulation of its top-ranking target gene, mitogen-activated protein kinase, kinase 2. Further bioinformatics analyses revealed that 45 of the gene targets of gga-miR-20b-5p were involved in signal transduction and immune system-related pathways, and 35 of these targets were predicted to interact with each other. Conclusion Our study is a novel report of miRNA expression in Fayoumi chickens, and could be very useful in understanding the role of differentially expressed miRNAs in a NE disease model. PMID:28111433

Mining, identification and function analysis of microRNAs and target genes in peanut (Arachis hypogaea L.).

PubMed

Zhang, Tingting; Hu, Shuhao; Yan, Caixia; Li, Chunjuan; Zhao, Xiaobo; Wan, Shubo; Shan, Shihua

2017-02-01

In the present investigation, a total of 60 conserved peanut (Arachis hypogaea L.) microRNA (miRNA) sequences, belonging to 16 families, were identified using bioinformatics methods. There were 392 target gene sequences, identified from 58 miRNAs with Target-align software and BLASTx analyses. Gene Ontology (GO) functional analysis suggested that these target genes were involved in mediating peanut growth and development, signal transduction and stress resistance. There were 55 miRNA sequences, verified employing a poly (A) tailing test, with a success rate of up to 91.67%. Twenty peanut target gene sequences were randomly selected, and the 5' rapid amplification of the cDNA ends (5'-RACE) method were used to validate the cleavage sites of these target genes. Of these, 14 (70%) peanut miRNA targets were verified by means of gel electrophoresis, cloning and sequencing. Furthermore, functional analysis and homologous sequence retrieval were conducted for target gene sequences, and 26 target genes were chosen as the objects for stress resistance experimental study. Real-time fluorescence quantitative PCR (qRT-PCR) technology was applied to measure the expression level of resistance-associated miRNAs and their target genes in peanut exposed to Aspergillus flavus (A. flavus) infection and drought stress, respectively. In consequence, 5 groups of miRNAs & targets were found accorded with the mode of miRNA negatively controlling the expression of target genes. This study, preliminarily determined the biological functions of some resistance-associated miRNAs and their target genes in peanut. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Quantitative Differential Expression Analysis Reveals Mir-7 As Major Islet MicroRNA

PubMed Central

Bravo-Egana, Valia; Rosero, Samuel; Molano, R. Damaris; Pileggi, Antonello; Ricordi, Camillo; Domínguez-Bendala, Juan; Pastori, Ricardo L.

2008-01-01

MicroRNAs (miRNAs) are non-coding gene products that regulate gene expression through specific binding to target mRNAs. Cell-specific patterns of miRNAs are associated with the acquisition and maintenance of a given phenotype, such as endocrine pancreas (islets). We hypothesized that a subset of miRNAs could be differentially expressed in the islets. Using miRNA microarray technology and quantitative RT-PCR we identified a subset of miRNAs that are the most differentially expressed islet miRNAs (ratio islet/acinar >150-fold), mir-7 being the most abundant. A similarly high ratio for mir-7 was observed in human islets. The ratio islet/acinar for mir-375, a previously described islet miRNA, was <10, and is 2.5X more abundant in the islets than mir-7. Therefore, we conclude that mir-7 is the most abundant endocrine miRNA in islets while mir-375 is the most abundant intra-islet miRNA. Our results may offer new insights into regulatory pathways of islet gene expression. PMID:18086561

Effect of β-hydroxy-β-methylbutyrate on miRNA expression in differentiating equine satellite cells exposed to hydrogen peroxide.

PubMed

Chodkowska, Karolina A; Ciecierska, Anna; Majchrzak, Kinga; Ostaszewski, Piotr; Sadkowski, Tomasz

2018-01-01

Skeletal muscle injury activates satellite cells to initiate processes of proliferation, differentiation, and hypertrophy in order to regenerate muscle fibers. The number of microRNAs and their target genes are engaged in satellite cell activation. β-Hydroxy-β-methylbutyrate (HMB) is known to prevent exercise-induced muscle damage. The purpose of this study was to evaluate the effect of HMB on miRNA and relevant target gene expression in differentiating equine satellite cells exposed to H 2 O 2 . We hypothesized that HMB may regulate satellite cell activity, proliferation, and differentiation, hence attenuate the pathological processes induced during an in vitro model of H 2 O 2 -related injury by changing the expression of miRNAs. Equine satellite cells (ESC) were isolated from the samples of skeletal muscle collected from young horses. ESC were treated with HMB (24 h) and then exposed to H 2 O 2 (1 h). For the microRNA and gene expression assessment microarrays, technique was used. Identified miRNAs and genes were validated using real-time qPCR. Cell viability, oxidative stress, and cell damage were measured using colorimetric method and flow cytometry. Analysis of miRNA and gene profile in differentiating ESC pre-incubated with HMB and then exposed to H 2 O 2 revealed difference in the expression of 27 miRNAs and 4740 genes, of which 344 were potential target genes for identified miRNAs. Special attention was focused on differentially expressed miRNAs and their target genes involved in processes related to skeletal muscle injury. Western blot analysis showed protein protection in HMB-pre-treated group compared to control. The viability test confirmed that HMB enhanced cell survival after the hydrogen peroxide exposition. Our results suggest that ESC pre-incubated with HMB and exposed to H 2 O 2 could affect expression on miRNA levels responsible for skeletal muscle development, cell proliferation and differentiation, and activation of tissue repair after injury. Enrichment analyses for targeted genes revealed that a large group of genes was associated with the regulation of signaling pathways crucial for muscle tissue development, protein metabolism, muscle injury, and regeneration, as well as with oxidative stress response.

Genome organization and characteristics of soybean microRNAs

PubMed Central

2012-01-01

Background microRNAs (miRNAs) are key regulators of gene expression and play important roles in many aspects of plant biology. The role(s) of miRNAs in nitrogen-fixing root nodules of leguminous plants such as soybean is not well understood. We examined a library of small RNAs from Bradyrhizobium japonicum-inoculated soybean roots and identified novel miRNAs. In order to enhance our understanding of miRNA evolution, diversification and function, we classified all known soybean miRNAs based on their phylogenetic conservation (conserved, legume- and soybean-specific miRNAs) and examined their genome organization, family characteristics and target diversity. We predicted targets of these miRNAs and experimentally validated several of them. We also examined organ-specific expression of selected miRNAs and their targets. Results We identified 120 previously unknown miRNA genes from soybean including 5 novel miRNA families. In the soybean genome, genes encoding miRNAs are primarily intergenic and a small percentage were intragenic or less than 1000 bp from a protein-coding gene, suggesting potential co-regulation between the miRNA and its parent gene. Difference in number and orientation of tandemly duplicated miRNA genes between orthologous genomic loci indicated continuous evolution and diversification. Conserved miRNA families are often larger in size and produce less diverse mature miRNAs than legume- and soybean-specific families. In addition, the majority of conserved and legume-specific miRNA families produce 21 nt long mature miRNAs with distinct nucleotide distribution and regulate a more conserved set of target mRNAs compared to soybean-specific families. A set of nodule-specific target mRNAs and their cognate regulatory miRNAs had inverse expression between root and nodule tissues suggesting that spatial restriction of target gene transcripts by miRNAs might govern nodule-specific gene expression in soybean. Conclusions Genome organization of soybean miRNAs suggests that they are actively evolving. Distinct family characteristics of soybean miRNAs suggest continuous diversification of function. Inverse organ-specific expression between selected miRNAs and their targets in the roots and nodules, suggested a potential role for these miRNAs in regulating nodule development. PMID:22559273

Deregulation of cancer-related miRNAs is a common event in both benign and malignant human breast tumors.

PubMed

Tahiri, Andliena; Leivonen, Suvi-Katri; Lüders, Torben; Steinfeld, Israel; Ragle Aure, Miriam; Geisler, Jürgen; Mäkelä, Rami; Nord, Silje; Riis, Margit L H; Yakhini, Zohar; Kleivi Sahlberg, Kristine; Børresen-Dale, Anne-Lise; Perälä, Merja; Bukholm, Ida R K; Kristensen, Vessela N

2014-01-01

MicroRNAs (miRNAs) are endogenous non-coding RNAs, which play an essential role in the regulation of gene expression during carcinogenesis. The role of miRNAs in breast cancer has been thoroughly investigated, and although many miRNAs are identified as cancer related, little is known about their involvement in benign tumors. In this study, we investigated miRNA expression profiles in the two most common types of human benign tumors (fibroadenoma/fibroadenomatosis) and in malignant breast tumors and explored their role as oncomirs and tumor suppressor miRNAs. Here, we identified 33 miRNAs with similar deregulated expression in both benign and malignant tumors compared with the expression levels of those in normal tissue, including breast cancer-related miRNAs such as let-7, miR-21 and miR-155. Additionally, messenger RNA (mRNA) expression profiles were obtained for some of the same samples. Using integrated mRNA/miRNA expression analysis, we observed that overexpression of certain miRNAs co-occurred with a significant downregulation of their candidate target mRNAs in both benign and malignant tumors. In support of these findings, in vitro functional screening of the downregulated miRNAs in non-malignant and breast cancer cell lines identified several possible tumor suppressor miRNAs, including miR-193b, miR-193a-3p, miR-126, miR-134, miR-132, miR-486-5p, miR-886-3p, miR-195 and miR-497, showing reduced growth when re-expressed in cancer cells. The finding of deregulated expression of oncomirs and tumor suppressor miRNAs in benign breast tumors is intriguing, indicating that they may play a role in proliferation. A role of cancer-related miRNAs in the early phases of carcinogenesis and malignant transformation can, therefore, not be ruled out.

microRNA analysis of Taenia crassiceps cysticerci under praziquantel treatment and genome-wide identification of Taenia solium miRNAs.

PubMed

Pérez, Matías Gastón; Macchiaroli, Natalia; Lichtenstein, Gabriel; Conti, Gabriela; Asurmendi, Sebastián; Milone, Diego Humberto; Stegmayer, Georgina; Kamenetzky, Laura; Cucher, Marcela; Rosenzvit, Mara Cecilia

2017-09-01

MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as important regulators of gene expression and perform critical functions in development and disease. In spite of the increased interest in miRNAs from helminth parasites, no information is available on miRNAs from Taenia solium, the causative agent of cysticercosis, a neglected disease affecting millions of people worldwide. Here we performed a comprehensive analysis of miRNAs from Taenia crassiceps, a laboratory model for T. solium studies, and identified miRNAs in the T. solium genome. Moreover, we analysed the effect of praziquantel, one of the two main drugs used for cysticercosis treatment, on the miRNA expression profile of T. crassiceps cysticerci. Using small RNA-seq and two independent algorithms for miRNA prediction, as well as northern blot validation, we found transcriptional evidence of 39 miRNA loci in T. crassiceps. Since miRNAs were mapped to the T. solium genome, these miRNAs are considered common to both parasites. The miRNA expression profile of T. crassiceps was biased to the same set of highly expressed miRNAs reported in other cestodes. We found a significant altered expression of miR-7b under praziquantel treatment. In addition, we searched for miRNAs predicted to target genes related to drug response. We performed a detailed target prediction for miR-7b and found genes related to drug action. We report an initial approach to study the effect of sub-lethal drug treatment on miRNA expression in a cestode parasite, which provides a platform for further studies of miRNA involvement in drug effects. The results of our work could be applied to drug development and provide basic knowledge of cysticercosis and other neglected helminth infections. Copyright © 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

Ago2 and Dicer1 are involved in METH-induced locomotor sensitization in mice via biogenesis of miRNA.

PubMed

Liu, Dan; Zhu, Li; Ni, Tong; Guan, Fang-Lin; Chen, Yan-Jiong; Ma, Dong-Liang; Goh, Eyleen L K; Chen, Teng

2018-03-08

microRNA (miRNA) play important roles in drug addiction and act as a post-transcriptional regulator of gene expression. We previously reported extensive downregulation of miRNAs in the nucleus accumbens (NAc) of methamphetamine (METH)-sensitized mice. However, the regulatory mechanism of this METH-induced downregulation of miRNAs has yet to be elucidated. Thus, we examined METH-induced changes in the expression of miRNAs and their precursors, as well as the expression levels of mRNA and the proteins involved in miRNA biogenesis such as Dicer1 and Ago2, in the nucleus accumbens of METH-induced locomotor sensitized mice. miRNAs and Ago2 were significantly downregulated, while the expression of miRNA precursors remained unchanged or upregulated, which suggests that the downregulation of miRNAs was likely due to a reduction in Ago2-mediated splicing but unlikely to be regulated at the transcription level. Interestingly, the expression level of Dicer1, which is a potential target of METH-induced decreased miRNAs, such as miR-124, miR-212 and miR-29b, was significantly increased. In conclusion, this study indicates that miRNA biogenesis (such as Ago2 and Dicer1) and their miRNA products may have a role in the development of METH addiction. © 2018 Society for the Study of Addiction.

Identification of Differentially Expressed miRNAs between White and Black Hair Follicles by RNA-Sequencing in the Goat (Capra hircus)

PubMed Central

Wu, Zhenyang; Fu, Yuhua; Cao, Jianhua; Yu, Mei; Tang, Xiaohui; Zhao, Shuhong

2014-01-01

MicroRNAs (miRNAs) play a key role in many biological processes by regulating gene expression at the post-transcriptional level. A number of miRNAs have been identified from livestock species. However, compared with other animals, such as pigs and cows, the number of miRNAs identified in goats is quite low, particularly in hair follicles. In this study, to investigate the functional roles of miRNAs in goat hair follicles of goats with different coat colors, we sequenced miRNAs from two hair follicles samples (white and black) using Solexa sequencing. A total of 35,604,016 reads were obtained, which included 30,878,637 clean reads (86.73%). MiRDeep2 software identified 214 miRNAs. Among them, 205 were conserved among species and nine were novel miRNAs. Furthermore, DESeq software identified six differentially expressed miRNAs. Quantitative PCR confirmed differential expression of two miRNAs, miR-10b and miR-211. KEGG pathways were analyzed using the DAVID website for the predicted target genes of the differentially expressed miRNAs. Several signaling pathways including Notch and MAPK pathways may affect the process of coat color formation. Our study showed that the identified miRNAs might play an essential role in black and white follicle formation in goats. PMID:24879525

PUFA diets alter the microRNA expression profiles in an inflammation rat model.

PubMed

Zheng, Zheng; Ge, Yinlin; Zhang, Jinyu; Xue, Meilan; Li, Quan; Lin, Dongliang; Ma, Wenhui

2015-06-01

Omega‑3 and ‑6 polyunsaturated fatty acids (PUFAs) can directly or indirectly regulate immune homeostasis via inflammatory pathways, and components of these pathways are crucial targets of microRNAs (miRNAs). However, no study has examined the changes in the miRNA transcriptome during PUFA‑regulated inflammatory processes. Here, we established PUFA diet‑induced autoimmune‑prone (AP) and autoimmune‑averse (AA) rat models, and studied their physical characteristics and immune status. Additionally, miRNA expression patterns in the rat models were compared using microarray assays and bioinformatic methods. A total of 54 miRNAs were differentially expressed in common between the AP and the AA rats, and the changes in rno‑miR‑19b‑3p, ‑146b‑5p and ‑183‑5p expression were validated using stem‑loop reverse transcription‑quantitative polymerase chain reaction. To better understand the mechanisms underlying PUFA‑regulated miRNA changes during inflammation, computational algorithms and biological databases were used to identify the target genes of the three validated miRNAs. Furthermore, Gene Ontology (GO) term annotation and KEGG pathway analyses of the miRNA targets further allowed to explore the potential implication of the miRNAs in inflammatory pathways. The predicted PUFA‑regulated inflammatory pathways included the Toll‑like receptor (TLR), T cell receptor (TCR), NOD‑like receptor (NLR), RIG‑I‑like receptor (RLR), mitogen‑activated protein kinase (MAPK) and the transforming growth factor‑β (TGF‑β) pathway. This study is the first report, to the best of our knowledge, on in vivo comparative profiling of miRNA transcriptomes in PUFA diet‑induced inflammatory rat models using a microarray approach. The results provide a useful resource for future investigation of the role of PUFA‑regulated miRNAs in immune homeostasis.

Skin tumor formation in human papillomavirus 8 transgenic mice is associated with a deregulation of oncogenic miRNAs and their tumor suppressive targets.

PubMed

Hufbauer, Martin; Lazić, Daliborka; Reinartz, Markus; Akgül, Baki; Pfister, Herbert; Weissenborn, Sönke Jan

2011-10-01

Dysregulation of microRNA (miRNA) expression is regularly found in various types of cancer and contributes to tumorigenic processes. However, little is known about miRNA expression in non-melanoma skin cancer in which a pathogenic role of beta human papillomaviruses (HPV) is discussed. A carcinogenic potential of beta HPV8 could be demonstrated in a transgenic mouse model, expressing all early genes of HPV8 (HPV8-CER). A single UVA/B-dose induced oncogene expression and led to papilloma growth within three weeks. Expression of miRNAs and their targets during HPV8-mediated tumor formation in mice. Skin of untreated or UV-irradiated wild-type and HPV8-CER mice was analyzed for miRNA expression and localization by qPCR and in situ hybridization. MiRNA target protein expression was analyzed by immunohistochemical staining. Early steps in skin tumor formation in HPV8-CER mice were associated with an upregulation of the oncogenic miRNA-17-5p, -21 and -106a and a downregulation of the tumor-suppressive miRNA-155 and -206, which could be demonstrated by qPCR and in situ hybridization. The respective targets of miRNA-21 and -106a, the tumor suppressors PTEN, PDCD4 and Rb with their pivotal role in cell cycle regulation, apoptosis and proliferation were found to be downregulated. This is the first report demonstrating that a cutaneous HPV type deregulates the expression of miRNAs. These deregulations are closely related to the UV-induced upregulation of HPV8 oncogene levels, which suggest a direct or indirect HPV8-specific effect on miRNA expression. These data presume that HPV8 interferes with the miRNA mediated gene regulation to induce tumorigenesis. Copyright © 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Preoperative chemoradiotherapy for rectal cancer: the sensitizer role of the association between miR-375 and c-Myc

PubMed Central

Conde-Muiño, Raquel; Cano, Carlos; Sánchez-Martín, Victoria; Herrera, Antonio; Comino, Ana; Medina, Pedro P.; Palma, Pablo; Cuadros, Marta

2017-01-01

Administration of chemoradiation before tumor resection has revolutionized the management of locally advanced rectal cancer, but many patients have proven resistant to this preoperative therapy. Our group recently reported a negative correlation between c-Myc gene expression and this resistance. In the present study, integrated analysis of miRNA and mRNA expression profiles was conducted in 45 pre-treatment rectal tumors in order to analyze the expressions of miRNAs and c-Myc and their relationship with clinicopathological factors and patient survival. Twelve miRNAs were found to be differentially expressed by responders and non-responders to the chemoradiation. Functional classification revealed an association between the differentially expressed miRNAs and c-Myc. Quantitative real-time PCR results showed that miRNA-148 and miRNA-375 levels were both significantly lower in responders than in non-responders. Notably, a significant negative correlation was found between miRNA-375 expression and c-Myc expression. According to these findings, miRNA-375 and its targeted c-Myc may be useful as a predictive biomarker of the response to neoadjuvant treatment in patients with locally advanced rectal cancer. PMID:29137264

Identification and characterization of microRNAs in Phaseolus vulgaris by high-throughput sequencing

PubMed Central

2012-01-01

Background MicroRNAs (miRNAs) are endogenously encoded small RNAs that post-transcriptionally regulate gene expression. MiRNAs play essential roles in almost all plant biological processes. Currently, few miRNAs have been identified in the model food legume Phaseolus vulgaris (common bean). Recent advances in next generation sequencing technologies have allowed the identification of conserved and novel miRNAs in many plant species. Here, we used Illumina's sequencing by synthesis (SBS) technology to identify and characterize the miRNA population of Phaseolus vulgaris. Results Small RNA libraries were generated from roots, flowers, leaves, and seedlings of P. vulgaris. Based on similarity to previously reported plant miRNAs,114 miRNAs belonging to 33 conserved miRNA families were identified. Stem-loop precursors and target gene sequences for several conserved common bean miRNAs were determined from publicly available databases. Less conserved miRNA families and species-specific common bean miRNA isoforms were also characterized. Moreover, novel miRNAs based on the small RNAs were found and their potential precursors were predicted. In addition, new target candidates for novel and conserved miRNAs were proposed. Finally, we studied organ-specific miRNA family expression levels through miRNA read frequencies. Conclusions This work represents the first massive-scale RNA sequencing study performed in Phaseolus vulgaris to identify and characterize its miRNA population. It significantly increases the number of miRNAs, precursors, and targets identified in this agronomically important species. The miRNA expression analysis provides a foundation for understanding common bean miRNA organ-specific expression patterns. The present study offers an expanded picture of P. vulgaris miRNAs in relation to those of other legumes. PMID:22394504

Role of non-coding RNAs in maintaining primary airway smooth muscle cells

PubMed Central

2014-01-01

Background The airway smooth muscle (ASM) cell maintains its own proliferative rate and contributes to the inflammatory response in the airways, effects that are inhibited by corticosteroids, used in the treatment of airways diseases. Objective We determined the differential expression of mRNAs, microRNAs (miRNAs) and long noncoding RNA species (lncRNAs) in primary ASM cells following treatment with a corticosteroid, dexamethasone, and fetal calf serum (FCS). Methods mRNA, miRNA and lncRNA expression was measured by microarray and quantitative real-time PCR. Results A small number of miRNAs (including miR-150, −371-5p, −718, −940, −1181, −1207-5p, −1915, and −3663-3p) were decreased following exposure to dexamethasone and FCS. The mRNA targets of these miRNAs were increased in expression. The changes in mRNA expression were associated with regulation of ASM actin cytoskeleton. We also observed changes in expression of lncRNAs, including natural antisense, pseudogenes, intronic lncRNAs, and intergenic lncRNAs following dexamethasone and FCS. We confirmed the change in expression of three of these, LINC00882, LINC00883, PVT1, and its transcriptional activator, c-MYC. We propose that four of these lincRNAs (RP11-46A10.4, LINC00883, BCYRN1, and LINC00882) act as miRNA ‘sponges’ for 4 miRNAs (miR-150, −371-5p, −940, −1207-5p). Conclusion This in-vitro model of primary ASM cell phenotype was associated with the regulation of several ncRNAs. Their identification allows for in-vitro functional experimentation to establish causality with the primary ASM phenotype, and in airway diseases such as asthma and chronic obstructive pulmonary disease (COPD). PMID:24886442

Second generation sequencing of microRNA in Human Bone Cells treated with Parathyroid Hormone or Dexamethasone.

PubMed

Laxman, Navya; Rubin, Carl-Johan; Mallmin, Hans; Nilsson, Olle; Tellgren-Roth, Christian; Kindmark, Andreas

2016-03-01

We investigated the impact of treatment with parathyroid hormone (PTH) and dexamethasone (DEX) for 2 and 24h by RNA sequencing of miRNAs in primary human bone (HOB) cells. A total of 207 million reads were obtained, and normalized absolute expression retrieved for 373 most abundant miRNAs. In naïve control cells, 7 miRNAs were differentially expressed (FDR<0.05) between the two time points. Ten miRNAs exhibited differential expression (FDR <0.05) across two time points and treatments after adjusting for expression in controls and were selected for downstream analyses. Results show significant effects on miRNA expression when comparing PTH with DEX at 2h with even more pronounced effects at 24h. Interestingly, several miRNAs exhibiting differences in expression are predicted to target genes involved in bone metabolism e.g. miR-30c2, miR-203 and miR-205 targeting RUNX2, and miR-320 targeting β-catenin (CTNNB1) mRNA expression. CTNNB1and RUNX2 levels were decreased after DEX treatment and increased after PTH treatment. Our analysis also identified 2 putative novel miRNAs in PTH and DEX treated cells at 24h. RNA sequencing showed that PTH and DEX treatment affect miRNA expression in HOB cells and that regulated miRNAs in turn are correlated with expression levels of key genes involved in bone metabolism. Copyright © 2016 Elsevier Inc. All rights reserved.

Differential Expression of miRNAs in the Respiratory Tree of the Sea Cucumber Apostichopus japonicus Under Hypoxia Stress.

PubMed

Huo, Da; Sun, Lina; Li, Xiaoni; Ru, Xiaoshang; Liu, Shilin; Zhang, Libin; Xing, Lili; Yang, Hongsheng

2017-11-06

The sea cucumber, an important economic species, has encountered high mortality since 2013 in northern China because of seasonal environmental stress such as hypoxia, high temperature, and low salinity. MicroRNAs (miRNAs) are important in regulating gene expression in marine organisms in response to environmental change. In this study, high-throughput sequencing was used to investigate alterations in miRNA expression in the sea cucumber under different levels of dissolved oxygen (DO). Nine small RNA libraries were constructed from the sea cucumber respiratory trees. A total of 26 differentially expressed miRNAs, including 12 upregulated and 14 downregulated miRNAs, were observed in severe hypoxia (DO 2 mg/L) compared with mild hypoxia (DO 4 mg/L) and normoxic conditions (DO 8 mg/L). Twelve differentially expressed miRNAs were clustered in severe hypoxia. In addition, real-time PCR revealed that 14 randomly selected differentially expressed miRNAs showed significantly increased expressions in severe hypoxia and the expressions of nine miRNAs, including key miRNAs such as Aja-miR-1, Aja-miR-2008, and Aja-miR-184, were consistent with the sequencing results. Moreover, gene ontology and pathway analyses of putative target genes suggest that these miRNAs are important in redox, transport, transcription, and hydrolysis under hypoxia stress. Notably, novel-miR-1, novel-miR-2, and novel-miR-3 were specifically clustered and upregulated in severe hypoxia, which may provide new insights into novel "hypoxamiR" identification. These results will provide a basis for future studies of miRNA regulation and molecular adaptive mechanisms in sea cucumbers under hypoxia stress. Copyright © 2017 Huo et al.

Ewing's Sarcoma: An Analysis of miRNA Expression Profiles and Target Genes in Paraffin-Embedded Primary Tumor Tissue.

PubMed

Parafioriti, Antonina; Bason, Caterina; Armiraglio, Elisabetta; Calciano, Lucia; Daolio, Primo Andrea; Berardocco, Martina; Di Bernardo, Andrea; Colosimo, Alessia; Luksch, Roberto; Berardi, Anna C

2016-04-30

The molecular mechanism responsible for Ewing's Sarcoma (ES) remains largely unknown. MicroRNAs (miRNAs), a class of small non-coding RNAs able to regulate gene expression, are deregulated in tumors and may serve as a tool for diagnosis and prediction. However, the status of miRNAs in ES has not yet been thoroughly investigated. This study compared global miRNAs expression in paraffin-embedded tumor tissue samples from 20 ES patients, affected by primary untreated tumors, with miRNAs expressed in normal human mesenchymal stromal cells (MSCs) by microarray analysis. A miRTarBase database was used to identify the predicted target genes for differentially expressed miRNAs. The miRNAs microarray analysis revealed distinct patterns of miRNAs expression between ES samples and normal MSCs. 58 of the 954 analyzed miRNAs were significantly differentially expressed in ES samples compared to MSCs. Moreover, the qRT-PCR analysis carried out on three selected miRNAs showed that miR-181b, miR-1915 and miR-1275 were significantly aberrantly regulated, confirming the microarray results. Bio-database analysis identified BCL-2 as a bona fide target gene of the miR-21, miR-181a, miR-181b, miR-29a, miR-29b, miR-497, miR-195, miR-let-7a, miR-34a and miR-1915. Using paraffin-embedded tissues from ES patients, this study has identified several potential target miRNAs and one gene that might be considered a novel critical biomarker for ES pathogenesis.

A quantitative framework for the forward design of synthetic miRNA circuits.

PubMed

Bloom, Ryan J; Winkler, Sally M; Smolke, Christina D

2014-11-01

Synthetic genetic circuits incorporating regulatory components based on RNA interference (RNAi) have been used in a variety of systems. A comprehensive understanding of the parameters that determine the relationship between microRNA (miRNA) and target expression levels is lacking. We describe a quantitative framework supporting the forward engineering of gene circuits that incorporate RNAi-based regulatory components in mammalian cells. We developed a model that captures the quantitative relationship between miRNA and target gene expression levels as a function of parameters, including mRNA half-life and miRNA target-site number. We extended the model to synthetic circuits that incorporate protein-responsive miRNA switches and designed an optimized miRNA-based protein concentration detector circuit that noninvasively measures small changes in the nuclear concentration of β-catenin owing to induction of the Wnt signaling pathway. Our results highlight the importance of methods for guiding the quantitative design of genetic circuits to achieve robust, reliable and predictable behaviors in mammalian cells.

Identification of differentially expressed microRNA in the stems and leaves during sugar accumulation in sweet sorghum.

PubMed

Yu, Huilin; Cong, Ling; Zhu, Zhenxing; Wang, Chunyu; Zou, Jianqiu; Tao, Chengguang; Shi, Zhensheng; Lu, Xiaochun

2015-10-25

MicroRNAs (miRNAs) have been shown to play important roles in plant development, growth and stress response. Sweet sorghum [Sorghum bicolor (L.) Moench] is an important source of bioenergy due to the high sugar content in its stems. However, it is not clear how the miRNA is involved in sugar accumulation in sorghum stems. In order to identify the miRNAs in the stems and the leaves of sweet sorghum, we extracted RNAs of the stems and leaves of sweet sorghum (Rio) and grain sorghum (BTx623) at the heading and dough stages for high-throughput sequencing. A total of 179279048 reads were obtained from Illumina-based sequencing. Further analysis identified nine known miRNAs and twelve novel miRNAs that showed significantly and specifically differentially expressed in the stems of sweet sorghum. The target genes of the differentially expressed novel miRNAs include the transcription factor, glucosyltransferase, protein kinase, cytochrome P450, transporters etc. GO enrichment analysis showed that the predicted targets of these differentially expressed miRNAs participated in diverse physiological and metabolic processes. We performed RT-qRCR analysis on these miRNAs across eight different libraries to validate the miRNAs. Finally, we screened stem-specifically expressed novel miRNA and a leaf-specifically expressed novel miRNA in sweet sorghum comparing with grain sorghum. Our results provide a basis for further investigation of the potential role of these individual miRNAs in sugar accumulation. Copyright © 2015 Elsevier B.V. All rights reserved.

Characterization and differential expression of microRNAs elicited by sulfur deprivation in Chlamydomonas reinhardtii

PubMed Central

2012-01-01

Background microRNAs (miRNAs) have been found to play an essential role in the modulation of numerous biological processes in eukaryotes. Chlamydomonas reinhardtii is an ideal model organism for the study of many metabolic processes including responses to sulfur-deprivation. We used a deep sequencing platform to extensively profile and identify changes in the miRNAs expression that occurred under sulfur-replete and sulfur-deprived conditions. The aim of our research was to characterize the differential expression of Chlamydomonas miRNAs under sulfur-deprived conditions, and subsequently, the target genes of miRNA involved in sulfur-deprivation were further predicted and analyzed. Results By using high-throughput sequencing, we characterized the microRNA transcriptomes under sulphur-replete and sulfur-deprived conditions in Chlamydomonas reinhardtii. We predicted a total of 310 miRNAs which included 85 known miRNAs and 225 novel miRNAs. 13 miRNAs were the specific to the sulfur-deprived conditions. 47 miRNAs showed significantly differential expressions responding to sulfur-deprivation, and most were up-regulated in the small RNA libraries with sulfur-deprivation. Using a web-based integrated system (Web MicroRNAs Designer 3) and combing the former information from a transcriptome of Chlamydomonas reinhardtii, 22 miRNAs and their targets involved in metabolism regulation with sulfur-deprivation were verified. Conclusions Our results indicate that sulfur-deprivation may have a significant influence on small RNA expression patterns, and the differential expressions of miRNAs and interactions between miRNA and its targets might further reveal the molecular mechanism responding to sulfur-deprivation in Chlamydomonas reinhardtii. PMID:22439676

Genome-wide identification of heat stress-responsive small RNAs in tall fescue (Festuca arundinacea) by high-throughput sequencing.

PubMed

Li, Huiying; Hu, Tao; Amombo, Erick; Fu, Jinmin

2017-06-01

MicroRNAs (miRNAs) play vital roles in the adaptive response of plants to various abiotic and biotic stresses. Tall fescue (Festuca arundinacea Schreb.) is a major cool-season forage and turf grass species which is severely influenced by heat stress. To unravel possible heat stress-responsive miRNAs, high-throughput sequencing was employed for heat-tolerant PI578718 and heat-sensitive PI234881 genotypes growing in presence and absence of heat stress (40°C for 36h). By searching against the miRBase database, among 1421 reference monocotyledon miRNAs, more than 850 were identified in all samples. Among these miRNAs, 1.46% and 2.29% were differentially expressed in PI234881 and PI578718 under heat stress, respectively, and most of them were down-regulated. In addition, a total of 170 novel miRNAs belonging to 145 miRNA families were identified. Furthermore, putative targets of differentially expressed miRNAs were predicted. The regulation of selected miRNAs by heat stress was revalidated through quantitative reverse transcription PCR (qRT-PCR) analysis. Most of these miRNAs shared similar expression patterns; however, some showed distinct expression patterns under heat stress, with their putative targets displaying different transcription levels. This is the first genome-wide miRNA identification in tall fescue. miRNAs specific to PI578718, or those that exhibited differential expression profiles between the two genotypes under high temperature, were probably associated with the variation in thermotolerance of tall fescue. The differentially expressed miRNAs between these two tall fescue genotypes and their putative targeted genes will provide essential information for further study on miRNAs mediating heat response and facilitate to improve turf grass breeding. Copyright © 2017. Published by Elsevier GmbH.

Changes in microRNA expression during differentiation of embryonic and induced pluripotent stem cells to definitive endoderm.

PubMed

Francis, Natalie; Moore, Melanie; Asan, Simona G; Rutter, Guy A; Burns, Chris

2015-01-01

Pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have the potential to treat type 1 diabetes through cell replacement therapy. However, the protocols used to generate insulin-expressing cells in vitro frequently result in cells which have an immature phenotype and are functionally restricted. MicroRNAs (miRNAs) are now known to be important in cell fate specification, and a unique miRNA signature characterises pancreatic development at the definitive endoderm stage. Several studies have described differences in miRNA expression between ESCs and iPSCs. Here we have used microarray analysis both to identify miRNAs up- or down-regulated upon endoderm formation, and also miRNAs differentially expressed between ESCs and iPSCs. Several miRNAs fulfilling both these criteria were identified, suggesting that differences in the expression of these miRNAs may affect the ability of pluripotent stem cells to differentiate into definitive endoderm. The expression of these miRNAs was validated by qRT-PCR, and the relationship between one of these miRNAs, miR-151a-5p, and its predicted target gene, SOX17, was investigated by luciferase assay, and suggested an interaction between miR-151a-5p and this key transcription factor. In conclusion, these findings demonstrate a unique miRNA expression pattern for definitive endoderm derived from both embryonic and induced pluripotent stem cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Relative Expression of PBMC MicroRNA-133a Analysis in Patients Receiving Warfarin After Mechanical Heart Valve Replacement

PubMed Central

Kabiri Rad, Hamid; Mazaheri, Mahta; Dehghani Firozabadi, Ali

Background: MicroRNAs (miRNAs) are implicated in various biological processes including anticoagulation. However, the modulation of miRNA by pharmacological intervention such as warfarin treatment in patients receiving warfarin has not been disclosed yet. The aim of this study work was to assess the effect of warfarin drug on expression level of mir-133a-3p in patients with mechanical heart valve replacement. Methods: In this research, the expression level of miRNA-133a-3p was analyzed in Peripheral Blood Mononuclear Cells (PBMCs) from mechanical valve replacement patients who had received warfarin for at least 3 months continuously. Quantitative RT-PCR method was used for this assay. Results: Our findings indicated a significant diffrence between the rate of miR-133a-3p expression in individuals receiving warfarin and the control group (p<0.01). There was also a statistically significant difference in miR-133a-3p expression in patients with different ages (p<0.05) suggesting that the rate of miR-133a-3p expression in persons receiving warfarin is related to age. However, other variables like warfarin dose, International Normalized Ratio (INR), gender, and Body Mass Index (BMI) were not significantly effective on the miR-133a-3p experssion rate in individuals receving warfarin. Conclusion: Based on our results, it can be concluded that miR-133a-3p is involved in the coagulation pathway. The recent result indicates that warfarin affects the expression of miR-133a. This expression may be potentially important for treatment by anticoagulants. Awareness of the time course of miRNA expression profile can improve efficiency of response to warfarin. PMID:29296264

Characterization and Comparative Profiling of MiRNA Transcriptomes in Bighead Carp and Silver Carp

PubMed Central

Chi, Wei; Tong, Chaobo; Gan, Xiaoni; He, Shunping

2011-01-01

MicroRNAs (miRNAs) are small non-coding RNA molecules that are processed from large ‘hairpin’ precursors and function as post-transcriptional regulators of target genes. Although many individual miRNAs have recently been extensively studied, there has been very little research on miRNA transcriptomes in teleost fishes. By using high throughput sequencing technology, we have identified 167 and 166 conserved miRNAs (belonging to 108 families) in bighead carp (Hypophthalmichthys nobilis) and silver carp (Hypophthalmichthys molitrix), respectively. We compared the expression patterns of conserved miRNAs by means of hierarchical clustering analysis and log2 ratio. Results indicated that there is not a strong correlation between sequence conservation and expression conservation, most of these miRNAs have similar expression patterns. However, high expression differences were also identified for several individual miRNAs. Several miRNA* sequences were also found in our dataset and some of them may have regulatory functions. Two computational strategies were used to identify novel miRNAs from un-annotated data in the two carps. A first strategy based on zebrafish genome, identified 8 and 22 novel miRNAs in bighead carp and silver carp, respectively. We postulate that these miRNAs should also exist in the zebrafish, but the methodologies used have not allowed for their detection. In the second strategy we obtained several carp-specific miRNAs, 31 in bighead carp and 32 in silver carp, which showed low expression. Gain and loss of family members were observed in several miRNA families, which suggests that duplication of animal miRNA genes may occur through evolutionary processes which are similar to the protein-coding genes. PMID:21858165

A rapid, ratiometric, enzyme-free, and sensitive single-step miRNA detection using three-way junction based FRET probes

NASA Astrophysics Data System (ADS)

Luo, Qingying; Liu, Lin; Yang, Cai; Yuan, Jing; Feng, Hongtao; Chen, Yan; Zhao, Peng; Yu, Zhiqiang; Jin, Zongwen

2018-03-01

MicroRNAs (miRNAs) are single stranded endogenous molecules composed of only 18-24 nucleotides which are critical for gene expression regulating the translation of messenger RNAs. Conventional methods based on enzyme-assisted nucleic acid amplification techniques have many problems, such as easy contamination, high cost, susceptibility to false amplification, and tendency to have sequence mismatches. Here we report a rapid, ratiometric, enzyme-free, sensitive, and highly selective single-step miRNA detection using three-way junction assembled (or self-assembled) FRET probes. The developed strategy can be operated within the linear range from subnanomolar to hundred nanomolar concentrations of miRNAs. In comparison with the traditional approaches, our method showed high sensitivity for the miRNA detection and extreme selectivity for the efficient discrimination of single-base mismatches. The results reveal that the strategy paved a new avenue for the design of novel highly specific probes applicable in diagnostics and potentially in microscopic imaging of miRNAs in real biological environments.

Dysregulation of hepatic microRNA expression profiles with Clonorchis sinensis infection.

PubMed

Han, Su; Tang, Qiaoran; Lu, Xi; Chen, Rui; Li, Yihong; Shu, Jing; Zhang, Xiaoli; Cao, Jianping

2016-11-30

Clonorchiasis remains an important zoonotic parasitic disease worldwide. The molecular mechanisms of host-parasite interaction are not fully understood. Non-coding microRNAs (miRNAs) are considered to be key regulators in parasitic diseases. The regulation of miRNAs and host micro-environment may be involved in clonorchiasis, and require further investigation. MiRNA microarray technology and bioinformatic analysis were used to investigate the regulatory mechanisms of host miRNA and to compare miRNA expression profiles in the liver tissues of control and Clonorchis sinensis (C. sinensis)-infected rats. A total of eight miRNAs were downregulated and two were upregulated, which showed differentially altered expression profiles in the liver tissue of C. sinensis-infected rats. Further analysis of the differentially expressed miRNAs revealed that many important signal pathways were triggered after infection with C. sinensis, which were related to clonorchiasis pathogenesis, such as cell apoptosis and inflammation, as well as genes involved in signal transduction mechanisms, such as pathways in cancer and the Wnt and Mitogen-activated protein kinases (MAPK) signaling pathways. The present study revealed that the miRNA expression profiles of the host were changed by C. sinensis infection. This dysregulation in miRNA expression may contribute to the etiology and pathophysiology of clonorchiasis. These results also provide new insights into the regulatory mechanisms of miRNAs in clonorchiasis, which may present potential targets for future C. sinensis control strategies.

Inflammation-Mediated Regulation of MicroRNA Expression in Transplanted Pancreatic Islets

PubMed Central

Bravo-Egana, Valia; Rosero, Samuel; Klein, Dagmar; Jiang, Zhijie; Vargas, Nancy; Tsinoremas, Nicholas; Doni, Marco; Podetta, Michele; Ricordi, Camillo; Molano, R. Damaris; Pileggi, Antonello; Pastori, Ricardo L.

2012-01-01

Nonspecific inflammation in the transplant microenvironment results in β-cell dysfunction and death influencing negatively graft outcome. MicroRNA (miRNA) expression and gene target regulation in transplanted islets are not yet well characterized. We evaluated the impact of inflammation on miRNA expression in transplanted rat islets. Islets exposed in vitro to proinflammatory cytokines and explanted syngeneic islet grafts were evaluated by miRNA arrays. A subset of 26 islet miRNAs was affected by inflammation both in vivo and in vitro. Induction of miRNAs was dependent on NF-κB, a pathway linked with cytokine-mediated islet cell death. RT-PCR confirmed expression of 8 miRNAs. The association between these miRNAs and mRNA target-predicting algorithms in genome-wide RNA studies of β-cell inflammation identified 238 potential miRNA gene targets. Several genes were ontologically associated with regulation of insulin signaling and secretion, diabetes, and islet physiology. One of the most activated miRNAs was miR-21. Overexpression of miR-21 in insulin-secreting MIN6 cells downregulated endogenous expression of the tumor suppressor Pdcd4 and of Pclo, a Ca2+ sensor protein involved in insulin secretion. Bioinformatics identified both as potential targets. The integrated analysis of miRNA and mRNA expression profiles revealed potential targets that may identify molecular targets for therapeutic interventions. PMID:22655170

Prognostic and Clinical Significance of miRNA-205 in Endometrioid Endometrial Cancer.

PubMed

Wilczynski, Milosz; Danielska, Justyna; Dzieniecka, Monika; Szymanska, Bozena; Wojciechowski, Michal; Malinowski, Andrzej

2016-01-01

Endometrial cancer is one of the most common malignancies of the reproductive female tract, with endometrioid endometrial cancer being the most frequent type. Despite the relatively favourable prognosis in cases of endometrial cancer, there is a necessity to evaluate clinical and prognostic utility of new molecular markers. MiRNAs are small, non-coding RNA molecules that take part in RNA silencing and post-transcriptional regulation of gene expression. Altered expression of miRNAs may be associated with cancer initiation, progression and metastatic capabilities. MiRNA-205 seems to be one of the key regulators of gene expression in endometrial cancer. In this study, we investigated clinical and prognostic role of miRNA-205 in endometrioid endometrial cancer. After total RNA extraction from 100 archival formalin-fixed paraffin-embedded tissues, real-time quantitative RT-PCR was used to define miRNA-205 expression levels. The aim of the study was to evaluate miRNA-205 expression levels in regard to patients' clinical and histopathological features, such as: survival rate, recurrence rate, staging, myometrial invasion, grading and lymph nodes involvement. Higher levels of miRNA-205 expression were observed in tumours with less than half of myometrial invasion and non-advanced cancers. Kaplan-Maier analysis revealed that higher levels of miRNA-205 were associated with better overall survival (p = 0,034). These results indicate potential clinical utility of miRNA-205 as a prognostic marker.

Selection of reference genes is critical for miRNA expression analysis in human cardiac tissue. A focus on atrial fibrillation.

PubMed

Masè, Michela; Grasso, Margherita; Avogaro, Laura; D'Amato, Elvira; Tessarolo, Francesco; Graffigna, Angelo; Denti, Michela Alessandra; Ravelli, Flavia

2017-01-24

MicroRNAs (miRNAs) are emerging as key regulators of complex biological processes in several cardiovascular diseases, including atrial fibrillation (AF). Reverse transcription-quantitative polymerase chain reaction is a powerful technique to quantitatively assess miRNA expression profile, but reliable results depend on proper data normalization by suitable reference genes. Despite the increasing number of studies assessing miRNAs in cardiac disease, no consensus on the best reference genes has been reached. This work aims to assess reference genes stability in human cardiac tissue with a focus on AF investigation. We evaluated the stability of five reference genes (U6, SNORD48, SNORD44, miR-16, and 5S) in atrial tissue samples from eighteen cardiac-surgery patients in sinus rhythm and AF. Stability was quantified by combining BestKeeper, delta-C q , GeNorm, and NormFinder statistical tools. All methods assessed SNORD48 as the best and U6 as the worst reference gene. Applications of different normalization strategies significantly impacted miRNA expression profiles in the study population. Our results point out the necessity of a consensus on data normalization in AF studies to avoid the emergence of divergent biological conclusions.

Characterization and potential role of microRNA in the Chinese dominant malaria mosquito Anopheles sinensis (Diptera: Culicidae) throughout four different life stages.

PubMed

Feng, Xinyu; Wu, Jiatong; Zhou, Shuisen; Wang, Jingwen; Hu, Wei

2018-01-01

microRNAs (miRNAs) are one kind of small non-coding RNAs widely distributed in insects. Many studies have shown that miRNAs play critical roles in development, differentiation, apoptosis, and innate immunity. However, there are a few reports describing miRNAs in Anopheles sinensis , the most common, and one of the dominant malaria mosquito in China. Here, we investigated the global miRNA expression profile across four different developmental stages including embryo, larval, pupal, and adult stages using Illumina Hiseq 2500 sequencing. In total, 164 miRNAs were obtained out of 107.46 million raw sequencing reads. 99 of them identified as known miRNAs, and the remaining 65 miRNAs were considered as novel. By analyzing the read counts of miRNAs in all developmental stages, 95 miRNAs showed stage-specific expression (q < 0.01 and |log2 (fold change)| > 1) in consecutive stages, indicating that these miRNAs may be involved in critical physiological activity during development. Sixteen miRNAs were identified to be commonly dysregulated throughout four developmental stages. Many miRNAs showed stage-specific expression, such as asi-miR-2943 was exclusively expressed in the embryo stage, and asi-miR-1891 could not be detected in larval stage. The expression of six selected differentially expressed miRNAs identified by qRT-PCR were consistent with our sequencing results. Furthermore, 5296 and 1902 target genes were identified for the dysregulated 68 known and 27 novel miRNAs respectively by combining miRanda and RNAhybrid prediction. GO annotation and KEGG pathway analysis for the predicted genes of dysregulated miRNAs revealed that they might be involved in a broad range of biological processes related with the development, such as membrane, organic substance transport and several key pathways including protein processing in endoplasmic reticulum, propanoate metabolism and folate biosynthesis. Thirty-two key miRNAs were identified by microRNA-gene network analysis. The present study represents the first global characterization of An. sinensis miRNAs in its four developmental stages. The presence and differential expression of An. sinensis miRNAs imply that such miRNAs may play critical roles in An. sinensis life cycle. A better understanding of the functions of these miRNAs will have great implication for the effective control of vector population and therefore interrupting malaria transmission.

Characterization of microRNAs in Mud Crab Scylla paramamosain under Vibrio parahaemolyticus Infection

PubMed Central

Li, Chuanbiao; Zhang, Zhao; Zhou, Lizhen; Wang, Shijia; Wang, Shuqi; Zhang, Yueling; Wen, Xiaobo

2013-01-01

Background Infection of bacterial Vibrio parahaemolyticus is common in mud crab farms. However, the mechanisms of the crab’s response to pathogenic V. parahaemolyticus infection are not fully understood. MicroRNAs (miRNAs) are a class of small noncoding RNAs that function as regulators of gene expression and play essential roles in various biological processes. To understand the underlying mechanisms of the molecular immune response of the crab to the pathogens, high-throughput Illumina/Solexa deep sequencing technology was used to investigate the expression profiles of miRNAs in S . paramamosain under V. parahaemolyticus infection. Methodology/Principal Findings Two mixed RNA pools of 7 tissues (intestine, heart, liver, gill, brain, muscle and blood) were obtained from V. parahaemolyticus infected crabs and the control groups, respectively. By aligning the sequencing data with known miRNAs, we characterized 421 miRNA families, and 133 conserved miRNA families in mud crab S . paramamosain were either identical or very similar to existing miRNAs in miRBase. Stem-loop qRT-PCRs were used to scan the expression levels of four randomly chosen differentially expressed miRNAs and tissue distribution. Eight novel potential miRNAs were confirmed by qRT-PCR analysis and the precursors of these novel miRNAs were verified by PCR amplification, cloning and sequencing in S . paramamosain . 161 miRNAs (106 of which up-regulated and 55 down-regulated) were significantly differentially expressed during the challenge and the potential targets of these differentially expressed miRNAs were predicted. Furthermore, we demonstrated evolutionary conservation of mud crab miRNAs in the animal evolution process. Conclusions/Significance In this study, a large number of miRNAs were identified in S . paramamosain when challenged with V. parahaemolyticus, some of which were differentially expressed. The results show that miRNAs might play some important roles in regulating gene expression in mud crab under V. parahaemolyticus infection, providing a basis for further investigation of miRNA-modulating networks in innate immunity of mud crab. PMID:24023678

Expression Variations of miRNAs and mRNAs in Rice (Oryza sativa)

PubMed Central

Wen, Ming; Xie, Munan; He, Lian; Wang, Yushuai; Shi, Suhua; Tang, Tian

2016-01-01

Differences in expression levels are an important source of phenotypic variation within and between populations. MicroRNAs (miRNAs) are key players in post-transcriptional gene regulation that are important for plant development and stress responses. We surveyed expression variation of miRNAs and mRNAs of six accessions from two rice subspecies Oryza sativa L. ssp. indica and Oryza sativa L. ssp. japonica using deep sequencing. While more than half (53.7%) of the mature miRNAs exhibit differential expression between grains and seedlings of rice, only 11.0% show expression differences between subspecies, with an additional 2.2% differentiated for the development-by-subspecies interaction. Expression variation is greater for lowly conserved miRNAs than highly conserved miRNAs, whereas the latter show stronger negative correlation with their targets in expression changes between subspecies. Using a permutation test, we identified 51 miRNA–mRNA pairs that correlate negatively or positively in expression level among cultivated rice. Genes involved in various metabolic processes and stress responses are enriched in the differentially expressed genes between rice indica and japonica subspecies. Our results indicate that stabilizing selection is the major force governing miRNA expression in cultivated rice, albeit positive selection may be responsible for much of the between-subspecies expression divergence. PMID:27797952

Matrigel Basement Membrane Matrix influences expression of microRNAs in cancer cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Price, Karina J.; School of Medicine and Pharmacology, University of Western Australia, Nedlands, WA 6008; Tsykin, Anna

2012-10-19

Highlights: Black-Right-Pointing-Pointer Matrigel alters cancer cell line miRNA expression relative to culture on plastic. Black-Right-Pointing-Pointer Many identified Matrigel-regulated miRNAs are implicated in cancer. Black-Right-Pointing-Pointer miR-1290, -210, -32 and -29b represent a Matrigel-induced miRNA signature. Black-Right-Pointing-Pointer miR-32 down-regulates Integrin alpha 5 (ITGA5) mRNA. -- Abstract: Matrigel is a medium rich in extracellular matrix (ECM) components used for three-dimensional cell culture and is known to alter cellular phenotypes and gene expression. microRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression and have roles in cancer. While miRNA profiles of numerous cell lines cultured on plastic have been reported, the influence ofmore » Matrigel-based culture on cancer cell miRNA expression is largely unknown. This study investigated the influence of Matrigel on the expression of miRNAs that might facilitate ECM-associated cancer cell growth. We performed miRNA profiling by microarray using two colon cancer cell lines (SW480 and SW620), identifying significant differential expression of miRNAs between cells cultured in Matrigel and on plastic. Many of these miRNAs have previously been implicated in cancer-related processes. A common Matrigel-induced miRNA signature comprised of up-regulated miR-1290 and miR-210 and down-regulated miR-29b and miR-32 was identified using RT-qPCR across five epithelial cancer cell lines (SW480, SW620, HT-29, A549 and MDA-MB-231). Experimental modulation of these miRNAs altered expression of their known target mRNAs involved in cell adhesion, proliferation and invasion, in colon cancer cell lines. Furthermore, ITGA5 was identified as a novel putative target of miR-32 that may facilitate cancer cell interactions with the ECM. We propose that culture of cancer cell lines in Matrigel more accurately recapitulates miRNA expression and function in cancer than culture on plastic and thus is a valuable approach to the in vitro study of miRNAs.« less

Characterisation and expression of microRNAs in developing wings of the neotropical butterfly Heliconius melpomene

PubMed Central

2011-01-01

Background Heliconius butterflies are an excellent system for studies of adaptive convergent and divergent phenotypic traits. Wing colour patterns are used as signals to both predators and potential mates and are inherited in a Mendelian manner. The underlying genetic mechanisms of pattern formation have been studied for many years and shed light on broad issues, such as the repeatability of evolution. In Heliconius melpomene, the yellow hindwing bar is controlled by the HmYb locus. MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that have key roles in many biological processes, including development. miRNAs could act as regulators of genes involved in wing development, patterning and pigmentation. For this reason we characterised miRNAs in developing butterfly wings and examined differences in their expression between colour pattern races. Results We sequenced small RNA libraries from two colour pattern races and detected 142 Heliconius miRNAs with homology to others found in miRBase. Several highly abundant miRNAs were differentially represented in the libraries between colour pattern races. These candidates were tested further using Northern blots, showing that differences in expression were primarily due to developmental stage rather than colour pattern. Assembly of sequenced reads to the HmYb region identified hme-miR-193 and hme-miR-2788; located 2380 bp apart in an intergenic region. These two miRNAs are expressed in wings and show an upregulation between 24 and 72 hours post-pupation, indicating a potential role in butterfly wing development. A search for miRNAs in all available H. melpomene BAC sequences (~ 2.5 Mb) did not reveal any other miRNAs and no novel miRNAs were predicted. Conclusions Here we describe the first butterfly miRNAs and characterise their expression in developing wings. Some show differences in expression across developing pupal stages and may have important functions in butterfly wing development. Two miRNAs were located in the HmYb region and were expressed in developing pupal wings. Future work will examine the expression of these miRNAs in different colour pattern races and identify miRNA targets among wing patterning genes. PMID:21266089

Characterisation and expression of microRNAs in developing wings of the neotropical butterfly Heliconius melpomene.

PubMed

Surridge, Alison K; Lopez-Gomollon, Sara; Moxon, Simon; Maroja, Luana S; Rathjen, Tina; Nadeau, Nicola J; Dalmay, Tamas; Jiggins, Chris D

2011-01-26

Heliconius butterflies are an excellent system for studies of adaptive convergent and divergent phenotypic traits. Wing colour patterns are used as signals to both predators and potential mates and are inherited in a Mendelian manner. The underlying genetic mechanisms of pattern formation have been studied for many years and shed light on broad issues, such as the repeatability of evolution. In Heliconius melpomene, the yellow hindwing bar is controlled by the HmYb locus. MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that have key roles in many biological processes, including development. miRNAs could act as regulators of genes involved in wing development, patterning and pigmentation. For this reason we characterised miRNAs in developing butterfly wings and examined differences in their expression between colour pattern races. We sequenced small RNA libraries from two colour pattern races and detected 142 Heliconius miRNAs with homology to others found in miRBase. Several highly abundant miRNAs were differentially represented in the libraries between colour pattern races. These candidates were tested further using Northern blots, showing that differences in expression were primarily due to developmental stage rather than colour pattern. Assembly of sequenced reads to the HmYb region identified hme-miR-193 and hme-miR-2788; located 2380 bp apart in an intergenic region. These two miRNAs are expressed in wings and show an upregulation between 24 and 72 hours post-pupation, indicating a potential role in butterfly wing development. A search for miRNAs in all available H. melpomene BAC sequences (~2.5 Mb) did not reveal any other miRNAs and no novel miRNAs were predicted. Here we describe the first butterfly miRNAs and characterise their expression in developing wings. Some show differences in expression across developing pupal stages and may have important functions in butterfly wing development. Two miRNAs were located in the HmYb region and were expressed in developing pupal wings. Future work will examine the expression of these miRNAs in different colour pattern races and identify miRNA targets among wing patterning genes.

Human salivary microRNAs in Cancer

PubMed Central

Rapado-González, Óscar; Majem, Blanca; Muinelo-Romay, Laura; Álvarez-Castro, Ana; Santamaría, Anna; Gil-Moreno, Antonio; López-López, Rafael; Suárez-Cunqueiro, María Mercedes

2018-01-01

Circulating microRNAs (miRNAs) have emerged as excellent candidates for cancer biomarkers. Several recent studies have highlighted the potential use of saliva for the identification of miRNAs as novel biomarkers, which represents a great opportunity to improve diagnosis and monitor general health and disease. This review summarises the mechanisms of miRNAs deregulation in cancer, the value of targeting them with a therapeutic intention and the evidence of the potential clinical use of miRNAs expressed in saliva for the detection of different cancer types. We also provide a comprehensive review of the different methods for normalising the levels of specific miRNAs present in saliva, as this is a critical step in their analysis, and the challenge to validate salivary miRNAs as a reality to manage cancer patients. PMID:29556321

Human Milk and Matched Serum Demonstrate Concentration of Select miRNAs.

PubMed

Qin, Wenyi; Dasgupta, Santanu; Corradi, John; Sauter, Edward R

Pregnancy-associated breast cancers (PABCs), especially those diagnosed after childbirth, are often aggressive, with a poor prognosis. Factors influencing PABC are largely unknown. Micro(mi)RNAs are present in many human body fluids and shown to influence cancer development and/or growth. In six nursing mothers, we determined if breast cancer-associated miRNAs were (1) detectable in human breast milk and (2) if detectable, their relative expression in milk fractions compared to matched serum. We evaluated by quantitative PCR the expression of 11 cancer-associated miRNAs (10a-5p, 16, 21, 100, 140, 145, 155, 181, 199, 205, 212) in breast milk cells, fat and supernatant (skim milk), and matched serum. miRNA expression was detectable in all samples. For 10/11 miRNAs, mean relative expression compared to control (ΔCt) values was lowest in milk cells, the exception being miR205. Relative concentration was highest in the skim fraction of milk for all miRNAs. Expression was higher in skim milk than matched serum for 7/11 miRNAs and in serum for 4/11 miRNAs. miR205 expression was higher in all milk fractions than in matched serum. In conclusion, the expression of breast cancer-associated miRNAs is detectable in human breast milk and serum samples. The concentration is highest in skim milk, but is also detectable in milk fat and milk cells.

MicroRNA Expression Profiling to Identify and Validate Reference Genes for the Relative Quantification of microRNA in Rectal Cancer.

PubMed

Eriksen, Anne Haahr Mellergaard; Andersen, Rikke Fredslund; Pallisgaard, Niels; Sørensen, Flemming Brandt; Jakobsen, Anders; Hansen, Torben Frøstrup

2016-01-01

MicroRNAs (miRNAs) play important roles in regulating biological processes at the post-transcriptional level. Deregulation of miRNAs has been observed in cancer, and miRNAs are being investigated as potential biomarkers regarding diagnosis, prognosis and prediction in cancer management. Real-time quantitative polymerase chain reaction (RT-qPCR) is commonly used, when measuring miRNA expression. Appropriate normalisation of RT-qPCR data is important to ensure reliable results. The aim of the present study was to identify stably expressed miRNAs applicable as normaliser candidates in future studies of miRNA expression in rectal cancer. We performed high-throughput miRNA profiling (OpenArray®) on ten pairs of laser micro-dissected rectal cancer tissue and adjacent stroma. A global mean expression normalisation strategy was applied to identify the most stably expressed miRNAs for subsequent validation. In the first validation experiment, a panel of miRNAs were analysed on 25 pairs of micro dissected rectal cancer tissue and adjacent stroma. Subsequently, the same miRNAs were analysed in 28 pairs of rectal cancer tissue and normal rectal mucosa. From the miRNA profiling experiment, miR-645, miR-193a-5p, miR-27a and let-7g were identified as stably expressed, both in malignant and stromal tissue. In addition, NormFinder confirmed high expression stability for the four miRNAs. In the RT-qPCR based validation experiments, no significant difference between tumour and stroma/normal rectal mucosa was detected for the mean of the normaliser candidates miR-27a, miR-193a-5p and let-7g (first validation P = 0.801, second validation P = 0.321). MiR-645 was excluded from the data analysis, because it was undetected in 35 of 50 samples (first validation) and in 24 of 56 samples (second validation), respectively. Significant difference in expression level of RNU6B was observed between tumour and adjacent stromal (first validation), and between tumour and normal rectal mucosa (second validation). We recommend the mean expression of miR-27a, miR-193a-5p and let-7g as normalisation factor, when performing miRNA expression analyses by RT-qPCR on rectal cancer tissue.

Transcriptome and Small RNA Deep Sequencing Reveals Deregulation of miRNA Biogenesis in Human Glioma

PubMed Central

Moore, Lynette M.; Kivinen, Virpi; Liu, Yuexin; Annala, Matti; Cogdell, David; Liu, Xiuping; Liu, Chang-Gong; Sawaya, Raymond; Yli-Harja, Olli; Shmulevich, Ilya; Fuller, Gregory N.; Zhang, Wei; Nykter, Matti

2013-01-01

Altered expression of oncogenic and tumor-suppressing microRNAs (miRNAs) is widely associated with tumorigenesis. However, the regulatory mechanisms underlying these alterations are poorly understood. We sought to shed light on the deregulation of miRNA biogenesis promoting the aberrant miRNA expression profiles identified in these tumors. Using sequencing technology to perform both whole-transcriptome and small RNA sequencing of glioma patient samples, we examined precursor and mature miRNAs to directly evaluate the miRNA maturation process, and interrogated expression profiles for genes involved in the major steps of miRNA biogenesis. We found that ratios of mature to precursor forms of a large number of miRNAs increased with the progression from normal brain to low-grade and then to high-grade gliomas. The expression levels of genes involved in each of the three major steps of miRNA biogenesis (nuclear processing, nucleo-cytoplasmic transport, and cytoplasmic processing) were systematically altered in glioma tissues. Survival analysis of an independent data set demonstrated that the alteration of genes involved in miRNA maturation correlates with survival in glioma patients. Direct quantification of miRNA maturation with deep sequencing demonstrated that deregulation of the miRNA biogenesis pathway is a hallmark for glioma genesis and progression. PMID:23007860

Global miRNA expression profile reveals novel molecular players in aneurysmal subarachnoid haemorrhage.

PubMed

Lopes, Katia de Paiva; Vinasco-Sandoval, Tatiana; Vialle, Ricardo Assunção; Paschoal, Fernando Mendes; Bastos, Vanessa Albuquerque P Aviz; Bor-Seng-Shu, Edson; Teixeira, Manoel Jacobsen; Yamada, Elizabeth Sumi; Pinto, Pablo; Vidal, Amanda Ferreira; Ribeiro-Dos-Santos, Arthur; Moreira, Fabiano; Santos, Sidney; Paschoal, Eric Homero Albuquerque; Ribeiro-Dos-Santos, Ândrea

2018-06-08

The molecular mechanisms behind aneurysmal subarachnoid haemorrhage (aSAH) are still poorly understood. Expression patterns of miRNAs may help elucidate the post-transcriptional gene expression in aSAH. Here, we evaluate the global miRNAs expression profile (miRnome) of patients with aSAH to identify potential biomarkers. We collected 33 peripheral blood samples (27 patients with cerebral aneurysm, collected 7 to 10 days after the haemorrhage, when usually is the cerebral vasospasm risk peak, and six controls). Then, were performed small RNA sequencing using an Illumina Next Generation Sequencing (NGS) platform. Differential expression analysis identified eight differentially expressed miRNAs. Among them, three were identified being up-regulated, and five down-regulated. miR-486-5p was the most abundant expressed and is associated with poor neurological admission status. In silico miRNA gene target prediction showed 148 genes associated with at least two differentially expressed miRNAs. Among these, THBS1 and VEGFA, known to be related to thrombospondin and vascular endothelial growth factor. Moreover, MYC gene was found to be regulated by four miRNAs, suggesting an important role in aneurysmal subarachnoid haemorrhage. Additionally, 15 novel miRNAs were predicted being expressed only in aSAH, suggesting possible involvement in aneurysm pathogenesis. These findings may help the identification of novel biomarkers of clinical interest.

Association between the miRNA signatures in plasma and bronchoalveolar fluid in respiratory pathologies.

PubMed

Molina-Pinelo, Sonia; Suárez, Rocío; Pastor, María Dolores; Nogal, Ana; Márquez-Martín, Eduardo; Martín-Juan, José; Carnero, Amancio; Paz-Ares, Luis

2012-01-01

The identification of new less invasive biomarkers is necessary to improve the detection and prognostic outcome of respiratory pathological processes. The measurement of miRNA expression through less invasive techniques such as plasma and serum have been suggested to analysis of several lung malignancies including lung cancer. These studies are assuming a common deregulated miRNA expression both in blood and lung tissue. The present study aimed to obtain miRNA representative signatures both in plasma and bronchoalveolar cell fraction that could serve as biomarker in respiratory diseases. Ten patients were evaluated to assess the expression levels of 381 miRNAs. We found that around 50% miRNAs were no detected in both plasma and bronchoalveolar cell fraction and only 20% of miRNAs showed similar expression in both samples. These results show a lack of association of miRNA signatures between plasma and bronchoalveolar cytology in the same patient. The profiles are not comparable; however, there is a similarity in the relative expression in a very small subset of miRNAs (miR-17, miR-19b, miR-195 and miR-20b) between both biological samples in all patients. This finding supports that the miRNAs profiles obtained from different biological samples have to be carefully validated to link with respiratory diseases.

MicroRNA-15b deteriorates hypoxia/reoxygenation-induced cardiomyocyte apoptosis by downregulating Bcl-2 and MAPK3.

PubMed

Liu, Yaling; Yang, Liqun; Yin, Jiemin; Su, Diansan; Pan, Zhiying; Li, Peiying; Wang, Xiaodong

2018-01-01

To investigate the role of miRNA-15b in cardiomyocyte apoptosis after ischemia reperfusion injury in acute myocardial infarction (AMI), we conducted the AMI rat model by using left anterior descending ligation and performed hypoxia/reoxygenation experiments in H9c2 cells. MiRNA-15b was measured by quantitative reverse transcription PCR (qRT-PCR). Cardiomyocyte apoptosis was determined by terminal deoxynucleotide transferase dUTP nick end labeling staining. Synthesized miRNA-15b mimic and inhibitor were transfected into H9c2 cells by Lipofectamine regent. RNA expression of B cell lymphoma/leukemia-2 (Bcl-2) and mitogen-activated protein kinase 3 (MAPK3) was examined by qRT-PCR and their protein expression was determined by western blot. Ischemia reperfusion increased miRNA-15b expression in the ischemic rat heart and resulted more severe cardiomyocytes apoptosis. In H9c2 cells, hypoxia/reoxygenation induced increased miRNA-15b expression and augmented cardiomyocyte apoptosis observed at 24 hours after 24-hour hypoxia. Compared with the vehicle group, miRNA-15b mimic further raised miRNA-15b level and increased cardiomyocyte apoptosis, whereas miRNA-15b inhibitor suppressed miRNA-15b expression and protected cardiomyocytes from apoptosis. Although the mRNA expression of the target genes Bcl-2 and MAPK3 was not changed significantly, the protein expression of these two genes were markedly reduced after miRNA-15b mimic treatment and significantly increased after transfected with miRNA-15b inhibitors. In conclusion, miRNA-15b deteriorates cardiomyocyte apoptosis by post-transcriptionally downregulating the expression of Bcl-2 and MAPK3. © American Federation for Medical Research (unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Discovery of cashmere goat (Capra hircus) microRNAs in skin and hair follicles by Solexa sequencing.

PubMed

Yuan, Chao; Wang, Xiaolong; Geng, Rongqing; He, Xiaolin; Qu, Lei; Chen, Yulin

2013-07-28

MicroRNAs (miRNAs) are a large family of endogenous, non-coding RNAs, about 22 nucleotides long, which regulate gene expression through sequence-specific base pairing with target mRNAs. Extensive studies have shown that miRNA expression in the skin changes remarkably during distinct stages of the hair cycle in humans, mice, goats and sheep. In this study, the skin tissues were harvested from the three stages of hair follicle cycling (anagen, catagen and telogen) in a fibre-producing goat breed. In total, 63,109,004 raw reads were obtained by Solexa sequencing and 61,125,752 clean reads remained for the small RNA digitalisation analysis. This resulted in the identification of 399 conserved miRNAs; among these, 326 miRNAs were expressed in all three follicular cycling stages, whereas 3, 12 and 11 miRNAs were specifically expressed in anagen, catagen, and telogen, respectively. We also identified 172 potential novel miRNAs by Mireap, 36 miRNAs were expressed in all three cycling stages, whereas 23, 29 and 44 miRNAs were specifically expressed in anagen, catagen, and telogen, respectively. The expression level of five arbitrarily selected miRNAs was analyzed by quantitative PCR, and the results indicated that the expression patterns were consistent with the Solexa sequencing results. Gene Ontology and KEGG pathway analyses indicated that five major biological pathways (Metabolic pathways, Pathways in cancer, MAPK signalling pathway, Endocytosis and Focal adhesion) accounted for 23.08% of target genes among 278 biological functions, indicating that these pathways are likely to play significant roles during hair cycling. During all hair cycle stages of cashmere goats, a large number of conserved and novel miRNAs were identified through a high-throughput sequencing approach. This study enriches the Capra hircus miRNA databases and provides a comprehensive miRNA transcriptome profile in the skin of goats during the hair follicle cycle.

Direct quantification of microRNA at low pM level in sera of glioma patients using a competitive hybridization followed by amplified voltammetric detection

PubMed Central

Wang, Jianxiu; Yi, Xinyao; Tang, Hailin; Han, Hongxing; Wu, Minghua; Zhou, Feimeng

2012-01-01

MicroRNAs (miRNAs), acting as oncogenes or tumor suppressors in humans, play a key role in regulating gene expression and are believed to be important for developing novel therapeutic treatments and clinical prognoses. Due to their short lengths (17–25 nucleotides) and extremely low concentrations (typically < pM) in biological samples, quantification of miRNAs has been challenging to conventional biochemical methods, such as Northern blotting, microarray, and quantitative polymerase chain reaction (qPCR). In this work, a biotinylated miRNA (biotin-miRNA) whose sequence is the same as that of a miRNA target is introduced into samples of interest and allowed to compete with the miRNA target for the oligonucleotide (ODN) probe preimmobilized onto an electrode. Voltammetric quantification of the miRNA target was accomplished after complexation of the biotin-miRNA with ferrocene (Fc)-capped gold nanoparticle/streptavidin conjugates. The Fc oxidation current was found to be inversely proportional to the concentration of target miRNA between 10 fM and 2.0 pM. The method is highly reproducible (RSD < 5%), regenerable (at least 8 regeneration/assay cycles without discernible signal decrease) and selective (with sequence specificity down to a single nucleotide mismatch). The low detection levels (10 fM or 0.1 attomoles of miRNA in a 10-HL solution) allow the direct quantification of miRNA-182, a marker correlated to the progression of glioma in patients, to be performed in serum samples without sample pretreatment and RNA extraction and enrichment. The concentration of miRNA-182 in glioma patients was found to be 3.1 times as high as that in healthy persons, a conclusion in excellent agreement with a separate qPCR measurement of the expression level. The obviations of the requirement of an internal reference in qPCR, simplicity, and cost-effectiveness are other additional advantages of this method for detection of nucleic acids in clinical samples. PMID:22788545

Expression profiles of miRNAs from bovine mammary glands in response to Streptococcus agalactiae-induced mastitis.

PubMed

Pu, Junhua; Li, Rui; Zhang, Chenglong; Chen, Dan; Liao, Xiangxiang; Zhu, Yihui; Geng, Xiaohan; Ji, Dejun; Mao, Yongjiang; Gong, Yunchen; Yang, Zhangping

2017-08-01

This study aimed to describe the expression profiles of microRNAs (miRNAs) from mammary gland tissues collected from dairy cows with Streptococcus agalactiae-induced mastitis and to identify differentially expressed miRNAs related to mastitis. The mammary glands of Chinese Holstein cows were challenged with Streptococcus agalactiae to induce mastitis. Small RNAs were isolated from the mammary tissues of the test and control groups and then sequenced using the Solexa sequencing technology to construct two small RNA libraries. Potential target genes of these differentially expressed miRNAs were predicted using the RNAhybrid software, and KEGG pathways associated with these genes were analysed. A total of 18 555 913 and 20 847 000 effective reads were obtained from the test and control groups, respectively. In total, 373 known and 399 novel miRNAs were detected in the test group, and 358 known and 232 novel miRNAs were uncovered in the control group. A total of 35 differentially expressed miRNAs were identified in the test group compared to the control group, including 10 up-regulated miRNAs and 25 down-regulated miRNAs. Of these miRNAs, miR-223 exhibited the highest degree of up-regulation with an approximately 3-fold increase in expression, whereas miR-26a exhibited the most decreased expression level (more than 2-fold). The RNAhybrid software predicted 18 801 genes as potential targets of these 35 miRNAs. Furthermore, several immune response and signal transduction pathways, including the RIG-I-like receptor signalling pathway, cytosolic DNA sensing pathway and Notch signal pathway, were enriched in these predicted targets. In summary, this study provided experimental evidence for the mechanism underlying the regulation of bovine mastitis by miRNAs and showed that miRNAs might be involved in signal pathways during S. agalactiae-induced mastitis.

Analysis of plasma microRNA expression profiles revealed different cancer susceptibility in healthy young adult smokers and middle-aged smokers

PubMed Central

Shi, Bing; Gao, Hongmin; Zhang, Tianyang; Cui, Qinghua

2016-01-01

Cigarette smoking is a world-wide habit and an important risk factor for cancer. It was known that cigarette smoking can change the expression of circulating microRNAs (miRNAs) in healthy middle-aged adults. However, it remains unclear whether cigarette smoking can change the levels of circulating miRNAs in young healthy smokers and whether there are differences in cancer susceptibility for the two cases. In this study, the miRNA expression profiles of 28 smokers and 12 non-smokers were determined by Agilent human MicroRNA array. We further performed bioinformatics analysis for the differentially expressed miRNAs. The result showed that 35 miRNAs were differentially expressed. Among them, 24 miRNAs were up-regulated and 11 miRNAs were down-regulated in smokers. Functional enrichment analysis showed that the deregulated miRNAs are related to immune system and hormones regulation. Strikingly, the up-regulated miRNAs are mostly associated with hematologic cancers, such as lymphoma, leukemia. As a comparison, the up-regulated plasma miRNAs in middle-aged smokers are mostly associated with solid cancers, such as hepatocellular carcinoma and lung cancer, suggesting that smoking could have different influences on young adults and middle-aged adults. In a conclusion, we identified the circulating miRNAs deregulated by cigarette smoking and revealed that the age-dependent deregulated miRNAs tend to be mainly involved in different types of human cancers. PMID:26943588

Analysis of plasma microRNA expression profiles revealed different cancer susceptibility in healthy young adult smokers and middle-aged smokers.

PubMed

Shi, Bing; Gao, Hongmin; Zhang, Tianyang; Cui, Qinghua

2016-04-19

Cigarette smoking is a world-wide habit and an important risk factor for cancer. It was known that cigarette smoking can change the expression of circulating microRNAs (miRNAs) in healthy middle-aged adults. However, it remains unclear whether cigarette smoking can change the levels of circulating miRNAs in young healthy smokers and whether there are differences in cancer susceptibility for the two cases. In this study, the miRNA expression profiles of 28 smokers and 12 non-smokers were determined by Agilent human MicroRNA array. We further performed bioinformatics analysis for the differentially expressed miRNAs. The result showed that 35 miRNAs were differentially expressed. Among them, 24 miRNAs were up-regulated and 11 miRNAs were down-regulated in smokers. Functional enrichment analysis showed that the deregulated miRNAs are related to immune system and hormones regulation. Strikingly, the up-regulated miRNAs are mostly associated with hematologic cancers, such as lymphoma, leukemia. As a comparison, the up-regulated plasma miRNAs in middle-aged smokers are mostly associated with solid cancers, such as hepatocellular carcinoma and lung cancer, suggesting that smoking could have different influences on young adults and middle-aged adults. In a conclusion, we identified the circulating miRNAs deregulated by cigarette smoking and revealed that the age-dependent deregulated miRNAs tend to be mainly involved in different types of human cancers.

The role of microRNAs in myopia.

PubMed

Jiang, Bo; Huo, Yanan; Gu, Yangshun; Wang, Jianyong

2017-01-01

In recent years, research on microRNAs (miRNAs) has become popular because of the critical role these macromolecules play in post-transcriptional gene regulation. Recent efforts have been made to identify miRNAs and their possible roles in myopia. The aim of this review was to summarize the expression and function of miRNAs during the development of myopia. In this article, we reviewed the current research on the mechanisms that regulate miRNA expression, the potential for miRNAs as a diagnostic biomarker for myopia, and the mechanisms by which miRNAs promote the development of myopia. We also discussed the miRNA expression profiles in human fetal sclera. We summarized the miRNA expression profiles in myopia, including miR-328, miR-184, miR-29a, and miR-let-7i, and also the miRNA expression profiles in fetal sclera, including miR-214, miR-let-7, miR-103, miR-107, miR-29b, miR-328, and miR-98. Such knowledge could lead to more precise diagnosis, prognosis, and response predictions for future treatments for myopia, and the pace of discovery is expected to accelerate dramatically in the near future.

Microarray analysis of miRNA expression profiles following whole body irradiation in a mouse model.

PubMed

Aryankalayil, Molykutty J; Chopra, Sunita; Makinde, Adeola; Eke, Iris; Levin, Joel; Shankavaram, Uma; MacMillan, Laurel; Vanpouille-Box, Claire; Demaria, Sandra; Coleman, C Norman

2018-06-19

Accidental exposure to life-threatening radiation in a nuclear event is a major concern; there is an enormous need for identifying biomarkers for radiation biodosimetry to triage populations and treat critically exposed individuals. To identify dose-differentiating miRNA signatures from whole blood samples of whole body irradiated mice. Mice were whole body irradiated with X-rays (2 Gy-15 Gy); blood was collected at various time-points post-exposure; total RNA was isolated; miRNA microarrays were performed; miRNAs differentially expressed in irradiated vs. unirradiated controls were identified; feature extraction and classification models were applied to predict dose-differentiating miRNA signature. We observed a time and dose responsive alteration in the expression levels of miRNAs. Maximum number of miRNAs were altered at 24-h and 48-h time-points post-irradiation. A 23-miRNA signature was identified using feature selection algorithms and classifier models. An inverse correlation in the expression level changes of miR-17 members, and their targets were observed in whole body irradiated mice and non-human primates. Whole blood-based miRNA expression signatures might be used for predicting radiation exposures in a mass casualty nuclear incident.

Characterization and Expression Patterns of microRNAs Involved in Rice Grain Filling

PubMed Central

Du, Yanxiu; Zhang, Jing; Li, Junzhou; Liu, Yanxia; Zhao, Yafan; Zhao, Quanzhi

2013-01-01

MicroRNAs (miRNAs) are upstream gene regulators of plant development and hormone homeostasis through their directed cleavage or translational repression of the target mRNAs, which may play crucial roles in rice grain filling and determining the final grain weight and yield. In this study, high-throughput sequencing was performed to survey the dynamic expressions of miRNAs and their corresponding target genes at five distinct developmental stages of grain filling. In total, 445 known miRNAs and 45 novel miRNAs were detected with most of them expressed in a developmental stage dependent manner, and the majority of known miRNAs, which increased gradually with rice grain filling, showed negatively related to the grain filling rate. Detailed expressional comparisons revealed a clear negative correlation between most miRNAs and their target genes. It was found that specific miRNA cohorts are expressed in a developmental stage dependent manner during grain filling and the known functions of these miRNAs are involved in plant hormone homeostasis and starch accumulation, indicating that the expression dynamics of these miRNAs might play key roles in regulating rice grain filling. PMID:23365650

miRNA Expression Change in Dorsal Root Ganglia After Peripheral Nerve Injury.

PubMed

Chang, Hsueh-Ling; Wang, Hung-Chen; Chunag, Yi-Ta; Chou, Chao-Wen; Lin, I-Ling; Lai, Chung-Sheng; Chang, Lin-Li; Cheng, Kuang-I

2017-02-01

The role of microRNAs (miRNAs) in the regulation of nerve injury-induced neuropathic pain is unclear. The aims of this study were to assess and compare miRNA expression profiles in dorsal root ganglia (DRG) following three different kinds of peripheral nerve injury, including spinal nerve ligation (SNL), dorsal root transection (DRT), and ventral root transection (VRT), in Sprague-Dawley rats. Responses to thermal and mechanical stimuli were measured preoperatively and on postoperative days (PODs) 1, 4, and 7. A miRNA microarray analysis was used to detect the miRNA expression profiles in injured L5 DRG from SNL, DRT, and VRT on POD 7. Validation of miRNA expression was performed by qPCR and in situ hybridization. Rats receiving SNL displayed significantly higher mechanical hypersensitivity, but those receiving DRT developed higher thermal hypersensitivity. The number of miRNAs that were significantly upregulated in L5 DRG was 49 (7.2%), 25 (3.7%), and 146 (21.5%) following SNL, DRT, and VRT, respectively. On the other hand, 35 (5.1%) miRNAs were significantly downregulated in the SNL group, 21 (3.1%) miRNAs in the DRT group, and 41 (6.0%) miRNAs in the VRT group. Of the four miRNAs that were mutually aberrant in all three models, two were significantly upregulated (twofold), miR-21 and miR-31, and two were significantly downregulated, miR-668 and miR-672. Using in situ hybridization, miRNA-21, miRNA-31, miRNA-668, and miRNA-672 were found to localize to neurons in the DRG. Collectively, the mutual abnormal miRNA expression of miR-21, miR-31, miR-668, and miR-677 implied that these miRNAs may be therapeutic targets for alleviating multiple forms of neuropathic pain.

MicroRNA-320 family is downregulated in colorectal adenoma and affects tumor proliferation by targeting CDK6

PubMed Central

Tadano, Toshihiro; Kakuta, Yoichi; Hamada, Shin; Shimodaira, Yosuke; Kuroha, Masatake; Kawakami, Yoko; Kimura, Tomoya; Shiga, Hisashi; Endo, Katsuya; Masamune, Atsushi; Takahashi, Seiichi; Kinouchi, Yoshitaka; Shimosegawa, Tooru

2016-01-01

AIM: To investigate the microRNA (miRNA) expression during histological progression from colorectal normal mucosa through adenoma to carcinoma within a lesion. METHODS: Using microarray, the sequential changes in miRNA expression profiles were compared in colonic lesions from matched samples; histologically, non-neoplastic mucosa, adenoma, and submucosal invasive carcinoma were microdissected from a tissue sample. Cell proliferation assay was performed to observe the effect of miRNA, and its target genes were predicted using bioinformatics approaches and the expression profile of SW480 transfected with the miRNA mimics. mRNA and protein levels of the target gene in colon cancer cell lines with a mimic control or miRNA mimics were measured using qRT-PCR and Western blotting. The expression levels of miRNA and target gene in colorectal tissue samples were also measured. RESULTS: Microarray analysis identified that the miR-320 family, including miR-320a, miR-320b, miR-320c, miR-320d and miR-320e, were differentially expressed in adenoma and submucosal invasive carcinoma. The miR-320 family, which inhibits cell proliferation, is frequently downregulated in colorectal adenoma and submucosal invasive carcinoma tissues. Seven genes including CDK6 were identified to be common in the results of gene expression array and bioinformatics analyses performed to find the target gene of the miR-320 family. We confirmed that mRNA and protein levels of CDK6 were significantly suppressed in colon cancer cell lines with miR-320 family mimics. CDK6 expression was found to increase from non-neoplastic mucosa through adenoma to submucosal invasive carcinoma tissues and showed an inverse correlation with miR-320 family expression. CONCLUSION: MiR-320 family affects colorectal tumor proliferation by targeting CDK6, plays important role in its growth, and is considered to be a biomarker for its early detection. PMID:27559432

Identification and characterization of microRNAs in white and brown alpaca skin

PubMed Central

2012-01-01

Background MicroRNAs (miRNAs) are small, non-coding 21–25 nt RNA molecules that play an important role in regulating gene expression. Little is known about the expression profiles and functions of miRNAs in skin and their role in pigmentation. Alpacas have more than 22 natural coat colors, more than any other fiber producing species. To better understand the role of miRNAs in control of coat color we performed a comprehensive analysis of miRNA expression profiles in skin of white versus brown alpacas. Results Two small RNA libraries from white alpaca (WA) and brown alpaca (BA) skin were sequenced with the aid of Illumina sequencing technology. 272 and 267 conserved miRNAs were obtained from the WA and BA skin libraries, respectively. Of these conserved miRNAs, 35 and 13 were more abundant in WA and BA skin, respectively. The targets of these miRNAs were predicted and grouped based on Gene Ontology and KEGG pathway analysis. Many predicted target genes for these miRNAs are involved in the melanogenesis pathway controlling pigmentation. In addition to the conserved miRNAs, we also obtained 22 potentially novel miRNAs from the WA and BA skin libraries. Conclusion This study represents the first comprehensive survey of miRNAs expressed in skin of animals of different coat colors by deep sequencing analysis. We discovered a collection of miRNAs that are differentially expressed in WA and BA skin. The results suggest important potential functions of miRNAs in coat color regulation. PMID:23067000

Human embryos secrete microRNAs into culture media--a potential biomarker for implantation.

PubMed

Rosenbluth, Evan M; Shelton, Dawne N; Wells, Lindsay M; Sparks, Amy E T; Van Voorhis, Bradley J

2014-05-01

To determine whether human blastocysts secrete microRNA (miRNAs) into culture media and whether these reflect embryonic ploidy status and can predict in vitro fertilization (IVF) outcomes. Experimental study of human embryos and IVF culture media. Academic IVF program. 91 donated, cryopreserved embryos that developed into 28 tested blastocysts, from 13 couples who had previously completed IVF cycles. None. Relative miRNA expression in IVF culture media. Blastocysts were assessed by chromosomal comparative genomic hybridization analysis, and the culture media from 55 single-embryo transfer cycles was tested for miRNA expression using an array-based quantitative real-time polymerase chain reaction analysis. The expression of the identified miRNA was correlated with pregnancy outcomes. Ten miRNA were identified in the culture media; two were specific to spent media (miR-191 and miR-372), and one was only present in media before the embryos had been cultured (miR-645). MicroRNA-191 was more highly concentrated in media from aneuploid embryos, and miR-191, miR-372, and miR-645 were more highly concentrated in media from failed IVF/non-intracytoplasmic sperm injection cycles. Additionally, miRNA were found to be more highly concentrated in ICSI and day-5 media samples when compared with regularly inseminated and day-4 samples, respectively. MicroRNA can be detected in IVF culture media. Some of these miRNA are differentially expressed according to the fertilization method, chromosomal status, and pregnancy outcome, which makes them potential biomarkers for predicting IVF success. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

The Role of ADAR1 and ADAR2 in the Regulation of miRNA-21 in Idiopathic Pulmonary Fibrosis.

PubMed

Díaz-Piña, Gabriela; Ordoñez-Razo, Rosa Ma; Montes, Eduardo; Páramo, Ignacio; Becerril, Carina; Salgado, Alfonso; Santibañez-Salgado, J Alfredo; Maldonado, Mariel; Ruiz, Victor

2018-04-10

microRNAs (miRNAs) are small non-coding 1RNAs that post-transcriptionally regulate gene expression. Recent evidence shows that adenosine deaminases that act on RNA (ADAR) can edit miRNAs. miRNAs are involved in the development of different diseases, such as idiopathic pulmonary fibrosis (IPF). In IPF, about 40% of the miRNAs are differentially expressed with respect to controls. Among these miRNAs, miRNA-21 has been found over-expressed in IPF and its targets are anti-fibrosing molecules such as PELI1 and SPRY2. The objective of this study is to determine the role of ADAR1 and 2 on the expression of miRNA-21 in human lung fibroblasts trough quantification of gene expression, protein levels, and overexpression of ADAR1 and 2. Six control and six fibrotic primary fibroblast cell cultures were used for RNA extraction, ADAR1, ADAR2, PELI1, SPRY2, miRNA-21, and pri-miRNA-21 expression was measured. Subsequently, two fibrotic fibroblast cultures were used for overexpression of ADAR1 and ADAR2, and they were stimulated with TGFβ1. Real-time PCR and Western blot were performed. ADAR1 is significantly downregulated in IPF fibroblasts; the overexpression of ADAR1 and ADAR2 reestablishes the expression levels of miRNA-21, PELI1, and SPRY2 in fibroblasts of patients with IPF. These changes in the processing of miRNAs have great value in pathology diagnosis, including lung diseases, and play an important role in the understanding of molecular mechanisms involved in the development of different pathologies, as well as representing new therapeutic targets.

Modulation of microRNA expression in human lung cancer cells by the G9a histone methyltransferase inhibitor BIX01294

PubMed Central

PANG, ALAN LAP-YIN; TITLE, ALEXANDRA C.; RENNERT, OWEN M.

2014-01-01

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of their target genes at the post-transcriptional level. In cancer cells, miRNAs, depending on the biological functions of their target genes, may have a tumor-promoting or -suppressing effect. Treatment of cancer cells with inhibitors of DNA methylation and/or histone deacetylation modulates the expression level of miRNAs, which provides evidence for epigenetic regulation of miRNA expression. The consequences of inhibition of histone methyltransferase on miRNA expression, however, have not been thoroughly investigated. The present study examined the expression pattern of miRNAs in the non-small cell lung cancer cell line, H1299 with or without treatment of BIX01294, a potent chemical inhibitor of G9a methyltransferase that catalyzes the mono-and di-methylation of the lysine 9 residue of histone H3. By coupling microarray analysis with quantitative real-time polymerase chain reaction analysis, two miRNAs were identified that showed consistent downregulation following BIX01294 treatment. The results indicate that histone H3 methylation regulates miRNA expression in lung cancer cells, which may provide additional insight for future chemical treatment of lung cancer. PMID:24932239

miRNA-148a serves as a prognostic factor and suppresses migration and invasion through Wnt1 in non-small cell lung cancer.

PubMed

Chen, Yong; Min, Lingfeng; Ren, Chuanli; Xu, Xingxiang; Yang, Jianqi; Sun, Xinchen; Wang, Tao; Wang, Fang; Sun, Changjiang; Zhang, Xizhi

2017-01-01

Lung cancer is the leading cause of cancer death in the world, and aberrant expression of miRNA is a common feature during the cancer initiation and development. Our previous study showed that levels of miRNA-148a assessed by quantitative real-time polymerase chain reaction (qRT-PCR) were a good prognosis factor for non-small cell lung cancer (NSCLC) patients. In this study, we used high-throughput formalin-fixed and paraffin-embedded (FFPE) lung cancer tissue arrays and in situ hybridization (ISH) to determine the clinical significances of miRNA-148a and aimed to find novel target of miRNA-148a in lung cancer. Our results showed that there were 86 of 159 patients with low miRNA-148a expression and miRNA-148a was significantly down-regulated in primary cancer tissues when compared with their adjacent normal lung tissues. Low expression of miRNA-148a was strongly associated with high tumor grade, lymph node (LN) metastasis and a higher risk of tumor-related death in NSCLC. Lentivirus mediated overexpression of miRNA-148a inhibited migration and invasion of A549 and H1299 lung cancer cells. Furthermore, we validated Wnt1 as a direct target of miRNA-148a. Our data showed that the Wnt1 expression was negatively correlated with the expression of miRNA-148a in both primary cancer tissues and their corresponding adjacent normal lung tissues. In addition, overexpression of miRNA-148a inhibited Wnt1 protein expression in cancer cells. And knocking down of Wnt-1 by siRNA had the similar effect of miRNA-148a overexpression on cell migration and invasion in lung cancer cells. In conclusion, our results suggest that miRNA-148a inhibited cell migration and invasion through targeting Wnt1 and this might provide a new insight into the molecular mechanisms of lung cancer metastasis.

Temporal expression profiles indicate a primary function for microRNA during the peak of DNA replication after rat partial hepatectomy.

PubMed

Raschzok, Nathanael; Werner, Wiebke; Sallmon, Hannes; Billecke, Nils; Dame, Christof; Neuhaus, Peter; Sauer, Igor M

2011-06-01

The liver has the unique capacity to regenerate after surgical resection. However, the regulation of liver regeneration is not completely understood. Recent reports indicate an essential role for small noncoding microRNAs (miRNAs) in the regulation of hepatic development, carcinogenesis, and early regeneration. We hypothesized that miRNAs are critically involved in all phases of liver regeneration after partial hepatectomy. We performed miRNA microarray analyses after 70% partial hepatectomy in rats under isoflurane anesthesia at different time points (0 h to 5 days) and after sham laparotomy. Putative targets of differentially expressed miRNAs were determined using a bioinformatic approach. Two-dimensional (2D)-PAGE proteomic analyses and protein identification were performed on specimens at 0 and 24 h after resection. The temporal dynamics of liver regeneration were characterized by 5-bromo- 2-deoxyuridine, proliferating cell nuclear antigen, IL-6, and hepatocyte growth factor. We demonstrate that miRNA expression patterns changed during liver regeneration and that these changes were most evident during the peak of DNA replication at 24 h after resection. Expression of 13 miRNAs was significantly reduced 12-48 h after resection (>25% change), out of which downreguation was confirmed in isolated hepatocytes for 6 miRNAs at 24 h, whereas three miRNAs were significantly upregulated. Proteomic analysis revealed 65 upregulated proteins; among them, 23 represent putative targets of the differentially expressed miRNAs. We provide a temporal miRNA expression and proteomic dataset of the regenerating rat liver, which indicates a primary function for miRNA during the peak of DNA replication. These data will assist further functional studies on the role of miRNAs during liver regeneration.

Environmental contaminants and microRNA regulation: Transcription factors as regulators of toxicant-altered microRNA expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sollome, James; Martin, Elizabeth

MicroRNAs (miRNAs) regulate gene expression by binding mRNA and inhibiting translation and/or inducing degradation of the associated transcripts. Expression levels of miRNAs have been shown to be altered in response to environmental toxicants, thus impacting cellular function and influencing disease risk. Transcription factors (TFs) are known to be altered in response to environmental toxicants and play a critical role in the regulation of miRNA expression. To date, environmentally-responsive TFs that are important for regulating miRNAs remain understudied. In a state-of-the-art analysis, we utilized an in silico bioinformatic approach to characterize potential transcriptional regulators of environmentally-responsive miRNAs. Using the miRStart database,more » genomic sequences of promoter regions for all available human miRNAs (n = 847) were identified and promoter regions were defined as − 1000/+500 base pairs from the transcription start site. Subsequently, the promoter region sequences of environmentally-responsive miRNAs (n = 128) were analyzed using enrichment analysis to determine overrepresented TF binding sites (TFBS). While most (56/73) TFs differed across environmental contaminants, a set of 17 TFs was enriched for promoter binding among miRNAs responsive to numerous environmental contaminants. Of these, one TF was common to miRNAs altered by the majority of environmental contaminants, namely SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 3 (SMARCA3). These identified TFs represent candidate common transcriptional regulators of miRNAs perturbed by environmental toxicants. - Highlights: • Transcription factors that regulate environmentally-modulated miRNA expression are understudied • Transcription factor binding sites (TFBS) located within DNA promoter regions of miRNAs were identified. • Specific transcription factors may serve as master regulators of environmentally-mediated microRNA expression.« less

Expression profiling of microRNAs in human bone tissue from postmenopausal women.

PubMed

De-Ugarte, Laura; Serra-Vinardell, Jenny; Nonell, Lara; Balcells, Susana; Arnal, Magdalena; Nogues, Xavier; Mellibovsky, Leonardo; Grinberg, Daniel; Diez-Perez, Adolfo; Garcia-Giralt, Natalia

2018-01-01

Bone tissue is composed of several cell types, which express their own microRNAs (miRNAs) that will play a role in cell function. The set of total miRNAs expressed in all cell types configures the specific signature of the bone tissue in one physiological condition. The aim of this study was to explore the miRNA expression profile of bone tissue from postmenopausal women. Tissue was obtained from trabecular bone and was analyzed in fresh conditions (n = 6). Primary osteoblasts were also obtained from trabecular bone (n = 4) and human osteoclasts were obtained from monocyte precursors after in vitro differentiation (n = 5). MicroRNA expression profiling was obtained for each sample by microarray and a global miRNA analysis was performed combining the data acquired in all the microarray experiments. From the 641 miRNAs detected in bone tissue samples, 346 (54%) were present in osteoblasts and/or osteoclasts. The other 46% were not identified in any of the bone cells analyzed. Intersection of osteoblast and osteoclast arrays identified 101 miRNAs shared by both cell types, which accounts for 30-40% of miRNAs detected in these cells. In osteoblasts, 266 miRNAs were detected, of which 243 (91%) were also present in the total bone array, representing 38% of all bone miRNAs. In osteoclasts, 340 miRNAs were detected, of which 196 (58%) were also present in the bone tissue array, representing 31% of all miRNAs detected in total bone. These analyses provide an overview of miRNAs expressed in bone tissue, broadening our knowledge in the microRNA field.

Regulatory networks between neurotrophins and miRNAs in brain diseases and cancers

PubMed Central

Shi, Jian

2015-01-01

Neurotrophins are involved in many physiological and pathological processes in the nervous system. They regulate and modify signal transduction, transcription and translation in neurons. It is recently demonstrated that the neurotrophin expression is regulated by microRNAs (miRNAs), changing our views on neurotrophins and miRNAs. Generally, miRNAs regulate neurotrophins and their receptors in at least two ways: (1) miRNAs bind directly to the 3′ untranslated region (UTR) of isoform-specific mRNAs and post-transcriptionally regulate their expression; (2) miRNAs bind to the 3′ UTR of the regulatory factors of neurotrophins and regulate their expression. On the other hand, neurotrophins can regulate miRNAs. The results of BNDF research show that neurotrophins regulate miRNAs in at least three ways: (1) ERK stimulation enhances the activation of TRBP (HIV-1 TAR RNA-binding protein) and Dicer, leading to the upregulation of miRNA biogenesis; (2) ERK-dependent upregulation of Lin28a (RNA-binding proteins) blocks select miRNA biogenesis; (3) transcriptional regulation of miRNA expression through activation of transcription factors, including CREB and NF-κB. These regulatory processes integrate positive and negative regulatory loops in neurotrophin and miRNA signaling pathways, and also expand the function of neurotrophins and miRNAs. In this review, we summarize the current knowledge of the regulatory networks between neurotrophins and miRNAs in brain diseases and cancers, for which novel cutting edge therapeutic, delivery and diagnostic approaches are emerging. PMID:25544363

Integrative Analysis of miRNA and mRNA Profiles in Response to Ethylene in Rose Petals during Flower Opening

PubMed Central

Pei, Haixia; Ma, Nan; Chen, Jiwei; Zheng, Yi; Tian, Ji; Li, Jing; Zhang, Shuai; Fei, Zhangjun; Gao, Junping

2013-01-01

MicroRNAs play an important role in plant development and plant responses to various biotic and abiotic stimuli. As one of the most important ornamental crops, rose (Rosa hybrida) possesses several specific morphological and physiological features, including recurrent flowering, highly divergent flower shapes, colors and volatiles. Ethylene plays an important role in regulating petal cell expansion during rose flower opening. Here, we report the population and expression profiles of miRNAs in rose petals during flower opening and in response to ethylene based on high throughput sequencing. We identified a total of 33 conserved miRNAs, as well as 47 putative novel miRNAs were identified from rose petals. The conserved and novel targets to those miRNAs were predicted using the rose floral transcriptome database. Expression profiling revealed that expression of 28 known (84.8% of known miRNAs) and 39 novel (83.0% of novel miRNAs) miRNAs was substantially changed in rose petals during the earlier opening period. We also found that 28 known and 22 novel miRNAs showed expression changes in response to ethylene treatment. Furthermore, we performed integrative analysis of expression profiles of miRNAs and their targets. We found that ethylene-caused expression changes of five miRNAs (miR156, miR164, miR166, miR5139 and rhy-miRC1) were inversely correlated to those of their seven target genes. These results indicate that these miRNA/target modules might be regulated by ethylene and were involved in ethylene-regulated petal growth. PMID:23696879

Integrated miRNA-mRNA analysis reveals regulatory pathways underlying the curly fleece trait in Chinese tan sheep.

PubMed

Liu, Yufang; Zhang, Jibin; Xu, Qiao; Kang, Xiaolong; Wang, Kejun; Wu, Keliang; Fang, Meiying

2018-05-11

Tan sheep is an indigenous Chinese breed well known for its beautiful curly fleece. One prominent breed characteristic of this sheep breed is that the degree of curliness differs markedly between lambs and adults, but the molecular mechanisms regulating the shift are still not well understood. In this study, we identified 49 differentially expressed (DE) microRNAs (miRNAs) between Tan sheep at the two stages through miRNA-seq, and combined the data with that in our earlier Suppression Subtractive Hybridization cDNA (SSH) library study to elucidate the mechanisms underlying curly fleece formation. Thirty-six potential miRNA-mRNA target pairs were identified using computational methods, including 25 DE miRNAs and 10 DE genes involved in the MAPK signaling pathway, steroid biosynthesis and metabolic pathways. With the differential expressions between lambs and adults confirmed by qRT-PCR, some miRNAs were already annotated in the genome, but some were novel miRNAs. Inhibition of KRT83 expression by miR-432 was confirmed by both gene knockdown with siRNA and overexpression, which was consistent with the miRNAs and targets prediction results. Our study represents the comprehensive analysis of mRNA and miRNA in Tan sheep and offers detailed insight into the development of curly fleece as well as the potential mechanisms controlling curly hair formation in humans.

The Expression Pattern of microRNAs in Granulosa Cells of Subordinate and Dominant Follicles during the Early Luteal Phase of the Bovine Estrous Cycle

PubMed Central

Gebremedhn, Samuel; Sahadevan, Sudeep; Hossain, MD Munir; Rings, Franca; Hoelker, Michael; Tholen, Ernst; Neuhoff, Christiane; Looft, Christian; Schellander, Karl; Tesfaye, Dawit

2014-01-01

This study aimed to investigate the miRNA expression patterns in granulosa cells of subordinate (SF) and dominant follicle (DF) during the early luteal phase of the bovine estrous cycle. For this, miRNA enriched total RNA isolated from granulosa cells of SF and DF obtained from heifers slaughtered at day 3 and day 7 of the estrous cycle was used for miRNAs deep sequencing. The results revealed that including 17 candidate novel miRNAs, several known miRNAs (n = 291–318) were detected in SF and DF at days 3 and 7 of the estrous cycle of which 244 miRNAs were common to all follicle groups. The let-7 families, bta-miR-10b, bta-miR-26a, bta-miR-99b and bta-miR-27b were among abundantly expressed miRNAs in both SF and DF at both days of the estrous cycle. Further analysis revealed that the expression patterns of 16 miRNAs including bta-miR-449a, bta-miR-449c and bta-miR-222 were differentially expressed between the granulosa cells of SF and DF at day 3 of the estrous cycle. However, at day 7 of the estrous cycle, 108 miRNAs including bta-miR-409a, bta-miR-383 and bta-miR-184 were differentially expressed between the two groups of granulosa cell revealing the presence of distinct miRNA expression profile changes between the two follicular stages at day 7 than day 3 of the estrous cycle. In addition, unlike the SF, marked temporal miRNA expression dynamics was observed in DF groups between day 3 and 7 of the estrous cycle. Target gene prediction and pathway analysis revealed that major signaling associated with follicular development including Wnt signaling, TGF-beta signaling, oocyte meiosis and GnRH signaling were affected by differentially expressed miRNAs. Thus, this study highlights the miRNA expression patterns of granulosa cells in subordinate and dominant follicles that could be associated with follicular recruitment, selection and dominance during the early luteal phase of the bovine estrous cycle. PMID:25192015

Expression profiling and structural characterization of microRNAs in adipose tissues of hibernating ground squirrels.

PubMed

Wu, Cheng-Wei; Biggar, Kyle K; Storey, Kenneth B

2014-12-01

MicroRNAs (miRNAs) are small non-coding RNAs that are important in regulating metabolic stress. In this study, we determined the expression and structural characteristics of 20 miRNAs in brown (BAT) and white adipose tissue (WAT) during torpor in thirteen-lined ground squirrels. Using a modified stem-loop technique, we found that during torpor, expression of six miRNAs including let-7a, let-7b, miR-107, miR-150, miR-222 and miR-31 was significantly downregulated in WAT (P<0.05), which was 16%-54% of euthermic non-torpid control squirrels, whereas expression of three miRNAs including miR-143, miR-200a and miR-519d was found to be upregulated by 1.32-2.34-fold. Similarly, expression of more miRNAs was downregulated in BAT during torpor. We detected reduced expression of 6 miRNAs including miR-103a, miR-107, miR-125b, miR-21, miR-221 and miR-31 (48%-70% of control), while only expression of miR-138 was significantly upregulated (2.91±0.8-fold of the control, P<0.05). Interestingly, miRNAs found to be downregulated in WAT during torpor were similar to those dysregulated in obese humans for increased adipogenesis, whereas miRNAs with altered expression in BAT during torpor were linked to mitochondrial β-oxidation. miRPath target prediction analysis showed that miRNAs downregulated in both WAT and BAT were associated with the regulation of mitogen-activated protein kinase (MAPK) signaling, while the miRNAs upregulated in WAT were linked to transforming growth factor β (TGFβ) signaling. Compared to mouse sequences, no unique nucleotide substitutions within the stem-loop region were discovered for the associated pre-miRNAs for the miRNAs used in this study, suggesting no structure-influenced changes in pre-miRNA processing efficiency in the squirrel. As well, the expression of miRNA processing enzyme Dicer remained unchanged in both tissues during torpor. Overall, our findings suggest that changes of miRNA expression in adipose tissues may be linked to distinct biological roles in WAT and BAT during hibernation and may involve the regulation of signaling cascades. Copyright © 2014 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

Genome-wide identification and characterization of miRNAs in the hypocotyl and cotyledon of cauliflower (Brassica oleracea L. var. botrytis) seedlings.

PubMed

Geng, Meijuan; Li, Hui; Jin, Chuan; Liu, Qian; Chen, Chengbin; Song, Wenqin; Wang, Chunguo

2014-02-01

MicroRNAs (miRNAs) are a class of small endogenous, non-coding RNAs that have key regulatory functions in plant growth, development, and other biological processes. Hypocotyl and cotyledon are the two major tissues of cauliflower (Brassica oleracea L. var. botrytis) seedlings. Tissue culture experiments have indicated that the regenerative abilities of these two tissues are significantly different. However, the characterization of miRNAs and their roles in regulating organ development in cauliflower remain unexplored. In the present study, two small RNA libraries were sequenced by Solexa sequencing technology. 99 known miRNAs belonging to 28 miRNA families were identified, in which 6 miRNA families were detected only in Brassicaceae. A total of 162 new miRNA sequences with single nucleotide substitutions corresponding to the known miRNAs, and 32 potentially novel miRNAs were also first discovered. Comparative analysis indicated that 42 of 99 known miRNAs and 17 of 32 novel miRNAs exhibited significantly differential expression between hypocotyl and cotyledon, and the differential expression of several miRNAs was further validated by stem-loop RT-PCR. In addition, 235 targets for 89 known miRNAs and 198 targets for 24 novel miRNAs were predicted, and their functions were further discussed. The expression patterns of several representative targets were also confirmed by qRT-PCR analysis. The results identified that the transcriptional expression patterns of miRNAs were negatively correlated with their targets. These findings gave new insights into the characteristics of miRNAs in cauliflower, and provided important clues to elucidate the roles of miRNAs in the tissue differentiation and development of cauliflower.

High-throughput sequencing of small RNAs from pollen and silk and characterization of miRNAs as candidate factors involved in pollen-silk interactions in maize.

PubMed

Li, Xiao Ming; Sang, Ya Lin; Zhao, Xiang Yu; Zhang, Xian Sheng

2013-01-01

In angiosperms, successful pollen-pistil interactions are the prerequisite and guarantee of subsequent fertilization and seed production. Recent profile analyses have helped elucidate molecular mechanisms underlying these processes at both transcriptomic and proteomic levels, but the involvement of miRNAs in pollen-pistil interactions is still speculative. In this study, we sequenced four small RNA libraries derived from mature pollen, in vitro germinated pollen, mature silks, and pollinated silks of maize (Zea mays L.). We identified 161 known miRNAs belonging to 27 families and 82 novel miRNAs. Of these, 40 conserved and 16 novel miRNAs showed different expression levels between mature and germinated pollen, and 30 conserved and eight novel miRNAs were differentially expressed between mature and pollinated silks. As candidates for factors associated with pollen-silk (pistil) interactions, expression patterns of the two sets of differentially expressed miRNAs were confirmed by stem-loop real-time RT-PCR. Transcript levels of 22 predicted target genes were also validated using real-time RT-PCR; most of these exhibited expression patterns contrasting with those of their corresponding miRNAs. In addition, GO analysis of target genes of differentially expressed miRNAs revealed that functional categories related to auxin signal transduction and gene expression regulation were overrepresented. These results suggest that miRNA-mediated auxin signal transduction and transcriptional regulation have roles in pollen-silk interactions. The results of our study provide novel information for understanding miRNA regulatory roles in pollen-pistil interactions.

Effects of space radiation and microgravity on miRNA expression profile in Caenorhabditis elegans

NASA Astrophysics Data System (ADS)

Xu, Dan; Sun, Yeqing; Lei, Huang; Gao, Ying

2012-07-01

Living organisms experience a shock and subsequent adaption when they are subjected to space radiation and microgravity during spaceflight. Such changes have been already documented for some biological consequences including skeletal muscle alterations, reduced immune function and bone loss. Recent advancement in the field of molecular biology has demonstrated that small non-coding microRNA (miRNA) can have a broad effect on gene expression networks, and play a key role in cellular response to environmental stresses. However, little is known about how radiation exposure and altered gravity affect miRNA expression. In the present study, we explored the changes in expression of miRNA and related genes from Caenorhabditis elegans (C.elegans) flown on spaceflight. We used wild-type (N2) and dys-1 mutant (deletion of dys-1) stains of C.elegans, which were cultured to Dauer stage and transferred to special SIMbox in the experiment container. These worms taken by Shenzhou VIII spacecraft experienced the 16.5-day shuttle spaceflight. During spaceflight, they suffered space radiation and underwent static zero gravity (microgravity) or imitated earth gravity (1g) in the rotating condition. In contrast, these worms live under static earth gravity (1g) in ground-based controls. To evaluate the effects of space radiation and microgravity on miRNA expression profile, we performed miRNA microarray expression analysis and found that a set of miRNAs in N2 groups were significantly upregulated or downregualted in radiation and microgravity conditions. Among these altered miRNAs, there are two up-regulated and four down-regulated miRNAs in space radiation conditions; one down-regulated miRNAs in microgravity condition. Expression of several miRNAs in N2 groups was only changed significantly in the imitated earth gravity (1g) conditions, presenting these altered miRNAs were affected by radiation exposure alone. Notably, dys-1 mutant is not sensitive to altered gravity due to muscle protein dystrophin deletion. Compared with those miRNAs in N2 groups, altered miRNAs in dys-1 mutant groups may play a role in the general class of myopathies. To confirm whether these altered miRNA expression correlates with gene expression and functional changes of C.elegans, we performed DNA microarray and found that expression of some muscle-related proteins and age-related factors were altered in radiation and microgravity conditions, accompanied with changes in biological processes such as oxidation, and signaling pathways. Our study suggested that molecular changes at the gene and miRNA levels might compromise the functional changes of C.elegans in response to radiation and microgravity.

Deep Sequencing Analysis of miRNA Expression in Breast Muscle of Fast-Growing and Slow-Growing Broilers

PubMed Central

Ouyang, Hongjia; He, Xiaomei; Li, Guihuan; Xu, Haiping; Jia, Xinzheng; Nie, Qinghua; Zhang, Xiquan

2015-01-01

Growth performance is an important economic trait in chicken. MicroRNAs (miRNAs) have been shown to play important roles in various biological processes, but their functions in chicken growth are not yet clear. To investigate the function of miRNAs in chicken growth, breast muscle tissues of the two-tail samples (highest and lowest body weight) from Recessive White Rock (WRR) and Xinghua Chickens (XH) were performed on high throughput small RNA deep sequencing. In this study, a total of 921 miRNAs were identified, including 733 known mature miRNAs and 188 novel miRNAs. There were 200, 279, 257 and 297 differentially expressed miRNAs in the comparisons of WRRh vs. WRRl, WRRh vs. XHh, WRRl vs. XHl, and XHh vs. XHl group, respectively. A total of 22 highly differentially expressed miRNAs (fold change > 2 or < 0.5; p-value < 0.05; q-value < 0.01), which also have abundant expression (read counts > 1000) were found in our comparisons. As far as two analyses (WRRh vs. WRRl, and XHh vs. XHl) are concerned, we found 80 common differentially expressed miRNAs, while 110 miRNAs were found in WRRh vs. XHh and WRRl vs. XHl. Furthermore, 26 common miRNAs were identified among all four comparisons. Four differentially expressed miRNAs (miR-223, miR-16, miR-205a and miR-222b-5p) were validated by quantitative real-time RT-PCR (qRT-PCR). Regulatory networks of interactions among miRNAs and their targets were constructed using integrative miRNA target-prediction and network-analysis. Growth hormone receptor (GHR) was confirmed as a target of miR-146b-3p by dual-luciferase assay and qPCR, indicating that miR-34c, miR-223, miR-146b-3p, miR-21 and miR-205a are key growth-related target genes in the network. These miRNAs are proposed as candidate miRNAs for future studies concerning miRNA-target function on regulation of chicken growth. PMID:26193261

Deep Sequencing Analysis of miRNA Expression in Breast Muscle of Fast-Growing and Slow-Growing Broilers.

PubMed

Ouyang, Hongjia; He, Xiaomei; Li, Guihuan; Xu, Haiping; Jia, Xinzheng; Nie, Qinghua; Zhang, Xiquan

2015-07-17

Growth performance is an important economic trait in chicken. MicroRNAs (miRNAs) have been shown to play important roles in various biological processes, but their functions in chicken growth are not yet clear. To investigate the function of miRNAs in chicken growth, breast muscle tissues of the two-tail samples (highest and lowest body weight) from Recessive White Rock (WRR) and Xinghua Chickens (XH) were performed on high throughput small RNA deep sequencing. In this study, a total of 921 miRNAs were identified, including 733 known mature miRNAs and 188 novel miRNAs. There were 200, 279, 257 and 297 differentially expressed miRNAs in the comparisons of WRRh vs. WRRl, WRRh vs. XHh, WRRl vs. XHl, and XHh vs. XHl group, respectively. A total of 22 highly differentially expressed miRNAs (fold change > 2 or < 0.5; p-value < 0.05; q-value < 0.01), which also have abundant expression (read counts > 1000) were found in our comparisons. As far as two analyses (WRRh vs. WRRl, and XHh vs. XHl) are concerned, we found 80 common differentially expressed miRNAs, while 110 miRNAs were found in WRRh vs. XHh and WRRl vs. XHl. Furthermore, 26 common miRNAs were identified among all four comparisons. Four differentially expressed miRNAs (miR-223, miR-16, miR-205a and miR-222b-5p) were validated by quantitative real-time RT-PCR (qRT-PCR). Regulatory networks of interactions among miRNAs and their targets were constructed using integrative miRNA target-prediction and network-analysis. Growth hormone receptor (GHR) was confirmed as a target of miR-146b-3p by dual-luciferase assay and qPCR, indicating that miR-34c, miR-223, miR-146b-3p, miR-21 and miR-205a are key growth-related target genes in the network. These miRNAs are proposed as candidate miRNAs for future studies concerning miRNA-target function on regulation of chicken growth.

Expression profile analysis of circulating microRNAs and their effects on ion channels in Chinese atrial fibrillation patients.

PubMed

Lu, Yingmin; Hou, Shuxin; Huang, Damin; Luo, Xiaohan; Zhang, Jinchun; Chen, Jian; Xu, Weiping

2015-01-01

To investigate the changes in expression profile of circulating microRNAs (miRNAs) and the regulatory effect of atrial fibrilation (AF)-related miRNAs on ion channels. 112 patients with AF were assigned into observation group, and another 112 non-AF people were assigned into control group. Total plasma RNAs were extracted from patients' blood samples. Differentially expressed miRNA-1s were transfected into primary-cultured neonatal rat cardiac myocytes. Compared with control group, significant differences were observed in 15 kinds of miRNAs in observation group. Down-regulation of the expression of miRNAs included hsa-miR-328, hsa-miR-145, hsa-miR-222, hsa-miR-1, hsa-miR-162, hsa-miR-432, and hsa-miR-493b; Up-regulation of the expression included hsa-miR634, hsa-miR-664, hsa-miR-9, hsa-miR-152, hsa-miR-19, hsa-miR-454, hsa-miR-146, and hsa-miR-374a. The expression level of CACNB2 protein in miRNA-1 group was significantly lower than that in blank control group, negative control group, MTmiRNA-1 group, AMO-1 group and miRNA-1+AMO-1 cotransfection group (P < 0.05), while in AMO-1 group, the expression level of CACNB2 protein was significantly higher than that in other groups (P < 0.05). These results indicated that transfected miRNA-1 could significantly inhibit the expression of CACNB2 protein. Circulating miRNAs can be used in studies concerning on the regulation mechanism of the occurrence and development of AF. MiRNA-1 can decrease the intracellular Ca(2+) concentration and prevent the AF.

Hantaviruses induce cell type- and viral species-specific host microRNA expression signatures

PubMed Central

Shin, Ok Sarah; Kumar, Mukesh; Yanagihara, Richard; Song, Jin-Won

2014-01-01

The mechanisms of hantavirus-induced modulation of host cellular immunity remain poorly understood. Recently, microRNAs (miRNAs) have emerged as a class of essential regulators of host immune response genes. To ascertain if differential host miRNA expression toward representative hantavirus species correlated with immune response genes, miRNA expression profiles were analyzed in human endothelial cells, macrophages and epithelial cells infected with pathogenic and nonpathogenic rodent- and shrew-borne hantaviruses. Distinct miRNA expression profiles were observed in a cell type- and viral species-specific pattern. A subset of miRNAs, including miR-151-5p and miR-1973, were differentially expressed between Hantaan virus and Prospect Hill virus. Pathway analyses confirmed that the targets of selected miRNAs were associated with inflammatory responses and innate immune receptor-mediated signaling pathways. Our data suggest that differential immune responses following hantavirus infection may be regulated in part by cellular miRNA through dysregulation of genes critical to the inflammatory process. PMID:24074584

Isolation and identification of gene-specific microRNAs.

PubMed

Lin, Shi-Lung; Chang, Donald C; Ying, Shao-Yao

2013-01-01

Computer programming has identified hundreds of genomic hairpin sequences, many with functions remain to be determined. Because direct transfection of hairpin-like miRNA precursors (pre)-miRNAs in mammalian cells is not always sufficient to trigger effective RNA-induced gene silencing complex (RISC) assembly, a key step for RNA interference (RNAi)-related gene silencing, we developed an intronic miRNA-expressing system to overcome this problem by inserting a hairpin-like pre-miRNA structure into the intron region of a gene and successfully increased the efficiency and effectiveness of miRNA-associated RNAi induction in vitro and in vivo. This intronic miRNA biogenesis has been found to depend on a coupled interaction of nascent precursor messenger RNA transcription and intron excision within a specific nuclear region proximal to genomic perichromatin fibrils. The intronic miRNA was transcribed by RNA type II polymerases, coexpressed with a primary gene transcript, and excised out of its encoding gene transcript by intracellular RNA splicing and processing mechanisms. Currently, some ribonuclease III endonucleases have been found to be involved in the processing of spliced introns and probably facilitating the intronic miRNA maturation. Using this miRNA generation system, we have shown for the first time that the intron-derived miRNAs were able to induce strong RNAi effects in not only human and mouse cells but also zebrafishes, chicken embryos, and adult mice. We have also developed an miRNA isolation protocol, based on the complementarity between the designed miRNA and its target gene sequence, to purify and identify the mature miRNAs generated by the intronic miRNA-expressing system. Several intronic miRNA identities and structures are currently confirmed to be active in vitro and in vivo. According to this proven-of-principle method, we now have full knowledge to design pre-miRNA inserts that are more efficient and effective for the intronic miRNA-expressing systems.

MicroRNA expression profiles of drug-resistance breast cancer cells and their exosomes.

PubMed

Zhong, Shanliang; Chen, Xiu; Wang, Dandan; Zhang, Xiaohui; Shen, Hongyu; Yang, Sujin; Lv, Mengmeng; Tang, Jinhai; Zhao, Jianhua

2016-04-12

Exosomes have been shown to transmit drug resistance through delivering miRNAs. We aimed to explore their roles in breast cancer. Three resistant sublines were established by exposing parental MDA-MB-231 cell line to docetaxel, epirubicin and vinorelbine, respectively. Preneoadjuvant chemotherapy biopsies and paired surgically-resected specimens embedded in paraffin from 23 breast cancer patients were collected. MiRNA expression profiles of the cell lines and their exosomes were evaluated using microarray. The result showed that most miRNAs in exosomes had a lower expression level than that in cells, however, some miRNAs expressed higher in exosomes than in cells, suggesting a number of miRNAs is concentrated in exosomes. Among the dysregulated miRNAs, 22 miRNAs were consistently up-regulated in exosomes and their cells of origin. We further found that 12 of the 22 miRNAs were significantly up-regulated after preneoadjuvant chemotherapy. Further study of the role of these 12 miRNAs in acquisition of drug resistance is needed to clarify their contribution to chemoresistance.

Liver microRNA profile of induced allograft tolerance

PubMed Central

Vitalone, Matthew James; Wai, Liang; Fujiki, Masato; Lau, Audrey H.; Littau, Erik; Esquivel, Carlos; Martinez, Olivia M.; Krams, Sheri M.

2016-01-01

Introduction Although the liver is less immunogenic than other solid organs, most liver transplant recipients receive lifelong immunosuppression. In both experimental models and clinical transplantation, total Lymphoid Irradiation (TLI) has been shown to induce allograft tolerance. Our goal was to identify the microRNAs (miRNAs) expressed in tolerant liver allograft recipients in an experimental model of TLI-induced tolerance. Methods To identify the miRNAs associated with TLI-induced tolerance we examined syngeneic recipients (Lewis→Lewis) and allogeneic recipients (DA→Lewis) of orthotropic liver transplants that received post-transplant TLI, allogeneic recipients that were not treated post-transplantation and experienced acute rejection, and native DA livers. QPCR miRNA array cards were used to profile liver grafts. Results We identified 12 miRNAs that were specifically and significantly increased during acute rejection. In early tolerance, 33 miRNAs were altered compared to syngeneic livers, with 80% of the miRNAs increased. In established tolerance 42 miRNAs were altered. In addition, miR-142-5p and miR-181a demonstrated increased expression in tolerant livers (both early and established tolerance) as compared to syngeneic livers. A principal component analysis of all miRNAs assayed, demonstrated a profile in established tolerance that was closely related to that seen in syngeneic livers. Conclusions The miRNA profile of established tolerant allografts is very similar to syngeneic grafts suggesting tolerance may be a return to an immunological state of quiescence. PMID:26950716

Identification, characterization and expression analysis of pigeonpea miRNAs in response to Fusarium wilt.

PubMed

Hussain, Khalid; Mungikar, Kanak; Kulkarni, Abhijeet; Kamble, Avinash

2018-05-05

Upon confrontation with unfavourable conditions, plants invoke a very complex set of biochemical and physiological reactions and alter gene expression patterns to combat the situations. MicroRNAs (miRNAs), a class of small non-coding RNA, contribute extensively in regulation of gene expression through translation inhibition or degradation of their target mRNAs during such conditions. Therefore, identification of miRNAs and their targets holds importance in understanding the regulatory networks triggered during stress. Structure and sequence similarity based in silico prediction of miRNAs in Cajanus cajan L. (Pigeonpea) draft genome sequence has been carried out earlier. These annotations also appear in related GenBank genome sequence entries. However, there are no reports available on context dependent miRNA expression and their targets in pigeonpea. Therefore, in the present study we addressed these questions computationally, using pigeonpea EST sequence information. We identified five novel pigeonpea miRNA precursors, their mature forms and targets. Interestingly, only one of these miRNAs (miR169i-3p) was identified earlier in draft genome sequence. We then validated expression of these miRNAs, experimentally. It was also observed that these miRNAs show differential expression patterns in response to Fusarium inoculation indicating their biotic stress responsive nature. Overall these results will help towards better understanding the regulatory network of defense during pigeonpea -pathogen interactions and role of miRNAs in the process. Copyright © 2018 Elsevier B.V. All rights reserved.

Overexpression of miR-9 in mast cells is associated with invasive behavior and spontaneous metastasis

PubMed Central

2014-01-01

Background While microRNA (miRNA) expression is known to be altered in a variety of human malignancies contributing to cancer development and progression, the potential role of miRNA dysregulation in malignant mast cell disease has not been previously explored. The purpose of this study was to investigate the potential contribution of miRNA dysregulation to the biology of canine mast cell tumors (MCTs), a well-established spontaneous model of malignant mast cell disease. Methods We evaluated the miRNA expression profiles from biologically low-grade and biologically high-grade primary canine MCTs using real-time PCR-based TaqMan Low Density miRNA Arrays and performed real-time PCR to evaluate miR-9 expression in primary canine MCTs, malignant mast cell lines, and normal bone marrow-derived mast cells (BMMCs). Mouse mast cell lines and BMMCs were transduced with empty or pre-miR-9 expressing lentiviral constructs and cell proliferation, caspase 3/7 activity, and invasion were assessed. Transcriptional profiling of cells overexpressing miR-9 was performed using Affymetrix GeneChip Mouse Gene 2.0 ST arrays and real-time PCR was performed to validate changes in mRNA expression. Results Our data demonstrate that unique miRNA expression profiles correlate with the biological behavior of primary canine MCTs and that miR-9 expression is increased in biologically high grade canine MCTs and malignant cell lines compared to biologically low grade tumors and normal canine BMMCs. In transformed mouse malignant mast cell lines expressing either wild-type (C57) or activating (P815) KIT mutations and mouse BMMCs, miR-9 overexpression significantly enhanced invasion but had no effect on cell proliferation or apoptosis. Transcriptional profiling of normal mouse BMMCs and P815 cells possessing enforced miR-9 expression demonstrated dysregulation of several genes, including upregulation of CMA1, a protease involved in activation of matrix metalloproteases and extracellular matrix remodeling. Conclusions Our findings demonstrate that unique miRNA expression profiles correlate with the biological behavior of canine MCTs. Furthermore, dysregulation of miR-9 is associated with MCT metastasis potentially through the induction of an invasive phenotype, identifying a potentially novel pathway for therapeutic intervention. PMID:24517413

Identification and functional analysis of flowering related microRNAs in common wild rice (Oryza rufipogon Griff.).

PubMed

Chen, Zongxiang; Li, Fuli; Yang, Songnan; Dong, Yibo; Yuan, Qianhua; Wang, Feng; Li, Weimin; Jiang, Ying; Jia, Shirong; Pei, Xinwu

2013-01-01

MicroRNAs (miRNAs) is a class of non-coding RNAs involved in post- transcriptional control of gene expression, via degradation and/or translational inhibition. Six-hundred sixty-one rice miRNAs are known that are important in plant development. However, flowering-related miRNAs have not been characterized in Oryza rufipogon Griff. It was approved by supervision department of Guangdong wild rice protection. We analyzed flowering-related miRNAs in O. rufipogon using high-throughput sequencing (deep sequencing) to understand the changes that occurred during rice domestication, and to elucidate their functions in flowering. Three O. rufipogon sRNA libraries, two vegetative stage (CWR-V1 and CWR-V2) and one flowering stage (CWR-F2) were sequenced using Illumina deep sequencing. A total of 20,156,098, 21,531,511 and 20,995,942 high quality sRNA reads were obtained from CWR-V1, CWR-V2 and CWR-F2, respectively, of which 3,448,185, 4,265,048 and 2,833,527 reads matched known miRNAs. We identified 512 known rice miRNAs in 214 miRNA families and predicted 290 new miRNAs. Targeted functional annotation, GO and KEGG pathway analyses predicted that 187 miRNAs regulate expression of flowering-related genes. Differential expression analysis of flowering-related miRNAs showed that: expression of 95 miRNAs varied significantly between the libraries, 66 are flowering-related miRNAs, such as oru-miR97, oru-miR117, oru-miR135, oru-miR137, et al. 17 are early-flowering -related miRNAs, including osa-miR160f, osa-miR164d, osa-miR167d, osa-miR169a, osa-miR172b, oru-miR4, et al., induced during the floral transition. Real-time PCR revealed the same expression patterns as deep sequencing. miRNAs targets were confirmed for cleavage by 5'-RACE in vivo, and were negatively regulated by miRNAs. This is the first investigation of flowering miRNAs in wild rice. The result indicates that variation in miRNAs occurred during rice domestication and lays a foundation for further study of phase change and flowering in O. rufipogon. Complicated regulatory networks mediated by multiple miRNAs regulate the expression of flowering genes that control the induction of flowering.

Expression Variations of miRNAs and mRNAs in Rice (Oryza sativa).

PubMed

Wen, Ming; Xie, Munan; He, Lian; Wang, Yushuai; Shi, Suhua; Tang, Tian

2016-12-31

Differences in expression levels are an important source of phenotypic variation within and between populations. MicroRNAs (miRNAs) are key players in post-transcriptional gene regulation that are important for plant development and stress responses. We surveyed expression variation of miRNAs and mRNAs of six accessions from two rice subspecies Oryza sativa L. ssp. indica and Oryza sativa L. ssp. japonica using deep sequencing. While more than half (53.7%) of the mature miRNAs exhibit differential expression between grains and seedlings of rice, only 11.0% show expression differences between subspecies, with an additional 2.2% differentiated for the development-by-subspecies interaction. Expression variation is greater for lowly conserved miRNAs than highly conserved miRNAs, whereas the latter show stronger negative correlation with their targets in expression changes between subspecies. Using a permutation test, we identified 51 miRNA-mRNA pairs that correlate negatively or positively in expression level among cultivated rice. Genes involved in various metabolic processes and stress responses are enriched in the differentially expressed genes between rice indica and japonica subspecies. Our results indicate that stabilizing selection is the major force governing miRNA expression in cultivated rice, albeit positive selection may be responsible for much of the between-subspecies expression divergence. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

BioVLAB-MMIA-NGS: microRNA-mRNA integrated analysis using high-throughput sequencing data.

PubMed

Chae, Heejoon; Rhee, Sungmin; Nephew, Kenneth P; Kim, Sun

2015-01-15

It is now well established that microRNAs (miRNAs) play a critical role in regulating gene expression in a sequence-specific manner, and genome-wide efforts are underway to predict known and novel miRNA targets. However, the integrated miRNA-mRNA analysis remains a major computational challenge, requiring powerful informatics systems and bioinformatics expertise. The objective of this study was to modify our widely recognized Web server for the integrated mRNA-miRNA analysis (MMIA) and its subsequent deployment on the Amazon cloud (BioVLAB-MMIA) to be compatible with high-throughput platforms, including next-generation sequencing (NGS) data (e.g. RNA-seq). We developed a new version called the BioVLAB-MMIA-NGS, deployed on both Amazon cloud and on a high-performance publicly available server called MAHA. By using NGS data and integrating various bioinformatics tools and databases, BioVLAB-MMIA-NGS offers several advantages. First, sequencing data is more accurate than array-based methods for determining miRNA expression levels. Second, potential novel miRNAs can be detected by using various computational methods for characterizing miRNAs. Third, because miRNA-mediated gene regulation is due to hybridization of an miRNA to its target mRNA, sequencing data can be used to identify many-to-many relationship between miRNAs and target genes with high accuracy. http://epigenomics.snu.ac.kr/biovlab_mmia_ngs/. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Epigallocatechin-3-O-gallate modulates global microRNA expression in interleukin-1β-stimulated human osteoarthritis chondrocytes: potential role of EGCG on negative co-regulation of microRNA-140-3p and ADAMTS5.

PubMed

Rasheed, Zafar; Rasheed, Naila; Al-Shaya, Osama

2018-04-01

MicroRNAs (miRNAs) are short, non-coding RNAs involved in almost all cellular processes. Epigallocatechin-3-O-gallate (EGCG) is a green tea polyphenol and is known to exert anti-arthritic effects by inhibiting genes associated with osteoarthritis (OA). This study was undertaken to investigate the global effect of EGCG on interleukin-1β (IL-1β)-induced expression of miRNAs in human chondrocytes. Human chondrocytes were derived from OA cartilage and then treated with EGCG and IL-1β. Human miRNA microarray technology was used to determine the expression profile of 1347 miRNAs. Microarray results were verified by taqman assays and transfection of chondrocytes with miRNA inhibitors. Out of 1347 miRNAs, EGCG up-regulated expression of 19 miRNAs and down-regulated expression of 17 miRNAs, whereas expression of 1311 miRNAs remains unchanged in IL-1β-stimulated human OA chondrocytes. Bioinformatics approach showed that 3`UTR of ADAMTS5 mRNA contains the 'seed-matched-sequence' for hsa-miR-140-3p. IL-1β-induced expression of ADAMTS5 correlated with down-regulation of hsa-miR-140-3p. Importantly, EGCG inhibited IL-1β-induced ADAMTS5 expression and up-regulated the expression of hsa-miR-140-3p. This EGCG-induced co-regulation between ADAMTS5 and hsa-miR-140-3p becomes reversed in OA chondrocytes transfected with anti-miR-140-3p. This study provides an important insight into the molecular basis of the reported anti-arthritic effects of EGCG. Our data indicate that the potential of EGCG in OA chondrocytes may be related to its ability to globally inhibit inflammatory response via modulation of miRNAs expressions.

miRNAome expression profiles in the gonads of adult Melopsittacus undulatus

PubMed Central

Jiang, Lan; Wang, Qingqing; Yu, Jue; Gowda, Vinita; Johnson, Gabriel; Yang, Jianke

2018-01-01

The budgerigar (Melopsittacus undulatus) is one of the most widely studied parrot species, serving as an excellent animal model for behavior and neuroscience research. Until recently, it was unknown how sexual differences in the behavior, physiology, and development of organisms are regulated by differential gene expression. MicroRNAs (miRNAs) are endogenous short non-coding RNA molecules that can post-transcriptionally regulate gene expression and play a critical role in gonadal differentiation as well as early development of animals. However, very little is known about the role gonadal miRNAs play in the early development of birds. Research on the sex-biased expression of miRNAs in avian gonads are limited, and little is known about M. undulatus. In the current study, we sequenced two small non-coding RNA libraries made from the gonads of adult male and female budgerigars using Illumina paired-end sequencing technology. We obtained 254 known and 141 novel miRNAs, and randomly validated five miRNAs. Of these, three miRNAs were differentially expressed miRNAs and 18 miRNAs involved in sexual differentiation as determined by functional analysis with GO annotation and KEGG pathway analysis. In conclusion, this work is the first report of sex-biased miRNAs expression in the budgerigar, and provides additional sequences to the avian miRNAome database which will foster further functional genomic research. PMID:29666766

Differential Expression of microRNAs in the Ovaries from Letrozole-Induced Rat Model of Polycystic Ovary Syndrome.

PubMed

Li, Dandan; Li, Chunjin; Xu, Ying; Xu, Duo; Li, Hongjiao; Gao, Liwei; Chen, Shuxiong; Fu, Lulu; Xu, Xin; Liu, Yongzheng; Zhang, Xueying; Zhang, Jingshun; Ming, Hao; Zheng, Lianwen

2016-04-01

Polycystic ovary syndrome (PCOS) is a complex and heterogeneous endocrine disorder. To understand the pathogenesis of PCOS, we established rat models of PCOS induced by letrozole and employed deep sequencing to screen the differential expression of microRNAs (miRNAs) in PCOS rats and control rats. We observed vaginal smear and detected ovarian pathological alteration and hormone level changes in PCOS rats. Deep sequencing showed that a total of 129 miRNAs were differentially expressed in the ovaries from letrozole-induced rat model compared with the control, including 49 miRNAs upregulated and 80 miRNAs downregulated. Furthermore, the differential expression of miR-201-5p, miR-34b-5p, miR-141-3p, and miR-200a-3p were confirmed by real-time polymerase chain reaction. Bioinformatic analysis revealed that these four miRNAs were predicted to target a large set of genes with different functions. Pathway analysis supported that the miRNAs regulate oocyte meiosis, mitogen-activated protein kinase (MAPK) signaling, phosphoinositide 3-kinase/Akt (PI3K-Akt) signaling, Rap1 signaling, and Notch signaling. These data indicate that miRNAs are differentially expressed in rat PCOS model and the differentially expressed miRNA are involved in the etiology and pathophysiology of PCOS. Our findings will help identify miRNAs as novel diagnostic markers and therapeutic targets for PCOS.

Robust Selection Algorithm (RSA) for Multi-Omic Biomarker Discovery; Integration with Functional Network Analysis to Identify miRNA Regulated Pathways in Multiple Cancers.

PubMed

Sehgal, Vasudha; Seviour, Elena G; Moss, Tyler J; Mills, Gordon B; Azencott, Robert; Ram, Prahlad T

2015-01-01

MicroRNAs (miRNAs) play a crucial role in the maintenance of cellular homeostasis by regulating the expression of their target genes. As such, the dysregulation of miRNA expression has been frequently linked to cancer. With rapidly accumulating molecular data linked to patient outcome, the need for identification of robust multi-omic molecular markers is critical in order to provide clinical impact. While previous bioinformatic tools have been developed to identify potential biomarkers in cancer, these methods do not allow for rapid classification of oncogenes versus tumor suppressors taking into account robust differential expression, cutoffs, p-values and non-normality of the data. Here, we propose a methodology, Robust Selection Algorithm (RSA) that addresses these important problems in big data omics analysis. The robustness of the survival analysis is ensured by identification of optimal cutoff values of omics expression, strengthened by p-value computed through intensive random resampling taking into account any non-normality in the data and integration into multi-omic functional networks. Here we have analyzed pan-cancer miRNA patient data to identify functional pathways involved in cancer progression that are associated with selected miRNA identified by RSA. Our approach demonstrates the way in which existing survival analysis techniques can be integrated with a functional network analysis framework to efficiently identify promising biomarkers and novel therapeutic candidates across diseases.

Using microRNA profiling in urine samples to develop a non-invasive test for bladder cancer.

PubMed

Mengual, Lourdes; Lozano, Juan José; Ingelmo-Torres, Mercedes; Gazquez, Cristina; Ribal, María José; Alcaraz, Antonio

2013-12-01

Current standard methods used to detect and monitor bladder urothelial cell carcinoma (UCC) are invasive or have low sensitivity. The incorporation into clinical practice of a non-invasive tool for UCC assessment would enormously improve patients' quality of life and outcome. This study aimed to examine the microRNA (miRNA) expression profiles in urines of UCC patients in order to develop a non-invasive accurate and reliable tool to diagnose and provide information on the aggressiveness of the tumor. We performed a global miRNA expression profiling analysis of the urinary cells from 40 UCC patients and controls using TaqMan Human MicroRNA Array followed by validation of 22 selected potentially diagnostic and prognostic miRNAs in a separate cohort of 277 samples using a miRCURY LNA qPCR system. miRNA-based signatures were developed by multivariate logistic regression analysis and internally cross-validated. In the initial cohort of patients, we identified 40 and 30 aberrantly expressed miRNA in UCC compared with control urines and in high compared with low grade tumors, respectively. Quantification of 22 key miRNAs in an independent cohort resulted in the identification of a six miRNA diagnostic signature with a sensitivity of 84.8% and specificity of 86.5% (AUC = 0.92) and a two miRNA prognostic model with a sensitivity of 84.95% and a specificity of 74.14% (AUC = 0.83). Internal cross-validation analysis confirmed the accuracy rates of both models, reinforcing the strength of our findings. Although the data needs to be externally validated, miRNA analysis in urine appears to be a valuable tool for the non-invasive assessment of UCC. Copyright © 2013 UICC.

Determining miRNA Expression Levels in Degraded RNA Samples Using Real-Time RT-qPCR and Microarray Technologies

PubMed Central

Tighe, S.; Holbrook, J.; Nadella, V.; Carmical, R.; Sol-Church, K.; Yueng, A.T.; Chittur, S.

2011-01-01

The Nucleic Acid Research Group (NARG) has previously conducted studies evaluating the impact of RNA integrity and priming strategies on cDNA synthesis and real-time RT-qPCR. The results of last year's field study as it relates to degraded RNA will be presented. In continuation of the RNA integrity theme, this year's study was designed to evaluate the impact of RNA integrity on the analysis of miRNA expression using real-time RT-qPCR. Target section was based on data obtained by the Microarray Research Group (MARG) and other published data from next gen sequencing. These 9 miRNAs represent three groups of miRNA that are expressed at low, medium or high levels in the First Choice human brain reference RNA sample. Two popular RT priming strategies tested in this study include the Megaplex miRNA TaqMan assay (ABI) and the RT2 miRNA qPCR assay (Qiagen/SA Biosciences). The basis for the ABI assay design is a target-specific stem-loop structure and reverse-transcription primer, while the Qiagen design combines poly(A) tailing and a universal reverse transcription in one cDNA synthesis reaction. For this study, the human brain reference RNA was subject to controlled degradation using RNase A to RIN (RNA Integrity Number) values of 7 (good), 4 (moderately degraded), and 2 (severely degraded).These templates were then used to assess both RT methods. In addition to this real-time RT-qPCR data, the same RNA templates were further analyzed using universal poly(A) tailing and hybridization to Affymetrix miRNA GeneChips. This talk will provide insights into RT priming strategies for miRNA and contrast the qPCR results obtained using different technologies.

Human papilloma virus, DNA methylation and microRNA expression in cervical cancer (Review).

PubMed

Jiménez-Wences, Hilda; Peralta-Zaragoza, Oscar; Fernández-Tilapa, Gloria

2014-06-01

Cancer is a complex disease caused by genetic and epigenetic abnormalities that affect gene expression. The progression from precursor lesions to invasive cervical cancer is influenced by persistent human papilloma virus (HPV) infection, which induces changes in the host genome and epigenome. Epigenetic alterations, such as aberrant miRNA expression and changes in DNA methylation status, favor the expression of oncogenes and the silencing of tumor-suppressor genes. Given that some miRNA genes can be regulated through epigenetic mechanisms, it has been proposed that alterations in the methylation status of miRNA promoters could be the driving mechanism behind their aberrant expression in cervical cancer. For these reasons, we assessed the relationship among HPV infection, cellular DNA methylation and miRNA expression. We conclude that alterations in the methylation status of protein-coding genes and various miRNA genes are influenced by HPV infection, the viral genotype, the physical state of the viral DNA, and viral oncogenic risk. Furthermore, HPV induces deregulation of miRNA expression, particularly at loci near fragile sites. This deregulation occurs through the E6 and E7 proteins, which target miRNA transcription factors such as p53.

Comparison of Total RNA Isolation Methods for Analysis of Immune-Related microRNAs in Market Milks

PubMed Central

2015-01-01

Bovine milk provides essential nutrients, including immunologically important molecules, as the primary source of nutrition to newborns. Recent studies showed that RNAs from bovine milk contain immune-related microRNAs (miRNA) that regulate various immune systems. To evaluate the biological and immunological activity of miRNAs from milk products, isolation methods need to be established. Six methods for extracting total RNAs from bovine colostrums were adopted to evaluate the isolating efficiency and expression of miRNAs. Total RNA from milk was presented in formulation of small RNAs, rather than ribosomal RNAs. Column-combined phenol isolating methods showed high recovery of total RNAs, especially the commercial columns for biofluid samples, which demonstrated outstanding efficiency for recovering miRNAs. We also evaluated the quantity of five immune-related miRNAs (miR-93, miR-106a, miR-155, miR-181a, miR-451) in milk processed by temperature treatments including low temperature for long time (LTLT, 63℃ for 30 min)-, high temperature for short time (HTST, 75℃ for 15 s)-, and ultra heat treatment (UHT, 120-130℃ for 0.5-4 s). All targeted miRNAs had significantly reduced levels in processed milks compared to colostrum and raw mature milk. Interestingly, the amount of immune-related miRNAs from HTST milk was more resistant than those of LTLT and UHT milks. Our present study examined defined methods of RNA isolation and quantification of immune-specific miRNAs from small volumes of milk for use in further analysis. PMID:26761866

Comparison of Total RNA Isolation Methods for Analysis of Immune-Related microRNAs in Market Milks.

PubMed

Oh, Sangnam; Park, Mi Ri; Son, Seok Jun; Kim, Younghoon

2015-01-01

Bovine milk provides essential nutrients, including immunologically important molecules, as the primary source of nutrition to newborns. Recent studies showed that RNAs from bovine milk contain immune-related microRNAs (miRNA) that regulate various immune systems. To evaluate the biological and immunological activity of miRNAs from milk products, isolation methods need to be established. Six methods for extracting total RNAs from bovine colostrums were adopted to evaluate the isolating efficiency and expression of miRNAs. Total RNA from milk was presented in formulation of small RNAs, rather than ribosomal RNAs. Column-combined phenol isolating methods showed high recovery of total RNAs, especially the commercial columns for biofluid samples, which demonstrated outstanding efficiency for recovering miRNAs. We also evaluated the quantity of five immune-related miRNAs (miR-93, miR-106a, miR-155, miR-181a, miR-451) in milk processed by temperature treatments including low temperature for long time (LTLT, 63℃ for 30 min)-, high temperature for short time (HTST, 75℃ for 15 s)-, and ultra heat treatment (UHT, 120-130℃ for 0.5-4 s). All targeted miRNAs had significantly reduced levels in processed milks compared to colostrum and raw mature milk. Interestingly, the amount of immune-related miRNAs from HTST milk was more resistant than those of LTLT and UHT milks. Our present study examined defined methods of RNA isolation and quantification of immune-specific miRNAs from small volumes of milk for use in further analysis.

Transduction of hematopoietic stem cells to stimulate RNA interference against feline infectious peritonitis.

PubMed

Anis, Eman A; Dhar, Madhu; Legendre, Alfred M; Wilkes, Rebecca P

2017-06-01

Objectives The goals of the study were: (1) to develop and evaluate non-replicating lentivirus vectors coding for feline coronavirus (FCoV)-specific micro (mi)RNA as a potential antiviral therapy for feline infectious peritonitis (FIP); (2) to assess the feasibility of transducing hematopoietic stem cells (HSCs) with ex vivo introduction of the miRNA-expressing lentivirus vector; and (3) to assess the ability of the expressed miRNA to inhibit FCoV replication in HSCs in vitro. Methods HSCs were obtained from feline bone marrow and replicated in vitro. Three lentiviruses were constructed, each expressing a different anti-FCoV miRNA. HSCs were stably transduced with the miRNA-expressing lentivirus vector that produced the most effective viral inhibition in a feline cell line. The effectiveness of the transduction and the expression of anti-FCoV miRNA were tested by infecting the HSCs with two different strains of FCoV. The inhibition of coronavirus replication was determined by relative quantification of the inhibition of intracellular viral genomic RNA synthesis using real-time, reverse-transcription PCR. The assessment of virus replication inhibition was determined via titration of extracellular virus using the TCID 50 assay. Results Inhibition of FCoV was most significant in feline cells expressing miRNA-L2 that targeted the viral leader sequence, 48 h postinfection. miRNA-L2 expression in stably transduced HSCs resulted in 90% and 92% reductions in FIPV WSU 79-1146 genomic RNA synthesis and extracellular virus production, respectively, as well as 74% and 80% reduction in FECV WSU 79-1683 genomic RNA synthesis and extracellular virus production, respectively, as compared with an infected negative control sample producing non-targeting miRNA. Conclusions and relevance These preliminary results show that genetic modification of HSCs for constitutive production of anti-coronavirus miRNA will reduce FCoV replication.

In Silico Analysis of Small RNAs Suggest Roles for Novel and Conserved miRNAs in the Formation of Epigenetic Memory in Somatic Embryos of Norway Spruce.

PubMed

Yakovlev, Igor A; Fossdal, Carl G

2017-01-01

Epigenetic memory in Norway spruce affects the timing of bud burst and bud set, vitally important adaptive traits for this long-lived forest species. Epigenetic memory is established in response to the temperature conditions during embryogenesis. Somatic embryogenesis at different epitype inducing (EpI) temperatures closely mimics the natural processes of epigenetic memory formation in seeds, giving rise to epigenetically different clonal plants in a reproducible and predictable manner, with respect to altered bud phenology. MicroRNAs (miRNAs) and other small non-coding RNAs (sRNAs) play an essential role in the regulation of plant gene expression and may affect this epigenetic mechanism. We used NGS sequencing and computational in silico methods to identify and profile conserved and novel miRNAs among small RNAs in embryogenic tissues of Norway spruce at three EpI temperatures (18, 23 and 28°C). We detected three predominant classes of sRNAs related to a length of 24 nt, followed by a 21-22 nt class and a third 31 nt class of sRNAs. More than 2100 different miRNAs within the prevailing length 21-22 nt were identified. Profiling these putative miRNAs allowed identification of 1053 highly expressed miRNAs, including 523 conserved and 530 novels. 654 of these miRNAs were found to be differentially expressed (DEM) depending on EpI temperature. For most DEMs, we defined their putative mRNA targets. The targets represented mostly by transcripts of multiple-repeats proteins, like TIR, NBS-LRR, PPR and TPR repeat, Clathrin/VPS proteins, Myb-like, AP2, etc. Notably, 124 DE miRNAs targeted 203 differentially expressed epigenetic regulators. Developing Norway spruce embryos possess a more complex sRNA structure than that reported for somatic tissues. A variety of the predicted miRNAs showed distinct EpI temperature dependent expression patterns. These putative EpI miRNAs target spruce genes with a wide range of functions, including genes known to be involved in epigenetic regulation, which in turn could provide a feedback process leading to the formation of epigenetic marks. We suggest that TIR, NBS and LRR domain containing proteins could fulfill more general functions for signal transduction from external environmental stimuli and conversion them into molecular response. Fine-tuning of the miRNA production likely participates in both developmental regulation and epigenetic memory formation in Norway spruce.

Analysis of secondary structural elements in human microRNA hairpin precursors.

PubMed

Liu, Biao; Childs-Disney, Jessica L; Znosko, Brent M; Wang, Dan; Fallahi, Mohammad; Gallo, Steven M; Disney, Matthew D

2016-03-01

MicroRNAs (miRNAs) regulate gene expression by targeting complementary mRNAs for destruction or translational repression. Aberrant expression of miRNAs has been associated with various diseases including cancer, thus making them interesting therapeutic targets. The composite of secondary structural elements that comprise miRNAs could aid the design of small molecules that modulate their function. We analyzed the secondary structural elements, or motifs, present in all human miRNA hairpin precursors and compared them to highly expressed human RNAs with known structures and other RNAs from various organisms. Amongst human miRNAs, there are 3808 are unique motifs, many residing in processing sites. Further, we identified motifs in miRNAs that are not present in other highly expressed human RNAs, desirable targets for small molecules. MiRNA motifs were incorporated into a searchable database that is freely available. We also analyzed the most frequently occurring bulges and internal loops for each RNA class and found that the smallest loops possible prevail. However, the distribution of loops and the preferred closing base pairs were unique to each class. Collectively, we have completed a broad survey of motifs found in human miRNA precursors, highly expressed human RNAs, and RNAs from other organisms. Interestingly, unique motifs were identified in human miRNA processing sites, binding to which could inhibit miRNA maturation and hence function.

Identification of highly expressed host microRNAs that respond to white spot syndrome virus infection in the Pacific white shrimp Litopenaeus vannamei (Penaeidae).

PubMed

Zeng, D G; Chen, X L; Xie, D X; Zhao, Y Z; Yang, Q; Wang, H; Li, Y M; Chen, X H

2015-05-11

MicroRNAs (miRNAs) are known to play an important role in regulating both adaptive and innate immunity. Pacific white shrimp (Litopenaeus vannamei) is the most widely farmed crustacean species in the world. However, little is known about the role miRNAs play in shrimp immunity. To understand the impact of viral infection on miRNA expression in shrimp, we used high-throughput sequencing technology to sequence two small RNA libraries prepared from L. vannamei under normal and white spot syndrome virus (WSSV) challenged conditions. Approximately 19,312,189 and 39,763,551 raw reads corresponding to 17,414,787 and 28,633,379 high-quality mappable reads were obtained from the two libraries, respectively. Twelve conserved miRNAs and one novel miRNA that were highly expressed (>100 RPM) in L. vannamei were identified. Of the identified miRNAs, 8 were differentially expressed in response to the virus infection, of which 1 was upregulated and 7 were downregulated. The prediction of miRNA targets showed that the target genes of the differentially expressed miRNAs were related to immunity, apoptosis, and development functions. Our study provides the first characterization of L. vannamei miRNAs in response to WSSV infection, which will help to reveal the roles of miRNAs in the antiviral mechanisms of shrimp.

Association between the miRNA Signatures in Plasma and Bronchoalveolar Fluid in Respiratory Pathologies

PubMed Central

Molina-Pinelo, Sonia; Suárez, Rocío; Pastor, María Dolores; Nogal, Ana; Márquez-Martín, Eduardo; Martín-Juan, José; Carnero, Amancio; Paz-Ares, Luis

2012-01-01

The identification of new less invasive biomarkers is necessary to improve the detection and prognostic outcome of respiratory pathological processes. The measurement of miRNA expression through less invasive techniques such as plasma and serum have been suggested to analysis of several lung malignancies including lung cancer. These studies are assuming a common deregulated miRNA expression both in blood and lung tissue. The present study aimed to obtain miRNA representative signatures both in plasma and bronchoalveolar cell fraction that could serve as biomarker in respiratory diseases. Ten patients were evaluated to assess the expression levels of 381 miRNAs. We found that around 50% miRNAs were no detected in both plasma and bronchoalveolar cell fraction and only 20% of miRNAs showed similar expression in both samples. These results show a lack of association of miRNA signatures between plasma and bronchoalveolar cytology in the same patient. The profiles are not comparable; however, there is a similarity in the relative expression in a very small subset of miRNAs (miR-17, miR-19b, miR-195 and miR-20b) between both biological samples in all patients. This finding supports that the miRNAs profiles obtained from different biological samples have to be carefully validated to link with respiratory diseases. PMID:22430188

Generation of a stable cell line for constitutive miRNA expression.

PubMed

Lieber, Diana

2013-01-01

miRNAs have in recent years emerged as novel players in virus-host interactions. While individual miRNAs are capable of regulating many targets simultaneously, not much is known about the role of distinct host or viral miRNAs in the context of infection. Analysis of the function of a miRNA is often hampered by the complexity of virus-host interactions and the enormous changes in the host cell during infection. Many viral miRNAs as for example from Kaposi sarcoma-associated Herpesvirus (KSHV) are probably exclusively expressed in latent infection. This might lead to a steady-state situation with offense and defense mechanisms counteracting each other. Cellular miRNAs involved in defense against pathogens on the other hand might be suppressed in infection. A cell culture system allowing for constitutive expression of individual miRNAs at high levels is a useful tool to enhance miRNA-specific functions and to uncouple viral miRNA function from other infection-related mechanisms. Here, a protocol is described to generate stable cell lines for constitutive expression of single cellular or viral miRNA precursors in absence of infection. The procedure comprises cloning of the precursor sequence, generation of the lentiviral expression vector, transduction of the cells of interest, selection for polyclonal cell lines, and isolation of monoclonal cell lines by limiting dilution.

The miiuy croaker microRNA transcriptome and microRNA regulation of RIG-I like receptor signaling pathway after poly(I:C) stimulation.

PubMed

Han, Jingjing; Xu, Guoliang; Xu, Tianjun

2016-07-01

MicroRNAs (miRNAs) as endogenous small non-coding RNAs play key regulatory roles in diverse biological processes via degrading the target mRNAs or inhibiting protein translation. Previously many researchers have reported the identification, characteristic of miRNAs and the interaction with its target gene. But, the study on the regulation of miRNAs to biological processes via regulatory the key signaling pathway was still limited. In order to comprehend the regulatory mechanism of miRNAs, two small RNA libraries from the spleen of miiuy croaker individuals with or without poly(I:C) infection were constructed. The 197 conserved miRNAs and 75 novel miRNAs were identified, and 14 conserved and 8 novel miRNAs appeared significant variations. Those differently expressed miRNAs relate to immune regulation of miiuy croaker. Furthermore, expressions of four differently expressed miRNAs were validated by qRT-PCR, and the result was consistent with sequencing data. The target genes of the differently expressed miRNAs in the two libraries were predicted, and some candidate target genes were involved in the RIG-I-like receptor (RLR) signaling pathway. The negative regulation of miRNAs to target genes were confirmed by comparing the expression pattern of miRNAs and their target genes. The results of regulating target genes were that firstly directly or indirectly activating the downstream signaling cascades and subsequent inducting the type I interferon, inflammatory cytokines and apoptosis. These studies could help us to deeper understand the roles of miRNAs played in the fish immune system, and provide a new way to investigate the defense mechanism of fish. Copyright © 2016 Elsevier Ltd. All rights reserved.

MicroRNA Expression Profile in Penile Cancer Revealed by Next-Generation Small RNA Sequencing

PubMed Central

Zhang, Yuanwei; Xu, Bo; Zhou, Jun; Fan, Song; Hao, Zongyao; Shi, Haoqiang; Zhang, Xiansheng; Kong, Rui; Xu, Lingfan; Gao, Jingjing; Zou, Duohong; Liang, Chaozhao

2015-01-01

Penile cancer (PeCa) is a relatively rare tumor entity but possesses higher morbidity and mortality rates especially in developing countries. To date, the concrete pathogenic signaling pathways and core machineries involved in tumorigenesis and progression of PeCa remain to be elucidated. Several studies suggested miRNAs, which modulate gene expression at posttranscriptional level, were frequently mis-regulated and aberrantly expressed in human cancers. However, the miRNA profile in human PeCa has not been reported before. In this present study, the miRNA profile was obtained from 10 fresh penile cancerous tissues and matched adjacent non-cancerous tissues via next-generation sequencing. As a result, a total of 751 and 806 annotated miRNAs were identified in normal and cancerous penile tissues, respectively. Among which, 56 miRNAs with significantly different expression levels between paired tissues were identified. Subsequently, several annotated miRNAs were selected randomly and validated using quantitative real-time PCR. Compared with the previous publications regarding to the altered miRNAs expression in various cancers and especially genitourinary (prostate, bladder, kidney, testis) cancers, the most majority of deregulated miRNAs showed the similar expression pattern in penile cancer. Moreover, the bioinformatics analyses suggested that the putative target genes of differentially expressed miRNAs between cancerous and matched normal penile tissues were tightly associated with cell junction, proliferation, growth as well as genomic instability and so on, by modulating Wnt, MAPK, p53, PI3K-Akt, Notch and TGF-β signaling pathways, which were all well-established to participate in cancer initiation and progression. Our work presents a global view of the differentially expressed miRNAs and potentially regulatory networks of their target genes for clarifying the pathogenic transformation of normal penis to PeCa, which research resource also provides new insights into future investigations aimed to explore the in-depth mechanisms of miRNAs and other small RNAs including piRNAs in penile carcinogenesis regulation and effective target-specific theragnosis. PMID:26158897

Differential expression of miRNAs in the nervous system of a rat model of bilateral sciatic nerve chronic constriction injury.

PubMed

Li, Haixia; Shen, Le; Ma, Chao; Huang, Yuguang

2013-07-01

Chronic neuropathic pain is associated with global changes in gene expression in different areas of the nociceptive pathway. MicroRNAs (miRNAs) are small (~22 nt long) non-coding RNAs, which are able to regulate hundreds of different genes post-transcriptionally. The aim of this study was to determine the miRNA expression patterns in the different regions of the pain transmission pathway using a rat model of human neuropathic pain induced by bilateral sciatic nerve chronic constriction injury (bCCI). Using microarray analysis and quantitative reverse transcriptase-PCR, we observed a significant upregulation in miR-341 expression in the dorsal root ganglion (DRG), but not in the spinal dorsal horn (SDH), hippocampus or anterior cingulate cortex (ACC), in the rats with neuropathic pain compared to rats in the naïve and sham-operated groups. By contrast, the expression of miR-203, miR-181a-1* and miR-541* was significantly reduced in the SDH of rats with neuropathic pain. Our data indicate that miR-341 is upregulated in the DRG, whereas miR-203, miR-181a-1* and miR-541* are downregulated in the SDH under neuropathic pain conditions. Thus, the differential expression of miRNAs in the nervous system may play a role in the development of chronic pain. These observations may aid in the development of novel treatment methods for neuropathic pain, which may involve miRNA gene therapy in local regions.

Preparation of highly multiplexed small RNA sequencing libraries.

PubMed

Persson, Helena; Søkilde, Rolf; Pirona, Anna Chiara; Rovira, Carlos

2017-08-01

MicroRNAs (miRNAs) are ~22-nucleotide-long small non-coding RNAs that regulate the expression of protein-coding genes by base pairing to partially complementary target sites, preferentially located in the 3´ untranslated region (UTR) of target mRNAs. The expression and function of miRNAs have been extensively studied in human disease, as well as the possibility of using these molecules as biomarkers for prognostication and treatment guidance. To identify and validate miRNAs as biomarkers, their expression must be screened in large collections of patient samples. Here, we develop a scalable protocol for the rapid and economical preparation of a large number of small RNA sequencing libraries using dual indexing for multiplexing. Combined with the use of off-the-shelf reagents, more samples can be sequenced simultaneously on large-scale sequencing platforms at a considerably lower cost per sample. Sample preparation is simplified by pooling libraries prior to gel purification, which allows for the selection of a narrow size range while minimizing sample variation. A comparison with publicly available data from benchmarking of miRNA analysis platforms showed that this method captures absolute and differential expression as effectively as commercially available alternatives.

Combination of miRNA499 and miRNA133 Exerts a Synergic Effect on Cardiac Differentiation

PubMed Central

Pisano, Federica; Altomare, Claudia; Cervio, Elisabetta; Barile, Lucio; Rocchetti, Marcella; Ciuffreda, Maria Chiara; Malpasso, Giuseppe; Copes, Francesco; Mura, Manuela; Danieli, Patrizia; Viarengo, Gianluca; Zaza, Antonio; Gnecchi, Massimiliano

2015-01-01

Several studies have demonstrated that miRNA are involved in cardiac development, stem cell maintenance, and differentiation. In particular, it has been shown that miRNA133, miRNA1, and miRNA499 are involved in progenitor cell differentiation into cardiomyocytes. However, it is unknown whether different miRNA may act synergistically to improve cardiac differentiation. We used mouse P19 cells as a cardiogenic differentiation model. miRNA499, miRNA1, or miRNA133 were transiently over-expressed in P19 cells individually or in different combinations. The over-expression of miRNA499 alone increased the number of beating cells and the association of miRNA499 with miRNA133 exerted a synergistic effect, further increasing the number of beating cells. Real-time polymerase chain reaction showed that the combination of miRNA499 + 133 enhanced the expression of cardiac genes compared with controls. Western blot and immunocytochemistry for connexin43 and cardiac troponin T confirmed these findings. Importantly, caffeine responsiveness, a clear functional parameter of cardiac differentiation, was increased by miRNA499 in association with miRNA133 and was directly correlated with the activation of the cardiac troponin I isoform promoter. Cyclic contractions were reversibly abolished by extracellular calcium depletion, nifedipine, ryanodine, and IP3R blockade. Finally, we demonstrated that the use of miRNA499 + 133 induced cardiac differentiation even in the absence of dimethyl sulfoxide. Our results show that the areas spontaneously contracting possess electrophysiological and pharmacological characteristics compatible with true cardiac excitation-contraction coupling. The translational relevance of our findings was reinforced by the demonstration that the over-expression of miRNA499 and miRNA133 was also able to induce the differentiation of human mesenchymal stromal cells toward the cardiac lineage. Stem Cells 2015;33:1187–1199 PMID:25534971

Potential functions of microRNAs in starch metabolism and development revealed by miRNA transcriptome profiling of cassava cultivars and their wild progenitor.

PubMed

Chen, Xin; Xia, Jing; Xia, Zhiqiang; Zhang, Hefang; Zeng, Changying; Lu, Cheng; Zhang, Weixiong; Wang, Wenquan

2015-02-04

MicroRNAs (miRNAs) are small (approximately 21 nucleotide) non-coding RNAs that are key post-transcriptional gene regulators in eukaryotic organisms. More than 100 cassava miRNAs have been identified in a conservation analysis and a repertoire of cassava miRNAs have also been characterised by next-generation sequencing (NGS) in recent studies. Here, using NGS, we profiled small non-coding RNAs and mRNA genes in two cassava cultivars and their wild progenitor to identify and characterise miRNAs that are potentially involved in plant growth and starch biosynthesis. Six small RNA and six mRNA libraries from leaves and roots of the two cultivars, KU50 and Arg7, and their wild progenitor, W14, were subjected to NGS. Analysis of the sequencing data revealed 29 conserved miRNA families and 33 new miRNA families. Together, these miRNAs potentially targeted a total of 360 putative target genes. Whereas 16 miRNA families were highly expressed in cultivar leaves, another 13 miRNA families were highly expressed in storage roots of cultivars. Co-expression analysis revealed that the expression level of some targets had negative relationship with their corresponding miRNAs in storage roots and leaves; these targets included MYB33, ARF10, GRF1, RD19, APL2, NF-YA3 and SPL2, which are known to be involved in plant development, starch biosynthesis and response to environmental stimuli. The identified miRNAs, target mRNAs and target gene ontology annotation all shed light on the possible functions of miRNAs in Manihot species. The differential expression of miRNAs between cultivars and their wild progenitor, together with our analysis of GO annotation and confirmation of miRNA: target pairs, might provide insight into know the differences between wild progenitor and cultivated cassava.

High-throughput sequencing of small RNAs and analysis of differentially expressed microRNAs associated with pistil development in Japanese apricot

PubMed Central

2012-01-01

Background MicroRNAs (miRNAs) are a class of endogenous, small, non-coding RNAs that regulate gene expression by mediating gene silencing at transcriptional and post-transcriptional levels in high plants. However, the diversity of miRNAs and their roles in floral development in Japanese apricot (Prunus mume Sieb. et Zucc) remains largely unexplored. Imperfect flowers with pistil abortion seriously decrease production yields. To understand the role of miRNAs in pistil development, pistil development-related miRNAs were identified by Solexa sequencing in Japanese apricot. Results Solexa sequencing was used to identify and quantitatively profile small RNAs from perfect and imperfect flower buds of Japanese apricot. A total of 22,561,972 and 24,952,690 reads were sequenced from two small RNA libraries constructed from perfect and imperfect flower buds, respectively. Sixty-one known miRNAs, belonging to 24 families, were identified. Comparative profiling revealed that seven known miRNAs exhibited significant differential expression between perfect and imperfect flower buds. A total of 61 potentially novel miRNAs/new members of known miRNA families were also identified by the presence of mature miRNAs and corresponding miRNA*s in the sRNA libraries. Comparative analysis showed that six potentially novel miRNAs were differentially expressed between perfect and imperfect flower buds. Target predictions of the 13 differentially expressed miRNAs resulted in 212 target genes. Gene ontology (GO) annotation revealed that high-ranking miRNA target genes are those implicated in the developmental process, the regulation of transcription and response to stress. Conclusions This study represents the first comparative identification of miRNAomes between perfect and imperfect Japanese apricot flowers. Seven known miRNAs and six potentially novel miRNAs associated with pistil development were identified, using high-throughput sequencing of small RNAs. The findings, both computationally and experimentally, provide valuable information for further functional characterisation of miRNAs associated with pistil development in plants. PMID:22863067

The expanding roles of microRNAs in kidney pathophysiology.

PubMed

Metzinger-Le Meuth, Valérie; Fourdinier, Ophélie; Charnaux, Nathalie; Massy, Ziad A; Metzinger, Laurent

2018-05-25

MicroRNAs (miRNAs) are short single-stranded RNAs that control gene expression through base pairing with regions within the 3'-untranslated region of target mRNAs. These small non-coding RNAs are now increasingly known to be involved in kidney physiopathology. In this review we will describe how miRNAs were in recent years implicated in cellular and animal models of kidney disease but also in chronic kidney disease, haemodialysed and grafted patients, acute kidney injury patients and so on. At the moment miRNAs are considered as potential biomarkers in nephrology, but larger cohorts as well as the standardization of methods of measurement will be needed to confirm their usefulness. It will further be of the utmost importance to select specific tissues and biofluids to make miRNAs appropriate in day-to-day clinical practice. In addition, up- or down-regulating miRNAs that were described as deregulated in kidney diseases may represent innovative therapeutic methods to cure these disorders. We will enumerate in this review the most recent methods that can be used to deliver miRNAs in a specific and suitable way in kidney and other organs damaged by kidney failure, such as the cardiovascular system.

Plant polycistronic precursors containing non-homologous microRNAs target transcripts encoding functionally related proteins

PubMed Central

2009-01-01

Background MicroRNAs (miRNAs) are endogenous single-stranded small RNAs that regulate the expression of specific mRNAs involved in diverse biological processes. In plants, miRNAs are generally encoded as a single species in independent transcriptional units, referred to as MIRNA genes, in contrast to animal miRNAs, which are frequently clustered. Results We performed a comparative genomic analysis in three model plants (rice, poplar and Arabidopsis) and characterized miRNA clusters containing two to eight miRNA species. These clusters usually encode miRNAs of the same family and certain share a common evolutionary origin across monocot and dicot lineages. In addition, we identified miRNA clusters harboring miRNAs with unrelated sequences that are usually not evolutionarily conserved. Strikingly, non-homologous miRNAs from the same cluster were predicted to target transcripts encoding related proteins. At least four Arabidopsis non-homologous clusters were expressed as single transcriptional units. Overexpression of one of these polycistronic precursors, producing Ath-miR859 and Ath-miR774, led to the DCL1-dependent accumulation of both miRNAs and down-regulation of their different mRNA targets encoding F-box proteins. Conclusions In addition to polycistronic precursors carrying related miRNAs, plants also contain precursors allowing coordinated expression of non-homologous miRNAs to co-regulate functionally related target transcripts. This mechanism paves the way for using polycistronic MIRNA precursors as a new molecular tool for plant biologists to simultaneously control the expression of different genes. PMID:19951405

Regulation of Isoflavone Biosynthesis by miRNAs in Two Contrasting Soybean Genotypes at Different Seed Developmental Stages.

PubMed

Gupta, Om P; Nigam, Deepti; Dahuja, Anil; Kumar, Sanjeev; Vinutha, T; Sachdev, Archana; Praveen, Shelly

2017-01-01

Owing to the presence of nutritionally important, health-promoting bioactive compounds, especially isoflavones, soybean has acquired the status of a functional food. miRNAs are tiny riboregulator of gene expression by either decreasing and/or increasing the expression of their corresponding target genes. Despite several works on identification and functional characterization of plant miRNAs, the role of miRNAs in the regulation of isoflavones metabolism is still a virgin field. In the present study, we identified a total of 31 new miRNAs along with their 245 putative target genes from soybean seed-specific ESTs using computational approach. The Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that miRNA putatively regulates metabolism and genetic information processing. Out of that, a total of 5 miRNAs ( Gma -miRNA12, Gma -miRNA24, Gma -miRNA26, Gma -miRNA28, and Gma -miRNA29) were predicted and validated for their probable role during isoflavone biosynthesis. We also validated their five target genes using RA-PCR, which is as good as 5'RLM-RACE. Temporal regulation [35 days after flowering, 45, 55, and 65 DAF] of miRNAs and their targets showed differential expression schema. Differential expression of Gma -miR26 and Gma -miRNA28 along with their corresponding target genes ( Glyma.10G197900 and Glyma.09G127200 ) showed a direct relationship with the total isoflavone content. Therefore, understanding the miRNA-based genetic regulation of isoflavone pathway would assist in selection and manipulation to get high-performing soybean genotypes with better isoflavone yield.

MicroRNA Expression-Based Model Indicates Event-Free Survival in Pediatric Acute Myeloid Leukemia

PubMed Central

Lim, Emilia L.; Trinh, Diane L.; Ries, Rhonda E.; Wang, Jim; Gerbing, Robert B.; Ma, Yussanne; Topham, James; Hughes, Maya; Pleasance, Erin; Mungall, Andrew J.; Moore, Richard; Zhao, Yongjun; Aplenc, Richard; Sung, Lillian; Kolb, E. Anders; Gamis, Alan; Smith, Malcolm; Gerhard, Daniela S.; Alonzo, Todd A.; Meshinchi, Soheil; Marra, Marco A.

2017-01-01

Purpose Children with acute myeloid leukemia (AML) whose disease is refractory to standard induction chemotherapy therapy or who experience relapse after initial response have dismal outcomes. We sought to comprehensively profile pediatric AML microRNA (miRNA) samples to identify dysregulated genes and assess the utility of miRNAs for improved outcome prediction. Patients and Methods To identify miRNA biomarkers that are associated with treatment failure, we performed a comprehensive sequence-based characterization of the pediatric AML miRNA landscape. miRNA sequencing was performed on 1,362 samples—1,303 primary, 22 refractory, and 37 relapse samples. One hundred sixty-four matched samples—127 primary and 37 relapse samples—were analyzed by using RNA sequencing. Results By using penalized lasso Cox proportional hazards regression, we identified 36 miRNAs the expression levels at diagnosis of which were highly associated with event-free survival. Combined expression of the 36 miRNAs was used to create a novel miRNA-based risk classification scheme (AMLmiR36). This new miRNA-based risk classifier identifies those patients who are at high risk (hazard ratio, 2.830; P ≤ .001) or low risk (hazard ratio, 0.323; P ≤ .001) of experiencing treatment failure, independent of conventional karyotype or mutation status. The performance of AMLmiR36 was independently assessed by using 878 patients from two different clinical trials (AAML0531 and AAML1031). Our analysis also revealed that miR-106a-363 was abundantly expressed in relapse and refractory samples, and several candidate targets of miR-106a-5p were involved in oxidative phosphorylation, a process that is suppressed in treatment-resistant leukemic cells. Conclusion To assess the utility of miRNAs for outcome prediction in patients with pediatric AML, we designed and validated a miRNA-based risk classification scheme. We also hypothesized that the abundant expression of miR-106a could increase treatment resistance via modulation of genes that are involved in oxidative phosphorylation. PMID:29068783

Comparative analysis of miRNA expression during the development of insects of different metamorphosis modes and germ-band types.

PubMed

Ylla, Guillem; Piulachs, Maria-Dolors; Belles, Xavier

2017-10-11

Do miRNAs contribute to specify the germ-band type and the body structure in the insect embryo? Our goal was to address that issue by studying the changes in miRNA expression along the ontogeny of the German cockroach Blattella germanica, which is a short germ-band and hemimetabolan species. We sequenced small RNA libraries representing 11 developmental stages of B. germanica ontogeny (with especial emphasis on embryogenesis) and the changes in miRNA expression were examined. Data were compared with equivalent data for two long germ-band holometabolan species Drosophila melanogaster and Drosophila virilis, and the short germ-band holometabolan species Tribolium castaneum. The identification of B. germanica embryo small RNA sequences unveiled miRNAs not detected in previous studies, such as those of the MIR-309 family and 54 novel miRNAs. Four main waves of miRNA expression were recognized (with most miRNA changes occurring during the embryonic stages): the first from day 0 to day 1 of embryogenesis, the second during mid-embryogenesis (days 0-6), the third (with an acute expression peak) on day 2 of embryonic development, and the fourth during post-embryonic development. The second wave defined the boundaries of maternal-to-zygotic transition, with maternal mRNAs being cleared, presumably by Mir-309 and associated scavenger miRNAs. miRNAs follow well-defined patterns of expression over hemimetabolan ontogeny, patterns that are more diverse during embryonic development than during the nymphal stages. The results suggest that miRNAs play important roles in the developmental transitions between the embryonic stages of development (starting with maternal loading), during which they might influence the germ-band type and metamorphosis mode.

Effector and regulatory dendritic cells display distinct patterns of miRNA expression.

PubMed

Lombardi, Vincent; Luce, Sonia; Moussu, Hélène; Morizur, Lise; Gueguen, Claire; Neukirch, Catherine; Chollet-Martin, Sylvie; Mascarell, Laurent; Aubier, Michel; Baron-Bodo, Véronique; Moingeon, Philippe

2017-09-01

MicroRNAs (miRNAs) contribute to the regulation of dendritic cell (DC) polarization, thereby influencing the balance of adaptive immune responses. Herein, we studied the expression of miRNAs in polarized DCs and analyzed whether expression of these miRNAs could be associated with allergic rhinitis and allergen immunotherapy (AIT) outcome. Using specific culture conditions, we differentiated immature human monocyte-derived DCs into DC1, DC2, and DCreg subsets (supporting the differentiation of T H 1, T H 2 or regulatory T cells, respectively). Profiling of miRNA expression was performed in these DC subpopulations using microarrays. Levels of miRNAs specific for polarized DCs were then evaluated in a cohort of 58 patients with allergic rhinitis and 25 non-allergic controls, as well as in samples from 30 subjects treated with sublingual grass pollen tablets or placebo for four months. We successfully identified 16 miRNAs differentially regulated between immature DCs, DC1, DC2, and DCreg cells. In allergic rhinoconjunctivitis patients, the expression of two of those miRNAs (miR-132 and miR-155), was down-regulated compared to non-allergic individuals. However, the levels of these miRNAs were not significantly modified following four months of grass pollen immunotherapy. Studying polarized DCs and clinical samples from subjects with or without allergic rhinoconjunctivitis, we demonstrated that the expression of two miRNAs linked to effector DCs (i.e., DC1 and/or DC2 cells), was reduced in the blood of patients with allergic rhinoconjunctivitis. Nevertheless, these miRNAs did not represent relevant biomarkers to predict or follow-up AIT efficacy. © 2017 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.

Altered expression of four miRNA (miR-1238-3p, miR-202-3p, miR-630 and miR-766-3p) and their potential targets in peripheral blood from vitiligo patients.

PubMed

Shang, Zhiwei; Li, Hongwen

2017-10-01

Vitiligo is an acquired skin disease with pigmentary disorder. Autoimmune destruction of melanocytes is thought to be major factor in the etiology of vitiligo. miRNA-based regulators of gene expression have been reported to play crucial roles in autoimmune disease. Therefore, we attempt to profile the miRNA expressions and predict their potential targets, assessing the biological functions of differentially expressed miRNA. Total RNA was extracted from peripheral blood of vitiligo (experimental group, n = 5) and non-vitiligo (control group, n = 5) age-matched patients. Samples were hybridized to a miRNA array. Box, scatter and principal component analysis plots were performed, followed by unsupervised hierarchical clustering analysis to classify the samples. Quantitative reverse transcription polymerase chain reaction (RT-PCR) was conducted for validation of microarray data. Three different databases, TargetScan, PITA and microRNA.org, were used to predict the potential target genes. Gene ontology (GO) annotation and pathway analysis were performed to assess the potential functions of predicted genes of identified miRNA. A total of 100 (29 upregulated and 71 downregulated) miRNA were filtered by volcano plot analysis. Four miRNA were validated by quantitative RT-PCR as significantly downregulated in the vitiligo group. The functions of predicted target genes associated with differentially expressed miRNA were assessed by GO analysis, showing that the GO term with most significantly enriched target genes was axon guidance, and that the axon guidance pathway was most significantly correlated with these miRNA. In conclusion, we identified four downregulated miRNA in vitiligo and assessed the potential functions of target genes related to these differentially expressed miRNA. © 2017 Japanese Dermatological Association.

A novel polydopamine-based chemiluminescence resonance energy transfer method for microRNA detection coupling duplex-specific nuclease-aided target recycling strategy.

PubMed

Wang, Qian; Yin, Bin-Cheng; Ye, Bang-Ce

2016-06-15

MicroRNAs (miRNAs), functioning as oncogenes or tumor suppressors, play significant regulatory roles in regulating gene expression and become as biomarkers for disease diagnostics and therapeutics. In this work, we have coupled a polydopamine (PDA) nanosphere-assisted chemiluminescence resonance energy transfer (CRET) platform and a duplex-specific nuclease (DSN)-assisted signal amplification strategy to develop a novel method for specific miRNA detection. With the assistance of hemin, luminol, and H2O2, the horseradish peroxidase (HRP)-mimicking G-rich sequence in the sensing probe produces chemiluminescence, which is quickly quenched by the CRET effect between PDA as energy acceptor and excited luminol as energy donor. The target miRNA triggers DSN to partially degrade the sensing probe in the DNA-miRNA heteroduplex to repeatedly release G-quadruplex formed by G-rich sequence from PDA for the production of chemiluminescence. The method allows quantitative detection of target miRNA in the range of 80 pM-50 nM with a detection limit of 49.6 pM. The method also shows excellent specificity to discriminate single-base differences, and can accurately quantify miRNA in biological samples, with good agreement with the result from a commercial miRNA detection kit. The procedure requires no organic dyes or labels, and is a simple and cost-effective method for miRNA detection for early clinical diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

A tripartite clustering analysis on microRNA, gene and disease model.

PubMed

Shen, Chengcheng; Liu, Ying

2012-02-01

Alteration of gene expression in response to regulatory molecules or mutations could lead to different diseases. MicroRNAs (miRNAs) have been discovered to be involved in regulation of gene expression and a wide variety of diseases. In a tripartite biological network of human miRNAs, their predicted target genes and the diseases caused by altered expressions of these genes, valuable knowledge about the pathogenicity of miRNAs, involved genes and related disease classes can be revealed by co-clustering miRNAs, target genes and diseases simultaneously. Tripartite co-clustering can lead to more informative results than traditional co-clustering with only two kinds of members and pass the hidden relational information along the relation chain by considering multi-type members. Here we report a spectral co-clustering algorithm for k-partite graph to find clusters with heterogeneous members. We use the method to explore the potential relationships among miRNAs, genes and diseases. The clusters obtained from the algorithm have significantly higher density than randomly selected clusters, which means members in the same cluster are more likely to have common connections. Results also show that miRNAs in the same family based on the hairpin sequences tend to belong to the same cluster. We also validate the clustering results by checking the correlation of enriched gene functions and disease classes in the same cluster. Finally, widely studied miR-17-92 and its paralogs are analyzed as a case study to reveal that genes and diseases co-clustered with the miRNAs are in accordance with current research findings.

Inflammation-related microRNA expression level in the bovine milk is affected by mastitis.

PubMed

Lai, Yu-Chang; Fujikawa, Takuro; Maemura, Tadashi; Ando, Takaaki; Kitahara, Go; Endo, Yasuyuki; Yamato, Osamu; Koiwa, Masateru; Kubota, Chikara; Miura, Naoki

2017-01-01

MicroRNA (miRNA) in tissue and liquid samples have been shown to be associated with many diseases including inflammation. We aimed to identify inflammation-related miRNA expression level in the bovine mastitis milk. Expression level of inflammation-related miRNA in milk from mastitis-affected and normal cows was analyzed using qPCR. We found that expression level of miR-21, miR-146a, miR-155, miR-222, and miR-383 was significantly upregulated in California mastitis test positive (CMT+) milk. We further analyzed these miRNA using a chip-based QuantStudio Digital PCR System. The digital PCR results correlated with those of qPCR, demonstrating upregulation of miR-21, miR-146a, miR-155, miR-222, and miR-383 in CMT+ milk. In conclusion, we identified miRNA that are upregulated in CMT+ milk. These miRNA exhibited sensitivity and specificity greater than 80% for differentiating between CMT+ milk and normal milk. Our findings suggest that inflammation-related miRNA expression level in the bovine milk was affected by mastitis, and miRNA in milk have potential for use as biomarkers of bovine mastitis.

Characterization and expression patterns of small RNAs in synthesized Brassica hexaploids.

PubMed

Shen, Yanyue; Zhao, Qin; Zou, Jun; Wang, Wenliang; Gao, Yi; Meng, Jinling; Wang, Jianbo

2014-06-01

Polyploidy has played an important role in promoting plant evolution through genomic merging and doubling. We used high-throughput sequencing to compare miRNA expression profiles between Brassica hexaploid and its parents. A total of 613, 784 and 742 known miRNAs were identified in Brassica rapa, Brassica carinata, and Brassica hexaploid, respectively. We detected 618 miRNAs were differentially expressed (log(2)Ratio ≥ 1, P ≤ 0.05) between Brassica hexaploid and its parents, and 425 miRNAs were non-additively expressed in Brassica hexaploid, which suggest a trend of non-additive miRNA regulation following hybridization and polyploidization. Remarkably, majority of the non-additively expressed miRNAs in the Brassica hexaploid are repressed, and there was a bias toward repression of B. rapa miRNAs, which is consistent with the progenitor-biased gene repression in the synthetic allopolyploids. In addition, we identified 653 novel mature miRNAs in Brassica hexaploid and its parents. Finally, we found that almost all the non-additive accumulation of siRNA clusters exhibited a low-parent pattern in Brassica hexaploid. Non-additive small RNA regulation is involved in a range of biological pathways, probably providing a driving force for variation and adaptation in allopolyploids.

The Small-RNA Profiles of Almond (Prunus dulcis Mill.) Reproductive Tissues in Response to Cold Stress.

PubMed

Karimi, Marzieh; Ghazanfari, Farahnaz; Fadaei, Adeleh; Ahmadi, Laleh; Shiran, Behrouz; Rabei, Mohammad; Fallahi, Hossein

2016-01-01

Spring frost is an important environmental stress that threatens the production of Prunus trees. However, little information is available regarding molecular response of these plants to the frost stress. Using high throughput sequencing, this study was conducted to identify differentially expressed miRNAs, both the conserved and the non-conserved ones, in the reproductive tissues of almond tolerant H genotype under cold stress. Analysis of 50 to 58 million raw reads led to identification of 174 unique conserved and 59 novel microRNAs (miRNAs). Differential expression pattern analysis showed that 50 miRNA families were expressed differentially in one or both of almond reproductive tissues (anther and ovary). Out of these 50 miRNA families, 12 and 15 displayed up-regulation and down-regulation, respectively. The distribution of conserved miRNA families indicated that miR482f harbor the highest number of members. Confirmation of miRNAs expression patterns by quantitative real- time PCR (qPCR) was performed in cold tolerant (H genotype) alongside a sensitive variety (Sh12 genotype). Our analysis revealed differential expression for 9 miRNAs in anther and 3 miRNAs in ovary between these two varieties. Target prediction of miRNAs followed by differential expression analysis resulted in identification of 83 target genes, mostly transcription factors. This study comprehensively catalogued expressed miRNAs under different temperatures in two reproductive tissues (anther and ovary). Results of current study and the previous RNA-seq study, which was conducted in the same tissues by our group, provide a unique opportunity to understand the molecular basis of responses of almond to cold stress. The results can also enhance the possibility for gene manipulation to develop cold tolerant plants.

Microarray analysis of 6-mercaptopurine-induced-toxicity-related genes and microRNAs in the rat placenta.

PubMed

Taki, Kenji; Fukushima, Tamio; Ise, Ryota; Horii, Ikuo; Yoshida, Takemi

2013-02-01

MicroRNAs (miRNAs) are small single-stranded RNAs of 19-25 nucleotides and are important in posttranscriptional regulation of genes. Recently, the role of miRNAs in toxicity incidence is reported to be a regulator of key-stopper of gene expression, however the detailed mechanism of miRNAs is not well known yet. 6-Mercaptopurine (6-MP), the anti-leukemic and immunosuppressive drug, produced teratogenicity and pregnancy loss. We focused on the placenta to evaluate toxicity in embryo/fetal development produced by 6-MP treatment. MiRNA expression in the placenta was analyzed by miRNA microarray. Fifteen miRNAs were upregulated on GD13 and 5 miRNAs were downregulated on GD15 in 6-MP treatment rat placentas. Some miRNAs may have functions in apoptosis (miR-195, miR-21, miR-29c and miR-34a), inflammation (miR-146b), and ischemia (miR-144 and miR-451). In the maternal plasma, expression of miR-144 was significantly reduced by 6-MP treatment when examined by real-time RT-PCR. We determined toxicity-related gene expression in the rat placenta. Gene expression analysis was carried out by DNA oligo microarray using rat placenta total RNAs. Compared between predicted targets of miRNAs and microarray data in 6-MP-treated rat placenta, expressions of hormone receptor genes (estrogen receptor 1; Esr1, progesterone receptor; Pgr, and prolactin receptor; Prlr), xanthine oxidase (Xdh), Slc38a5 and Phlda2 genes were changed. The histopathologically found increase in trophoblastic giant cells and reduced placental growth by 6-MP treatment were well correlated to these gene expressions. These data suggest that some miRNAs may link to toxicological reactions in 6-MP-induced placental toxicity.

The Small-RNA Profiles of Almond (Prunus dulcis Mill.) Reproductive Tissues in Response to Cold Stress

PubMed Central

Shiran, Behrouz; Rabei, Mohammad; Fallahi, Hossein

2016-01-01

Spring frost is an important environmental stress that threatens the production of Prunus trees. However, little information is available regarding molecular response of these plants to the frost stress. Using high throughput sequencing, this study was conducted to identify differentially expressed miRNAs, both the conserved and the non-conserved ones, in the reproductive tissues of almond tolerant H genotype under cold stress. Analysis of 50 to 58 million raw reads led to identification of 174 unique conserved and 59 novel microRNAs (miRNAs). Differential expression pattern analysis showed that 50 miRNA families were expressed differentially in one or both of almond reproductive tissues (anther and ovary). Out of these 50 miRNA families, 12 and 15 displayed up-regulation and down-regulation, respectively. The distribution of conserved miRNA families indicated that miR482f harbor the highest number of members. Confirmation of miRNAs expression patterns by quantitative real- time PCR (qPCR) was performed in cold tolerant (H genotype) alongside a sensitive variety (Sh12 genotype). Our analysis revealed differential expression for 9 miRNAs in anther and 3 miRNAs in ovary between these two varieties. Target prediction of miRNAs followed by differential expression analysis resulted in identification of 83 target genes, mostly transcription factors. This study comprehensively catalogued expressed miRNAs under different temperatures in two reproductive tissues (anther and ovary). Results of current study and the previous RNA-seq study, which was conducted in the same tissues by our group, provide a unique opportunity to understand the molecular basis of responses of almond to cold stress. The results can also enhance the possibility for gene manipulation to develop cold tolerant plants. PMID:27253370

High-Throughput Sequencing of Small RNA Transcriptomes in Maize Kernel Identifies miRNAs Involved in Embryo and Endosperm Development.

PubMed

Xing, Lijuan; Zhu, Ming; Zhang, Min; Li, Wenzong; Jiang, Haiyang; Zou, Junjie; Wang, Lei; Xu, Miaoyun

2017-12-14

Maize kernel development is a complex biological process that involves the temporal and spatial expression of many genes and fine gene regulation at a transcriptional and post-transcriptional level, and microRNAs (miRNAs) play vital roles during this process. To gain insight into miRNA-mediated regulation of maize kernel development, a deep-sequencing technique was used to investigate the dynamic expression of miRNAs in the embryo and endosperm at three developmental stages in B73. By miRNA transcriptomic analysis, we characterized 132 known miRNAs and six novel miRNAs in developing maize kernel, among which, 15 and 14 miRNAs were commonly differentially expressed between the embryo and endosperm at 9 days after pollination (DAP), 15 DAP and 20 DAP respectively. Conserved miRNA families such as miR159, miR160, miR166, miR390, miR319, miR528 and miR529 were highly expressed in developing embryos; miR164, miR171, miR393 and miR2118 were highly expressed in developing endosperm. Genes targeted by those highly expressed miRNAs were found to be largely related to a regulation category, including the transcription, macromolecule biosynthetic and metabolic process in the embryo as well as the vitamin biosynthetic and metabolic process in the endosperm. Quantitative reverse transcription-PCR (qRT-PCR) analysis showed that these miRNAs displayed a negative correlation with the levels of their corresponding target genes. Importantly, our findings revealed that members of the miR169 family were highly and dynamically expressed in the developing kernel, which will help to exploit new players functioning in maize kernel development.

Arsenic responsive microRNAs in vivo and their potential involvement in arsenic-induced oxidative stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ren, Xuefeng, E-mail: xuefengr@buffalo.edu; Department of Pharmacology and Toxicology, School of Biomedical Sciences, The State University of New York, Buffalo, NY 14214; Gaile, Daniel P.

Arsenic exposure is postulated to modify microRNA (miRNA) expression, leading to changes of gene expression and toxicities, but studies relating the responses of miRNAs to arsenic exposure are lacking, especially with respect to in vivo studies. We utilized high-throughput sequencing technology and generated miRNA expression profiles of liver tissues from Sprague Dawley (SD) rats exposed to various concentrations of sodium arsenite (0, 0.1, 1, 10 and 100 mg/L) for 60 days. Unsupervised hierarchical clustering analysis of the miRNA expression profiles clustered the SD rats into different groups based on the arsenic exposure status, indicating a highly significant association between arsenicmore » exposure and cluster membership (p-value of 0.0012). Multiple miRNA expressions were altered by arsenic in an exposure concentration-dependent manner. Among the identified arsenic-responsive miRNAs, several are predicted to target Nfe2l2-regulated antioxidant genes, including glutamate–cysteine ligase (GCL) catalytic subunit (GCLC) and modifier subunit (GCLM) which are involved in glutathione (GSH) synthesis. Exposure to low concentrations of arsenic increased mRNA expression for Gclc and Gclm, while high concentrations significantly reduced their expression, which were correlated to changes in hepatic GCL activity and GSH level. Moreover, our data suggested that other mechanisms, e.g., miRNAs, rather than Nfe2l2-signaling pathway, could be involved in the regulation of mRNA expression of Gclc and Gclm post-arsenic exposure in vivo. Together, our findings show that arsenic exposure disrupts the genome-wide expression of miRNAs in vivo, which could lead to the biological consequence, such as an altered balance of antioxidant defense and oxidative stress. - Highlights: • Chronic arsenic exposure induces changes of hepatic miRNA expression profiles. • Hepatic GCL activity and GSH level in rats are altered following arsenic exposure. • Arsenic induced GCL expression change is independent to Nfe2l2-signaling pathway. • Expression of several miRNAs predicted to target GCL changed after arsenic exposure.« less

MicroRNA expression profiling in endometriosis-associated infertility and its relationship with endometrial receptivity evaluated by ultrasound.

PubMed

Xu, Xianfeng; Li, Zhenzhou; Liu, Jin; Yu, Sha; Wei, Zhaolian

2017-01-01

To investigate the microRNA expression profiling in endometriosis-associate infertility, and relationship between the microRNA expression and endometrial receptivity evaluated by ultrasound. First, miRNA expression profiling difference of ectopic endometrium between 8 endometriosis patients and 6 endometriosis-free patients were compared. Bioinformatics analyses detected 61 differentially expressed (DE) known miRNAs and 57 DE novel miRNAs. Next, other 24 patients were selected for checking the microRNAs in differential expression by RT-PCR. Among them, case and control groups include 14 endometriosis and 10 endometriosis-free infertility patients, respectively. Last, endometrial receptivity of other 20 endometriosis patients was evaluated by ultrasound. In this group of patients, 12 had high endometrial receptivity, in which infertility is caused by fallopian tube occlusion, and 8 had low endometrial receptivity. The study compared endometrial miRNAs expression between two groups, and also evaluated the relationship between the endometrial miRNAs expression and the endometrial receptivity. First, study indicated that "proteinaceous extracellular matrix," "laminin binding" and "extracellular matrix binding" were enriched in 6 up-regulated miRNA targets, while "cell proliferation" was enriched in the 4 down-regulated miRNA targets. Second, 10 miRNAs in different expression (miR-1304- 3p, miR-544b, miR-3684, miR-494-5p, miR-4683, miR-6747-3p; miR-3935, miR-4427, miR-652-5p, miR-205-5p) were detected by RT-PCR, and the results showed statistically significant differences between 2 groups in all 10 miRNAs. Third, the expression levels of miR-1304-3p, miR-494-5p, and miR-4427 were different between the two groups with different endometrial receptivity. But for the miR-544b, there was no statistically significant difference between two groups. The study provided a comprehensive understanding to the current knowledge in the field of miRNAs in endometriosis and the relationship between them and the endometrial receptivity. miRNAs could be used as diagnostic biomarkers and therapeutic agents for this disease. The combination of ultrasound and miRNAs detection could be a better choice for the diagnosis of infertility in the future.

Differentiation of Human Dental Stem Cells Reveal a Role for microRNA-218

PubMed Central

Gay, Isabel; Cavender, Adriana; Peto, David; Sun, Zhao; Speer, Aline; Cao, Huojun; Amendt, Brad A.

2013-01-01

Background Regeneration of the lost periodontium is the ultimate goal of periodontal therapy. Advances in tissue engineering have demonstrated the multilineage potential and plasticity of adult stem cells located in the periodontal apparatus. However, it remains unclear how epigenetic mechanisms controlling signals determine tissue specification and cell lineage decisions. To date, no data is available on micro-RNAs (miRNAs) activity behind human-derived dental stem cells. Methods In this study, we isolated periodontal ligament stem cells (PDLSCs), dental pulp stem cells (DPSCs), and gingival stem cells (GSCs) from extracted third molars; human bone marrow stem cells (BMSCs) were used as a positive control. The expression of OCT4A and NANOG was confirmed in these undifferentiated cells. All cells were cultured under osteogenic inductive conditions and RUNX2 expression was analyzed as a marker of mineralized tissue differentiation. A miRNA expression profile was obtained at baseline and after osteogenic induction in all cell types. Results RUNX2 expression demonstrated the successful osteogenic induction of all cell types, which was confirmed by alizarin red stain. The analysis of 765 miRNAs demonstrated a shift in miRNA expression occurred in all four stem cell types, including a decrease in hsa-mir-218 across all differentiated cell populations. Hsa-mir-218 targets RUNX2 and decreases RUNX2 expression in undifferentiated human dental stem cells (DSCs). DSC mineralized tissue type differentiation is associated with a decrease in hsa-mir-218 expression. Conclusions These data reveal a miRNA regulated pathway for the differentiation of human DSCs and a select network of human microRNAs that control DSC osteogenic differentiation. PMID:23662917

Identification of microRNAs in Caragana intermedia by high-throughput sequencing and expression analysis of 12 microRNAs and their targets under salt stress.

PubMed

Zhu, Jianfeng; Li, Wanfeng; Yang, Wenhua; Qi, Liwang; Han, Suying

2013-09-01

142 miRNAs were identified and 38 miRNA targets were predicted, 4 of which were validated, in C. intermedia . The expression of 12 miRNAs in salt-stressed leaves was assessed by qRT-PCR. MicroRNAs (miRNAs) are endogenous small RNAs that play important roles in various biological and metabolic processes in plants. Caragana intermedia is an important ecological and economic tree species prominent in the desert environment of west and northwest China. To date, no investigation into C. intermedia miRNAs has been reported. In this study, high-throughput sequencing of small RNAs and analysis of transcriptome data were performed to identify both conserved and novel miRNAs, and also their target mRNA genes in C. intermedia. Based on sequence similarity and hairpin structure prediction, 132 putative conserved miRNAs (12 of which were confirmed to form hairpin precursors) belonging to 31 known miRNA families were identified. Ten novel miRNAs (including the miRNA* sequences of three novel miRNAs) were also discovered. Furthermore, 36 potential target genes of 17 known miRNA families and 2 potential target genes of 1 novel miRNA were predicted; 4 of these were validated by 5' RACE. The expression of 12 miRNAs was validated in different tissues, and these and five target mRNAs were assessed by qRT-PCR after salt treatment. The expression levels of seven miRNAs (cin-miR157a, cin-miR159a, cin-miR165a, cin-miR167b, cin-miR172b, cin-miR390a and cin-miR396a) were upregulated, while cin-miR398a expression was downregulated after salt treatment. The targets of cin-miR157a, cin-miR165a, cin-miR172b and cin-miR396a were downregulated and showed an approximately negative correlation with their corresponding miRNAs under salt treatment. These results would help further understanding of miRNA regulation in response to abiotic stress in C. intermedia.

Identification of microRNAs in the Toxigenic Dinoflagellate Alexandrium catenella by High-Throughput Illumina Sequencing and Bioinformatic Analysis

PubMed Central

Geng, Huili; Sui, Zhenghong; Zhang, Shu; Du, Qingwei; Ren, Yuanyuan; Liu, Yuan; Kong, Fanna; Zhong, Jie; Ma, Qingxia

2015-01-01

Micro-ribonucleic acids (miRNAs) are a large group of endogenous, tiny, non-coding RNAs consisting of 19–25 nucleotides that regulate gene expression at either the transcriptional or post-transcriptional level by mediating gene silencing in eukaryotes. They are considered to be important regulators that affect growth, development, and response to various stresses in plants. Alexandrium catenella is an important marine toxic phytoplankton species that can cause harmful algal blooms (HABs). To date, identification and function analysis of miRNAs in A. catenella remain largely unexamined. In this study, high-throughput sequencing was performed on A. catenella to identify and quantitatively profile the repertoire of small RNAs from two different growth phases. A total of 38,092,056 and 32,969,156 raw reads were obtained from the two small RNA libraries, respectively. In total, 88 mature miRNAs belonging to 32 miRNA families were identified. Significant differences were found in the member number, expression level of various families, and expression abundance of each member within a family. A total of 15 potentially novel miRNAs were identified. Comparative profiling showed that 12 known miRNAs exhibited differential expression between the lag phase and the logarithmic phase. Real-time quantitative RT-PCR (qPCR) was performed to confirm the expression of two differentially expressed miRNAs that were one up-regulated novel miRNA (aca-miR-3p-456915), and one down-regulated conserved miRNA (tae-miR159a). The expression trend of the qPCR assay was generally consistent with the deep sequencing result. Target predictions of the 12 differentially expressed miRNAs resulted in 1813target genes. Gene ontology (GO) analysis and the Kyoto Encyclopedia of Genes and Genomes pathway database (KEGG) annotations revealed that some miRNAs were associated with growth and developmental processes of the alga. These results provide insights into the roles that miRNAs play in the growth of A. catenella, and they provide the basis for further studies of the molecular mechanisms that underlie bloom growth in red tides species. PMID:26398216

Evaluation of the capability of the PCV2 genome to encode miRNAs: lack of viral miRNA expression in an experimental infection.

PubMed

Núñez-Hernández, Fernando; Pérez, Lester J; Vera, Gonzalo; Córdoba, Sarai; Segalés, Joaquim; Sánchez, Armand; Núñez, José I

2015-05-01

Porcine circovirus type 2 (PCV2) is a ssDNA virus causing PCV2-systemic disease (PCV2-SD), one of the most important diseases in swine. MicroRNAs (miRNAs) are a new class of small non-coding RNAs that regulate gene expression post-transcriptionally. Viral miRNAs have recently been described and the number of viral miRNAs has been increasing in the past few years. In this study, small RNA libraries were constructed from two tissues of subclinically PCV2 infected pigs to explore if PCV2 can encode viral miRNAs. The deep sequencing data revealed that PCV2 does not express miRNAs in an in vivo subclinical infection.

[The role of miRNA in endometrial cancer in the context of miRNA 205].

PubMed

Wilczyński, Miłosz; Danielska, Justyna; Dzieniecka, Monika; Malinowski, Andrzej

2015-11-01

MiRNAs are small, non-coding molecules of ribonucleic acids of approximately 22 bp length, which serve as regulators of gene expression and protein translation due to interference with messenger RNA (mRNA). MiRNAs, which take part in the regulation of cell cycle and apoptosis, may be associated with carcinogenesis. Aberrant expression of miRNAs in endometrial cancer might contribute to the endometrial cancer initiation or progression, as well as metastasis formation, and may influence cancer invasiveness. Specific-miRNAs expressed in endometrial cancer tissues may serve as diagnostic markers of the disease, prognostic biomarkers, or play an important part in oncological therapy We aimed to describe the role of miRNAs in endometrial cancer with special consideration of miRNA 205.

Integrated analysis of miRNA and mRNA expression profiles in tilapia gonads at an early stage of sex differentiation.

PubMed

Tao, Wenjing; Sun, Lina; Shi, Hongjuan; Cheng, Yunying; Jiang, Dongneng; Fu, Beide; Conte, Matthew A; Gammerdinger, William J; Kocher, Thomas D; Wang, Deshou

2016-05-04

MicroRNAs (miRNAs) represent a second regulatory network that has important effects on gene expression and protein translation during biological process. However, the possible role of miRNAs in the early stages of fish sex differentiation is not well understood. In this study, we carried an integrated analysis of miRNA and mRNA expression profiles to explore their possibly regulatory patterns at the critical stage of sex differentiation in tilapia. We identified 279 pre-miRNA genes in tilapia genome, which were highly conserved in other fish species. Based on small RNA library sequencing, we identified 635 mature miRNAs in tilapia gonads, in which 62 and 49 miRNAs showed higher expression in XX and XY gonads, respectively. The predicted targets of these sex-biased miRNAs (e.g., miR-9, miR-21, miR-30a, miR-96, miR-200b, miR-212 and miR-7977) included genes encoding key enzymes in steroidogenic pathways (Cyp11a1, Hsd3b, Cyp19a1a, Hsd11b) and key molecules involved in vertebrate sex differentiation (Foxl2, Amh, Star1, Sf1, Dmrt1, and Gsdf). These genes also showed sex-biased expression in tilapia gonads at 5 dah. Some miRNAs (e.g., miR-96 and miR-737) targeted multiple genes involved in steroid synthesis, suggesting a complex miRNA regulatory network during early sex differentiation in this fish. The sequence and expression patterns of most miRNAs in tilapia are conserved in fishes, indicating the basic functions of vertebrate miRNAs might share a common evolutionary origin. This comprehensive analysis of miRNA and mRNA at the early stage of molecular sex differentiation in tilapia XX and XY gonads lead to the discovery of differentially expressed miRNAs and their putative targets, which will facilitate studies of the regulatory network of molecular sex determination and differentiation in fishes.

Regulatory role of miRNAs in polyamine gene expression in the prefrontal cortex of depressed suicide completers.

PubMed

Lopez, Juan Pablo; Fiori, Laura M; Gross, Jeffrey A; Labonte, Benoit; Yerko, Volodymyr; Mechawar, Naguib; Turecki, Gustavo

2014-01-01

MicroRNAs (miRNAs) are small, non-coding RNA molecules that play an important role in the post-transcriptional regulation of mRNA. These molecules have been the subject of growing interest as they are believed to control the regulation of a large number of genes, including those expressed in the brain. Evidence suggests that miRNAs could be involved in the pathogenesis of neuropsychiatric disorders. Alterations in metabolic enzymes of the polyamine system have been reported to play a role in predisposition to suicidal behaviour. We have previously shown the expression of the polyamine genes SAT1 and SMOX to be down-regulated in the brains of suicide completers. In this study, we hypothesized that the dysregulation of these genes in depressed suicide completers could be influenced by miRNA post-transcriptional regulation. Using a stringent target prediction analysis, we identified several miRNAs that target the 3'UTR of SAT1 and SMOX. We profiled the expression of 10 miRNAs in the prefrontal cortex (BA44) of suicide completers (N = 15) and controls (N = 16) using qRT-PCR. We found that several miRNAs showed significant up-regulation in the prefrontal cortex of suicide completers compared to psychiatric healthy controls. Furthermore, we demonstrated a significant correlation between these miRNAs and the expression levels of both SAT1 and SMOX. Our results suggest a relationship between miRNAs and polyamine gene expression in the suicide brain, and postulate a mechanism for SAT1 and SMOX down-regulation by post-transcriptional activity of miRNAs.

Serum miRNAs as Biomarkers for the Diagnosis and Prognosis of Thyroid Cancer: A Comprehensive Review of the Literature.

PubMed

Mahmoudian-Sani, Mohammad-Reza; Mehri-Ghahfarrokhi, Ameneh; Asadi-Samani, Majid; Mobini, Gholam-Reza

2017-07-01

Thyroid cancer is the most common endocrine malignancy and accounts for 1% of cancers. In recent years, there has been much interest in the feasibility of using miRNAs or miRNA panels as biomarkers for the diagnosis of thyroid cancer. miRNAs are noncoding RNAs with 21-23 nucleotides that are highly conserved during evolution. They have been proposed as regulators of gene expression, apoptosis, cancer, and cell growth and differentiation. The Directory of Open Access Journals (DOAJ), Google Scholar, PubMed (NLM), LISTA (EBSCO), and Web of Science were searched. The serum level of miRNAs (miRNA-375, 34a, 145b, 221, 222, 155, Let-7, 181b) can be used as molecular markers for the diagnosis and prognosis of thyroid cancer in the serum samples of patients with thyroid glands. Given that most common methods for the screening of thyroid cancer cannot detect the disease in its early stages, identifying miRNAs that are released in the bloodstream during the gradual progression of the disease is considered a key method in the early diagnosis of thyroid cancers.

Digital quantitative analysis of microRNA in single cell based on ligation-depended polymerase colony (Polony).

PubMed

Wang, Hui; Wang, Honghong; Duan, Xinrui; Liu, Chenghui; Li, Zhengping

2017-09-15

The ability to dissect cell-to-cell variations of microRNA (miRNA) expression with single-cell resolution has become a powerful tool to investigate the regulatory function of miRNAs in biological processes and the pathogenesis of miRNA-related diseases. Herein, we have developed a novel scheme for digital detection of miRNA in single cell by using the ligation-depended DNA polymerase colony (polony). Firstly, two simply designed target-specific DNA probes were ligated by using individual miRNA as the template. Then the ligated DNA probe acted as polony template that was amplified by PCR process in the thin polyacrylamide hydrogel. Due to the covalent attachment of a PCR primer on polyacrylamide matrix and the retarding effect of the polyacrylamide hydrogel matrix itself, as the polony reaction proceeds, the PCR products diffused radially near individual template molecule to form a bacteria colony-like spots of DNA molecules. The spots can be counted after staining the polyacrylamide gel with SYBR Green I and imaging with a microarray scanner. Our polony-based method is sensitive enough to detect 60 copies of miRNA molecules. Meanwhile, the new strategy has the capability of distinguishing singe-base difference. Due to its high sensitivity and specificity, the proposed method has been successfully applied to analysis of the expression profiling of miRNA in single cell. Copyright © 2017 Elsevier B.V. All rights reserved.

Selection of reference genes is critical for miRNA expression analysis in human cardiac tissue. A focus on atrial fibrillation

PubMed Central

Masè, Michela; Grasso, Margherita; Avogaro, Laura; D’Amato, Elvira; Tessarolo, Francesco; Graffigna, Angelo; Denti, Michela Alessandra; Ravelli, Flavia

2017-01-01

MicroRNAs (miRNAs) are emerging as key regulators of complex biological processes in several cardiovascular diseases, including atrial fibrillation (AF). Reverse transcription-quantitative polymerase chain reaction is a powerful technique to quantitatively assess miRNA expression profile, but reliable results depend on proper data normalization by suitable reference genes. Despite the increasing number of studies assessing miRNAs in cardiac disease, no consensus on the best reference genes has been reached. This work aims to assess reference genes stability in human cardiac tissue with a focus on AF investigation. We evaluated the stability of five reference genes (U6, SNORD48, SNORD44, miR-16, and 5S) in atrial tissue samples from eighteen cardiac-surgery patients in sinus rhythm and AF. Stability was quantified by combining BestKeeper, delta-Cq, GeNorm, and NormFinder statistical tools. All methods assessed SNORD48 as the best and U6 as the worst reference gene. Applications of different normalization strategies significantly impacted miRNA expression profiles in the study population. Our results point out the necessity of a consensus on data normalization in AF studies to avoid the emergence of divergent biological conclusions. PMID:28117343

MicroRNAome genome: a treasure for cancer diagnosis and therapy

PubMed Central

Berindan-Neagoe, Ioana; Monroig, Paloma; Pasculli, Barbara; Calin, George A.

2015-01-01

The interplay between abnormalities in genes coding for proteins and microRNAs (miRNAs) has been among the most exiting yet unexpected discoveries in oncology over the last decade. The complexity of this network has redefined cancer research as these molecules produced from what was once considered “genomic trash”, have shown to be crucial for cancer initiation, progression, and dissemination. Naturally occurring miRNAs are very short transcripts that never produce a protein or amino acid chain, but act by regulating protein expression during cellular processes such as growth, development and differentiation at the transcriptional, post-transcriptional and/or translational level. In this review article we present miRNAs as ubiquitous players involved in all cancer hallmarks. We also describe the most used methods to detect their expression, which have revealed through gene expression studies the identity of hundreds of miRNAs dysregulated in cancer cells or tumor microenvironment cells. Furthermore, we discuss the role of miRNAs as hormones and as reliable cancer biomarkers and predictors of treatment-response. Along with this, we explore current strategies in designing miRNA-targeting therapeutics, as well as the associated challenges that research envisions to overcome. Finally, we introduce a new wave in molecular oncology translational research, the study of long non-coding RNAs. PMID:25104502

Ensemble Methods for MiRNA Target Prediction from Expression Data.

PubMed

Le, Thuc Duy; Zhang, Junpeng; Liu, Lin; Li, Jiuyong

2015-01-01

microRNAs (miRNAs) are short regulatory RNAs that are involved in several diseases, including cancers. Identifying miRNA functions is very important in understanding disease mechanisms and determining the efficacy of drugs. An increasing number of computational methods have been developed to explore miRNA functions by inferring the miRNA-mRNA regulatory relationships from data. Each of the methods is developed based on some assumptions and constraints, for instance, assuming linear relationships between variables. For such reasons, computational methods are often subject to the problem of inconsistent performance across different datasets. On the other hand, ensemble methods integrate the results from individual methods and have been proved to outperform each of their individual component methods in theory. In this paper, we investigate the performance of some ensemble methods over the commonly used miRNA target prediction methods. We apply eight different popular miRNA target prediction methods to three cancer datasets, and compare their performance with the ensemble methods which integrate the results from each combination of the individual methods. The validation results using experimentally confirmed databases show that the results of the ensemble methods complement those obtained by the individual methods and the ensemble methods perform better than the individual methods across different datasets. The ensemble method, Pearson+IDA+Lasso, which combines methods in different approaches, including a correlation method, a causal inference method, and a regression method, is the best performed ensemble method in this study. Further analysis of the results of this ensemble method shows that the ensemble method can obtain more targets which could not be found by any of the single methods, and the discovered targets are more statistically significant and functionally enriched. The source codes, datasets, miRNA target predictions by all methods, and the ground truth for validation are available in the Supplementary materials.

Ensemble Methods for MiRNA Target Prediction from Expression Data

PubMed Central

Le, Thuc Duy; Zhang, Junpeng; Liu, Lin; Li, Jiuyong

2015-01-01

Background microRNAs (miRNAs) are short regulatory RNAs that are involved in several diseases, including cancers. Identifying miRNA functions is very important in understanding disease mechanisms and determining the efficacy of drugs. An increasing number of computational methods have been developed to explore miRNA functions by inferring the miRNA-mRNA regulatory relationships from data. Each of the methods is developed based on some assumptions and constraints, for instance, assuming linear relationships between variables. For such reasons, computational methods are often subject to the problem of inconsistent performance across different datasets. On the other hand, ensemble methods integrate the results from individual methods and have been proved to outperform each of their individual component methods in theory. Results In this paper, we investigate the performance of some ensemble methods over the commonly used miRNA target prediction methods. We apply eight different popular miRNA target prediction methods to three cancer datasets, and compare their performance with the ensemble methods which integrate the results from each combination of the individual methods. The validation results using experimentally confirmed databases show that the results of the ensemble methods complement those obtained by the individual methods and the ensemble methods perform better than the individual methods across different datasets. The ensemble method, Pearson+IDA+Lasso, which combines methods in different approaches, including a correlation method, a causal inference method, and a regression method, is the best performed ensemble method in this study. Further analysis of the results of this ensemble method shows that the ensemble method can obtain more targets which could not be found by any of the single methods, and the discovered targets are more statistically significant and functionally enriched. The source codes, datasets, miRNA target predictions by all methods, and the ground truth for validation are available in the Supplementary materials. PMID:26114448

Screening of miRNA profiles and construction of regulation networks in early and late lactation of dairy goat mammary glands.

PubMed

Ji, Zhibin; Liu, Zhaohua; Chao, Tianle; Hou, Lei; Fan, Rui; He, Rongyan; Wang, Guizhi; Wang, Jianmin

2017-09-20

In recent years, studies related to the expression profiles of miRNAs in the dairy goat mammary gland were performed, but regulatory mechanisms in the physiological environment and the dynamic homeostasis of mammary gland development and lactation are not clear. In the present study, sequencing data analysis of early and late lactation uncovered a total of 1,487 unique miRNAs, including 45 novel miRNA candidates and 1,442 known and conserved miRNAs, of which 758 miRNAs were co-expressed and 378 differentially expressed with P < 0.05. Moreover, 76 non-redundant target genes were annotated in 347 GO consortiums, with 3,143 candidate target genes grouped into 33 pathways. Additionally, 18 predicted target genes of 214 miRNAs were directly annotated in mammary gland development and used to construct regulatory networks based on GO annotation and the KEGG pathway. The expression levels of seven known miRNAs and three novel miRNAs were examined using quantitative real-time PCR. The results showed that miRNAs might play important roles in early and late lactation during dairy goat mammary gland development, which will be helpful to obtain a better understanding of the genetic control of mammary gland lactation and development.

Elsevier Trophoblast Research Award Lecture: origin, evolution and future of placenta miRNAs.

PubMed

Morales-Prieto, D M; Ospina-Prieto, S; Schmidt, A; Chaiwangyen, W; Markert, U R

2014-02-01

MicroRNAs (miRNAs) regulate the expression of a large number of genes in plants and animals. Placental miRNAs appeared late in evolution and can be found only in mammals. Nevertheless, these miRNAs are constantly under evolutionary pressure. As a consequence, miRNA sequences and their mRNA targets may differ between species, and some miRNAs can only be found in humans. Their expression can be tissue- or cell-specific and can vary time-dependently. Human placenta tissue exhibits a specific miRNA expression pattern that dynamically changes during pregnancy and is reflected in the maternal plasma. Some placental miRNAs are involved in or associated with major pregnancy disorders, such as preeclampsia, intrauterine growth restriction or preterm delivery and, therefore, have a strong potential for usage as sensitive and specific biomarkers. In this review we summarize current knowledge on the origin of placental miRNAs, their expression in humans with special regard to trophoblast cells, interspecies differences, and their future as biomarkers. It can be concluded that animal models for human reproduction have a different panel of miRNAs and targets, and can only partly reflect or predict the situation in humans. Copyright © 2013. Published by Elsevier Ltd.

A Diversity of Conserved and Novel Ovarian MicroRNAs in the Speckled Wood (Pararge aegeria)

PubMed Central

Quah, Shan; Breuker, Casper J.; Holland, Peter W. H.

2015-01-01

microRNAs (miRNAs) are important regulators of animal development and other processes, and impart robustness to living systems through post-transcriptional regulation of specific mRNA transcripts. It is postulated that newly emergent miRNAs are generally expressed at low levels and with spatiotemporally restricted expression domains, thus minimising effects of spurious targeting on animal transcriptomes. Here we present ovarian miRNA transcriptome data for two geographically distinct populations of the Speckled Wood butterfly (Pararge aegeria). A total of 74 miRNAs were identified, including 11 newly discovered and evolutionarily-young miRNAs, bringing the total of miRNA genes known from P. aegeria up to 150. We find a positive correlation between miRNA age and expression level. A common set of 55 miRNAs are expressed in both populations. From this set, we identify seven that are consistently either ovary-specific or highly upregulated in ovaries relative to other tissues. This ‘ovary set’ includes miRNAs with known contributions to ovarian function in other insect species with similar ovaries and mode of oogenesis, including miR-989 and miR-2763, plus new candidates for ovarian function. We also note that conserved miRNAs are overrepresented in the ovary relative to the whole body. PMID:26556800

MicroRNAs: regulators of gene expression and cell differentiation

PubMed Central

Shivdasani, Ramesh A.

2006-01-01

The existence and roles of a class of abundant regulatory RNA molecules have recently come into sharp focus. Micro-RNAs (miRNAs) are small (approximately 22 bases), non–protein-coding RNAs that recognize target sequences of imperfect complementarity in cognate mRNAs and either destabilize them or inhibit protein translation. Although mechanisms of miRNA biogenesis have been elucidated in some detail, there is limited appreciation of their biological functions. Reported examples typically focus on miRNA regulation of a single tissue-restricted transcript, often one encoding a transcription factor, that controls a specific aspect of development, cell differentiation, or physiology. However, computational algorithms predict up to hundreds of putative targets for individual miRNAs, single transcripts may be regulated by multiple miRNAs, and miRNAs may either eliminate target gene expression or serve to finetune transcript and protein levels. Theoretical considerations and early experimental results hence suggest diverse roles for miRNAs as a class. One appealing possibility, that miRNAs eliminate low-level expression of unwanted genes and hence refine unilineage gene expression, may be especially amenable to evaluation in models of hematopoiesis. This review summarizes current understanding of miRNA mechanisms, outlines some of the important outstanding questions, and describes studies that attempt to define miRNA functions in hematopoiesis. PMID:16882713

Viruses and miRNAs: More Friends than Foes.

PubMed

Bruscella, Patrice; Bottini, Silvia; Baudesson, Camille; Pawlotsky, Jean-Michel; Feray, Cyrille; Trabucchi, Michele

2017-01-01

There is evidence that eukaryotic miRNAs (hereafter called host miRNAs) play a role in the replication and propagation of viruses. Expression or targeting of host miRNAs can be involved in cellular antiviral responses. Most times host miRNAs play a role in viral life-cycles and promote infection through complex regulatory pathways. miRNAs can also be encoded by a viral genome and be expressed in the host cell. Viral miRNAs can share common sequences with host miRNAs or have totally different sequences. They can regulate a variety of biological processes involved in viral infection, including apoptosis, evasion of the immune response, or modulation of viral life-cycle phases. Overall, virus/miRNA pathway interaction is defined by a plethora of complex mechanisms, though not yet fully understood. This article review summarizes recent advances and novel biological concepts related to the understanding of miRNA expression, control and function during viral infections. The article also discusses potential therapeutic applications of this particular host-pathogen interaction.

Viruses and miRNAs: More Friends than Foes

PubMed Central

Bruscella, Patrice; Bottini, Silvia; Baudesson, Camille; Pawlotsky, Jean-Michel; Feray, Cyrille; Trabucchi, Michele

2017-01-01

There is evidence that eukaryotic miRNAs (hereafter called host miRNAs) play a role in the replication and propagation of viruses. Expression or targeting of host miRNAs can be involved in cellular antiviral responses. Most times host miRNAs play a role in viral life-cycles and promote infection through complex regulatory pathways. miRNAs can also be encoded by a viral genome and be expressed in the host cell. Viral miRNAs can share common sequences with host miRNAs or have totally different sequences. They can regulate a variety of biological processes involved in viral infection, including apoptosis, evasion of the immune response, or modulation of viral life-cycle phases. Overall, virus/miRNA pathway interaction is defined by a plethora of complex mechanisms, though not yet fully understood. This article review summarizes recent advances and novel biological concepts related to the understanding of miRNA expression, control and function during viral infections. The article also discusses potential therapeutic applications of this particular host–pathogen interaction. PMID:28555130

Characterization of microRNAs of Beta macrocarpa and their responses to Beet necrotic yellow vein virus infection.

PubMed

Liu, Jun-Ying; Fan, Hui-Yan; Wang, Ying; Zhang, Yong-Liang; Li, Da-Wei; Yu, Jia-Lin; Han, Cheng-Gui

2017-01-01

Plant microRNAs (miRNAs) are a class of non-coding RNAs that play important roles in plant development, defense, and symptom development. Here, 547 known miRNAs representing 129 miRNA families, and 282 potential novel miRNAs were identified in Beta macrocarpa using small RNA deep sequencing. A phylogenetic analysis was performed, and 8 Beta lineage-specific miRNAs were identified. Through a differential expression analysis, miRNAs associated with Beet necrotic yellow vein virus (BNYVV) infection were identified and confirmed using a microarray analysis and stem-loop RT-qPCR. In total, 103 known miRNAs representing 38 miRNA families, and 45 potential novel miRNAs were differentially regulated, with at least a two-fold change, in BNYVV-infected plants compared with that of the mock-inoculated control. Targets of these differentially expressed miRNAs were also predicted by degradome sequencing. These differentially expressed miRNAs were involved in hormone biosynthesis and signal transduction pathways, and enhanced axillary bud development and plant defenses. This work is the first to describe miRNAs of the plant genus Beta and may offer a reference for miRNA research in other species in the genus. It provides valuable information on the pathogenicity mechanisms of BNYVV.

Uncovering microRNA-mediated response to SO2 stress in Arabidopsis thaliana by deep sequencing.

PubMed

Li, Lihong; Xue, Meizhao; Yi, Huilan

2016-10-05

Sulfur dioxide (SO2) is a major air pollutant and has significant impacts on plants. MicroRNAs (miRNAs) are a class of gene expression regulators that play important roles in response to environmental stresses. In this study, deep sequencing was used for genome-wide identification of miRNAs and their expression profiles in response to SO2 stress in Arabidopsis thaliana shoots. A total of 27 conserved miRNAs and 5 novel miRNAs were found to be differentially expressed under SO2 stress. qRT-PCR analysis showed mostly negative correlation between miRNA accumulation and target gene mRNA abundance, suggesting regulatory roles of these miRNAs during SO2 exposure. The target genes of SO2-responsive miRNAs encode transcription factors and proteins that regulate auxin signaling and stress response, and the miRNAs-mediated suppression of these genes could improve plant resistance to SO2 stress. Promoter sequence analysis of genes encoding SO2-responsive miRNAs showed that stress-responsive and phytohormone-related cis-regulatory elements occurred frequently, providing additional evidence of the involvement of miRNAs in adaption to SO2 stress. This study represents a comprehensive expression profiling of SO2-responsive miRNAs in Arabidopsis and broads our perspective on the ubiquitous regulatory roles of miRNAs under stress conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

Genome-wide identification and characterization of microRNA genes and their targets in flax (Linum usitatissimum): Characterization of flax miRNA genes.

PubMed

Barvkar, Vitthal T; Pardeshi, Varsha C; Kale, Sandip M; Qiu, Shuqing; Rollins, Meaghen; Datla, Raju; Gupta, Vidya S; Kadoo, Narendra Y

2013-04-01

MicroRNAs (miRNAs) are small (20-24 nucleotide long) endogenous regulatory RNAs that play important roles in plant growth and development. They regulate gene expression at the post-transcriptional level by translational repression or target degradation and gene silencing. In this study, we identified 116 conserved miRNAs belonging to 23 families from the flax (Linum usitatissimum L.) genome using a computational approach. The precursor miRNAs varied in length; while most of the mature miRNAs were 21 nucleotide long, intergenic and showed conserved signatures of RNA polymerase II transcripts in their upstream regions. Promoter region analysis of the flax miRNA genes indicated prevalence of MYB transcription factor binding sites. Four miRNA gene clusters containing members of three phylogenetic groups were identified. Further, 142 target genes were predicted for these miRNAs and most of these represent transcriptional regulators. The miRNA encoding genes were expressed in diverse tissues as determined by digital expression analysis as well as real-time PCR. The expression of fourteen miRNAs and nine target genes was independently validated using the quantitative reverse transcription PCR (qRT-PCR). This study suggests that a large number of conserved plant miRNAs are also found in flax and these may play important roles in growth and development of flax.

MicroRNAs shape circadian hepatic gene expression on a transcriptome-wide scale

PubMed Central

Du, Ngoc-Hien; Arpat, Alaaddin Bulak; De Matos, Mara; Gatfield, David

2014-01-01

A considerable proportion of mammalian gene expression undergoes circadian oscillations. Post-transcriptional mechanisms likely make important contributions to mRNA abundance rhythms. We have investigated how microRNAs (miRNAs) contribute to core clock and clock-controlled gene expression using mice in which miRNA biogenesis can be inactivated in the liver. While the hepatic core clock was surprisingly resilient to miRNA loss, whole transcriptome sequencing uncovered widespread effects on clock output gene expression. Cyclic transcription paired with miRNA-mediated regulation was thus identified as a frequent phenomenon that affected up to 30% of the rhythmic transcriptome and served to post-transcriptionally adjust the phases and amplitudes of rhythmic mRNA accumulation. However, only few mRNA rhythms were actually generated by miRNAs. Overall, our study suggests that miRNAs function to adapt clock-driven gene expression to tissue-specific requirements. Finally, we pinpoint several miRNAs predicted to act as modulators of rhythmic transcripts, and identify rhythmic pathways particularly prone to miRNA regulation. DOI: http://dx.doi.org/10.7554/eLife.02510.001 PMID:24867642

MicroRNA expression profiling during the life cycle of the silkworm (Bombyx mori)

PubMed Central

Liu, Shiping; Zhang, Liang; Li, Qibin; Zhao, Ping; Duan, Jun; Cheng, Daojun; Xiang, Zhonghuai; Xia, Qingyou

2009-01-01

Background MicroRNAs (miRNAs) are expressed by a wide range of eukaryotic organisms, and function in diverse biological processes. Numerous miRNAs have been identified in Bombyx mori, but the temporal expression profiles of miRNAs corresponding to each stage transition over the entire life cycle of the silkworm remain to be established. To obtain a comprehensive overview of the correlation between miRNA expression and stage transitions, we performed a whole-life test and subsequent stage-by-stage examinations on nearly one hundred miRNAs in the silkworm. Results Our results show that miRNAs display a wide variety of expression profiles over the whole life of the silkworm, including continuous expression from embryo to adult (miR-184), up-regulation over the entire life cycle (let-7 and miR-100), down-regulation over the entire life cycle (miR-124), expression associated with embryogenesis (miR-29 and miR-92), up-regulation from early 3rd instar to pupa (miR-275), and complementary pulses in expression between miR-34b and miR-275. Stage-by-stage examinations revealed further expression patterns, such as emergence at specific time-points during embryogenesis and up-regulation of miRNA groups in late embryos (miR-1 and bantam), expression associated with stage transition between instar and molt larval stages (miR-34b), expression associated with silk gland growth and spinning activity (miR-274), continuous high expression from the spinning larval to pupal and adult stages (miR-252 and miR-31a), a coordinate expression trough in day 3 pupae of both sexes (miR-10b and miR-281), up-regulation in pupal metamorphosis of both sexes (miR-29b), and down-regulation in pupal metamorphosis of both sexes (miR-275). Conclusion We present the full-scale expression profiles of miRNAs throughout the life cycle of Bombyx mori. The whole-life expression profile was further investigated via stage-by-stage analysis. Our data provide an important resource for more detailed functional analysis of miRNAs in this animal. PMID:19785751

MicroRNA expression profiling during the life cycle of the silkworm (Bombyx mori).

PubMed

Liu, Shiping; Zhang, Liang; Li, Qibin; Zhao, Ping; Duan, Jun; Cheng, Daojun; Xiang, Zhonghuai; Xia, Qingyou

2009-09-28

MicroRNAs (miRNAs) are expressed by a wide range of eukaryotic organisms, and function in diverse biological processes. Numerous miRNAs have been identified in Bombyx mori, but the temporal expression profiles of miRNAs corresponding to each stage transition over the entire life cycle of the silkworm remain to be established. To obtain a comprehensive overview of the correlation between miRNA expression and stage transitions, we performed a whole-life test and subsequent stage-by-stage examinations on nearly one hundred miRNAs in the silkworm. Our results show that miRNAs display a wide variety of expression profiles over the whole life of the silkworm, including continuous expression from embryo to adult (miR-184), up-regulation over the entire life cycle (let-7 and miR-100), down-regulation over the entire life cycle (miR-124), expression associated with embryogenesis (miR-29 and miR-92), up-regulation from early 3rd instar to pupa (miR-275), and complementary pulses in expression between miR-34b and miR-275. Stage-by-stage examinations revealed further expression patterns, such as emergence at specific time-points during embryogenesis and up-regulation of miRNA groups in late embryos (miR-1 and bantam), expression associated with stage transition between instar and molt larval stages (miR-34b), expression associated with silk gland growth and spinning activity (miR-274), continuous high expression from the spinning larval to pupal and adult stages (miR-252 and miR-31a), a coordinate expression trough in day 3 pupae of both sexes (miR-10b and miR-281), up-regulation in pupal metamorphosis of both sexes (miR-29b), and down-regulation in pupal metamorphosis of both sexes (miR-275). We present the full-scale expression profiles of miRNAs throughout the life cycle of Bombyx mori. The whole-life expression profile was further investigated via stage-by-stage analysis. Our data provide an important resource for more detailed functional analysis of miRNAs in this animal.

Profiling microRNA expression during multi-staged date palm (Phoenix dactylifera L.) fruit development.

PubMed

Xin, Chengqi; Liu, Wanfei; Lin, Qiang; Zhang, Xiaowei; Cui, Peng; Li, Fusen; Zhang, Guangyu; Pan, Linlin; Al-Amer, Ali; Mei, Hailiang; Al-Mssallem, Ibrahim S; Hu, Songnian; Al-Johi, Hasan Awad; Yu, Jun

2015-04-01

MicroRNAs (miRNAs) play crucial roles in multiple stages of plant development and regulate gene expression at posttranscriptional and translational levels. In this study, we first identified 238 conserved miRNAs in date palm (Phoenix dactylifera) based on a high-quality genome assembly and defined 78 fruit-development-associated (FDA) miRNAs, whose expression profiles are variable at different fruit development stages. Using experimental data, we subsequently detected 276 novel P. dactylifera-specific FDA miRNAs and predicted their targets. We also revealed that FDA miRNAs function mainly in regulating genes involved in starch/sucrose metabolisms and other carbon metabolic pathways; among them, 221 FDA miRNAs exhibit negative correlation with their corresponding targets, which suggests their direct regulatory roles on mRNA targets. Our data define a comprehensive set of conserved and novel FDA miRNAs along with their expression profiles, which provide a basis for further experimentation in assigning discrete functions of these miRNAs in P. dactylifera fruit development. Copyright © 2015. Published by Elsevier Inc.

Genome-wide analysis of miRNAs in the ovaries of Jining Grey and Laiwu Black goats to explore the regulation of fecundity.

PubMed

Miao, Xiangyang; Luo, Qingmiao; Zhao, Huijing; Qin, Xiaoyu

2016-11-29

Goat fecundity is important for agriculture and varies depending on the genetic background of the goat. Two excellent domestic breeds in China, the Jining Grey and Laiwu Black goats, have different fecundity and prolificacies. To explore the potential miRNAs that regulate the expression of the genes involved in these prolific differences and to potentially discover new miRNAs, we performed a genome-wide analysis of the miRNAs in the ovaries from these two goats using RNA-Seq technology. Thirty miRNAs were differentially expressed between the Jining Grey and Laiwu Black goats. Gene Ontology and KEGG pathway analyses revealed that the target genes of the differentially expressed miRNAs were significantly enriched in several biological processes and pathways. A protein-protein interaction analysis indicated that the miRNAs and their target genes were related to the reproduction complex regulation network. The differential miRNA expression profiles found in the ovaries between the two distinctive breeds of goats studied here provide a unique resource for addressing fecundity differences in goats.

Identification and comparative analysis of the microRNA transcriptome in roots of two contrasting tobacco genotypes in response to cadmium stress

NASA Astrophysics Data System (ADS)

He, Xiaoyan; Zheng, Weite; Cao, Fangbin; Wu, Feibo

2016-09-01

Tobacco (Nicotiana tabacum L.) is more acclimated to cadmium (Cd) uptake and preferentially enriches Cd in leaves than other crops. MicroRNAs (miRNAs) play crucial roles in regulating expression of various stress response genes in plants. However, genome-wide expression of miRNAs and their target genes in response to Cd stress in tobacco are still unknown. Here, miRNA high-throughput sequencing technology was performed using two contrasting tobacco genotypes Guiyan 1 and Yunyan 2 of Cd-sensitive and tolerance. Comprehensive analysis of miRNA expression profiles in control and Cd treated plants identified 72 known (27 families) and 14 novel differentially expressed miRNAs in the two genotypes. Among them, 28 known (14 families) and 5 novel miRNAs were considered as Cd tolerance associated miRNAs, which mainly involved in cell growth, ion homeostasis, stress defense, antioxidant and hormone signaling. Finally, a hypothetical model of Cd tolerance mechanism in Yunyan 2 was presented. Our findings suggest that some miRNAs and their target genes and pathways may play critical roles in Cd tolerance.

[MicroRNAs in diagnosis and prognosis in lung cancer].

PubMed

Avila-Moreno, Federico; Urrea, Francisco; Ortiz-Quintero, Blanca

2011-01-01

MicroRNAs (miRNAs) are endogenous small non-coding RNA molecules that regulate gene expression at the posttranscriptional level by blocking translation or inducing degradation of messenger RNA targets. It has been shown that miRNAs participate in a wide spectrum of essential biologic processes including cell cycle, differentiation, development, apoptosis and hematopoiesis, revealing one of the major regulators of human gene expression. Recent studies have shown evidences of abnormal expression of miRNAs in solid and hematological tumors, as well as the association of altered miRNAs with oncogenic or tumor suppressor functions, suggesting a key role of miRNAs in carcinogenesis. Moreover, unique profiles of altered miRNAs expression seem to allow distinction from normal tissue, prediction of disease outcomes, and evaluation of tumor aggressiveness in several types of cancer, including lung cancer. These unique and highly stable miRNAs patterns seems not to depend of age and race, and these characteristics highlight their potential diagnostic and prognosis utility. These findings are particularly promising for lung cancer, a worldwide leading cause of cancer-related deaths with a poor survival rate, despite the discovery of novel therapies. This review describes the potential of miRNAs as biomarkers for diagnosis, cancer classification and estimation of prognosis in lung cancer; and the approaches used to detect and quantify these miRNAs; including the current information about circulating miRNAs as potential biomarkers in lung cancer. This review also provides a description of miRNAs biogenesis, nomenclature and available database for miRNA sequences.

MicroRNAs and cancer.

PubMed

Cowland, Jack B; Hother, Christoffer; Grønbaek, Kirsten

2007-10-01

MicroRNAs (miRNAs) are a recently discovered group of small RNA molecules involved in the regulation of gene expression. Analogously to mRNAs, the non-protein-encoding pri-miRNAs are synthesized by RNA polymerase II and post-transcriptionally modified by addition of a 5'-cap and a 3'-poly (A) tail. Subsequently, the pri-miRNA undergoes a number of processing steps in the nucleus and cytoplasm, and ends up as a mature approximately 22 nt miRNA, which can exert its function by binding to the 3'-untranslated region of a subset of mRNAs. Binding of the miRNA to the mRNA results in a reduced translation rate and/or increased degradation of the mRNA. In this way a large number of cellular pathways, such as cellular proliferation, differentiation, and apoptosis, are regulated by mi-RNAs. As corruption of these pathways is the hallmark of many cancers, dysregulation of miRNA biogenesis or expression levels may lead to tumorigenesis. The mechanisms that alter the expression of miRNAs are similar to those that change the expression levels of mRNAs of tumor suppressor- and oncogenes, i.e. gross genomic aberrations, epigenetic changes, and minor mutations affecting the expression level, processing, or target-interaction potential of the miRNA. Furthermore, expression profiling of miRNAs has been found to be useful for classification of different tumor types. Taken together, miRNAs can be classified as onco-miRs or tumor suppressor-miRs, and may turn out to be potential targets for cancer therapy.

Identification of candidate miRNAs and expression profile of yak oocytes before and after in vitro maturation by high-throughput sequencing.

PubMed

Xiong, X R; Lan, D L; Li, J; Zi, X D; Li, M Y

2016-12-01

Small RNA represents several unique non-coding RNA classes that have important function in a wide range of biological processes including development of germ cells and early embryonic, cell differentiation, cell proliferation and apoptosis in diverse organisms. However, little is known about their expression profiles and effects in yak oocytes maturation and early development. To investigate the function of small RNAs in the maturation process of yak oocyte and early development, two small RNA libraries of oocytes were constructed from germinal vesicle stage (GV) and maturation in vitro to metaphase II-arrested stage (M II) and then sequenced using small RNA high-throughput sequencing technology. A total of 9,742,592 and 12,168,523 clean reads were obtained from GV and M II oocytes, respectively. In total, 801 and 1,018 known miRNAs were acquired from GV and M II oocytes, and 75 miRNAs were found to be significantly differentially expressed: 47 miRNAs were upregulated and 28 miRNAs were downregulated in the M II oocytes compared to the GV stage. Among the upregulated miRNAs, miR-342 has the largest fold change (9.25-fold). Six highly expressed miRNAs (let-7i, miR-10b, miR-10c, miR-143, miR-146b and miR-148) were validated by real-time quantitative PCR (RT-qPCR) and consistent with the sequencing results. Furthermore, the expression patterns of two miRNAs and their potential targets were analysed in different developmental stages of oocytes and early embryos. This study provides the first miRNA profile in the mature process of yak oocyte. Seventy-five miRNAs are expressed differentially in GV and M II oocytes as well as among different development stages of early embryos, suggesting miRNAs involved in regulating oocyte maturation and early development of yak. These results showed specific miRNAs in yak oocytes had dynamic changes during meiosis. Further functional and mechanistic studies on the miRNAs during meiosis may beneficial to understanding the role of miRNAs on meiotic division. © 2016 Blackwell Verlag GmbH.

Global population-specific variation in miRNA associated with cancer risk and clinical biomarkers.

PubMed

Rawlings-Goss, Renata A; Campbell, Michael C; Tishkoff, Sarah A

2014-08-28

MiRNA expression profiling is being actively investigated as a clinical biomarker and diagnostic tool to detect multiple cancer types and stages as well as other complex diseases. Initial investigations, however, have not comprehensively taken into account genetic variability affecting miRNA expression and/or function in populations of different ethnic backgrounds. Therefore, more complete surveys of miRNA genetic variability are needed to assess global patterns of miRNA variation within and between diverse human populations and their effect on clinically relevant miRNA genes. Genetic variation in 1524 miRNA genes was examined using whole genome sequencing (60x coverage) in a panel of 69 unrelated individuals from 14 global populations, including European, Asian and African populations. We identified 33 previously undescribed miRNA variants, and 31 miRNA containing variants that are globally population-differentiated in frequency between African and non-African populations (PD-miRNA). The top 1% of PD-miRNA were significantly enriched for regulation of genes involved in glucose/insulin metabolism and cell division (p < 10(-7)), most significantly the mitosis pathway, which is strongly linked to cancer onset. Overall, we identify 7 PD-miRNAs that are currently implicated as cancer biomarkers or diagnostics: hsa-mir-202, hsa-mir-423, hsa-mir-196a-2, hsa-mir-520h, hsa-mir-647, hsa-mir-943, and hsa-mir-1908. Notably, hsa-mir-202, a potential breast cancer biomarker, was found to show significantly high allele frequency differentiation at SNP rs12355840, which is known to affect miRNA expression levels in vivo and subsequently breast cancer mortality. MiRNA expression profiles represent a promising new category of disease biomarkers. However, population specific genetic variation can affect the prevalence and baseline expression of these miRNAs in diverse populations. Consequently, miRNA genetic and expression level variation among ethnic groups may be contributing in part to health disparities observed in multiple forms of cancer, specifically breast cancer, and will be an essential consideration when assessing the utility of miRNA biomarkers for the clinic.

Interaction between Host MicroRNAs and the Gut Microbiota in Colorectal Cancer.

PubMed

Yuan, Ce; Burns, Michael B; Subramanian, Subbaya; Blekhman, Ran

2018-01-01

Although variation in gut microbiome composition has been linked with colorectal cancer (CRC), the factors that mediate the interactions between CRC tumors and the microbiome are poorly understood. MicroRNAs (miRNAs) are known to regulate CRC progression and are associated with patient survival outcomes. In addition, recent studies suggested that host miRNAs can also regulate bacterial growth and influence the composition of the gut microbiome. Here, we investigated the association between miRNA expression and microbiome composition in human CRC tumor and normal tissues. We identified 76 miRNAs as differentially expressed (DE) in tissue from CRC tumors and normal tissue, including the known oncogenic miRNAs miR-182, miR-503, and mir-17~92 cluster. These DE miRNAs were correlated with the relative abundances of several bacterial taxa, including Firmicutes , Bacteroidetes , and Proteobacteria . Bacteria correlated with DE miRNAs were enriched with distinct predicted metabolic categories. Additionally, we found that miRNAs that correlated with CRC-associated bacteria are predicted to regulate targets that are relevant for host-microbiome interactions and highlight a possible role for miRNA-driven glycan production in the recruitment of pathogenic microbial taxa. Our work characterized a global relationship between microbial community composition and miRNA expression in human CRC tissues. IMPORTANCE Recent studies have found an association between colorectal cancer (CRC) and the gut microbiota. One potential mechanism by which the microbiota can influence host physiology is through affecting gene expression in host cells. MicroRNAs (miRNAs) are small noncoding RNA molecules that can regulate gene expression and have important roles in cancer development. Here, we investigated the link between the gut microbiota and the expression of miRNA in CRC. We found that dozens of miRNAs are differentially regulated in CRC tumors and adjacent normal colon and that these miRNAs are correlated with the abundance of microbes in the tumor microenvironment. Moreover, we found that microbes that have been previously associated with CRC are correlated with miRNAs that regulate genes related to interactions with microbes. Notably, these miRNAs likely regulate glycan production, which is important for the recruitment of pathogenic microbial taxa to the tumor. This work provides a first systems-level map of the association between microbes and host miRNAs in the context of CRC and provides targets for further experimental validation and potential interventions.

Identification of rat lung-specific microRNAs by micoRNA microarray: valuable discoveries for the facilitation of lung research.

PubMed

Wang, Yang; Weng, Tingting; Gou, Deming; Chen, Zhongming; Chintagari, Narendranath Reddy; Liu, Lin

2007-01-24

An important mechanism for gene regulation utilizes small non-coding RNAs called microRNAs (miRNAs). These small RNAs play important roles in tissue development, cell differentiation and proliferation, lipid and fat metabolism, stem cells, exocytosis, diseases and cancers. To date, relatively little is known about functions of miRNAs in the lung except lung cancer. In this study, we utilized a rat miRNA microarray containing 216 miRNA probes, printed in-house, to detect the expression of miRNAs in the rat lung compared to the rat heart, brain, liver, kidney and spleen. Statistical analysis using Significant Analysis of Microarray (SAM) and Tukey Honestly Significant Difference (HSD) revealed 2 miRNAs (miR-195 and miR-200c) expressed specifically in the lung and 9 miRNAs co-expressed in the lung and another organ. 12 selected miRNAs were verified by Northern blot analysis. The identified lung-specific miRNAs from this work will facilitate functional studies of miRNAs during normal physiological and pathophysiological processes of the lung.

Functional analysis of neuronal microRNAs in Caenorhabditis elegans dauer formation by combinational genetics and Neuronal miRISC immunoprecipitation.

PubMed

Than, Minh T; Kudlow, Brian A; Han, Min

2013-06-01

Identifying the physiological functions of microRNAs (miRNAs) is often challenging because miRNAs commonly impact gene expression under specific physiological conditions through complex miRNA::mRNA interaction networks and in coordination with other means of gene regulation, such as transcriptional regulation and protein degradation. Such complexity creates difficulties in dissecting miRNA functions through traditional genetic methods using individual miRNA mutations. To investigate the physiological functions of miRNAs in neurons, we combined a genetic "enhancer" approach complemented by biochemical analysis of neuronal miRNA-induced silencing complexes (miRISCs) in C. elegans. Total miRNA function can be compromised by mutating one of the two GW182 proteins (AIN-1), an important component of miRISC. We found that combining an ain-1 mutation with a mutation in unc-3, a neuronal transcription factor, resulted in an inappropriate entrance into the stress-induced, alternative larval stage known as dauer, indicating a role of miRNAs in preventing aberrant dauer formation. Analysis of this genetic interaction suggests that neuronal miRNAs perform such a role partly by regulating endogenous cyclic guanosine monophosphate (cGMP) signaling, potentially influencing two other dauer-regulating pathways. Through tissue-specific immunoprecipitations of miRISC, we identified miRNAs and their likely target mRNAs within neuronal tissue. We verified the biological relevance of several of these miRNAs and found that many miRNAs likely regulate dauer formation through multiple dauer-related targets. Further analysis of target mRNAs suggests potential miRNA involvement in various neuronal processes, but the importance of these miRNA::mRNA interactions remains unclear. Finally, we found that neuronal genes may be more highly regulated by miRNAs than intestinal genes. Overall, our study identifies miRNAs and their targets, and a physiological function of these miRNAs in neurons. It also suggests that compromising other aspects of gene expression, along with miRISC, can be an effective approach to reveal miRNA functions in specific tissues under specific physiological conditions.

Comparative analysis of miRNAs of two rapeseed genotypes in response to acetohydroxyacid synthase-inhibiting herbicides by high-throughput sequencing.

PubMed

Hu, Maolong; Pu, Huiming; Gao, Jianqin; Long, Weihua; Chen, Feng; Zhang, Wei; Zhou, Xiaoyin; Peng, Qi; Chen, Song; Zhang, Jiefu

2017-01-01

Acetohydroxyacid synthase (AHAS), also called acetolactate synthase, is a key enzyme involved in the first step of the biosynthesis of the branched-chain amino acids valine, isoleucine and leucine. Acetohydroxyacid synthase-inhibiting herbicides (AHAS herbicides) are five chemical families of herbicides that inhibit AHAS enzymes, including imidazolinones (IMI), sulfonylureas (SU), pyrimidinylthiobenzoates, triazolinones and triazolopyrimidines. Five AHAS genes have been identified in rapeseed, but little information is available regarding the role of miRNAs in response to AHAS herbicides. In this study, an AHAS herbicides tolerant genotype and a sensitive genotype were used for miRNA comparative analysis. A total of 20 small RNA libraries were obtained of these two genotypes at three time points (0h, 24 h and 48 h) after spraying SU and IMI herbicides with two replicates. We identified 940 conserved miRNAs and 1515 novel candidate miRNAs in Brassica napus using high-throughput sequencing methods combined with computing analysis. A total of 3284 genes were predicted to be targets of these miRNAs, and their functions were shown using GO, KOG and KEGG annotations. The differentiation expression results of miRNAs showed almost twice as many differentiated miRNAs were found in tolerant genotype M342 (309 miRNAs) after SU herbicide application than in sensitive genotype N131 (164 miRNAs). In additiond 177 and 296 miRNAs defined as differentiated in sensitive genotype and tolerant genotype in response to SU herbicides. The miR398 family was observed to be associated with AHAS herbicide tolerance because their expression increased in the tolerant genotype but decreased in the sensitive genotype. Moreover, 50 novel miRNAs from 39 precursors were predicted. There were 8 conserved miRNAs, 4 novel miRNAs and 3 target genes were validated by quantitative real-time PCR experiment. This study not only provides novel insights into the miRNA content of AHAS herbicides tolerant rapeseed in response to AHAS herbicides, but also demonstrates that miRNAs may be involved in AHAS herbicides tolerance.

Comparative analysis of miRNAs of two rapeseed genotypes in response to acetohydroxyacid synthase-inhibiting herbicides by high-throughput sequencing

PubMed Central

Hu, Maolong; Pu, Huiming; Gao, Jianqin; Long, Weihua; Chen, Feng; Zhang, Wei; Zhou, Xiaoyin; Peng, Qi; Chen, Song; Zhang, Jiefu

2017-01-01

Acetohydroxyacid synthase (AHAS), also called acetolactate synthase, is a key enzyme involved in the first step of the biosynthesis of the branched-chain amino acids valine, isoleucine and leucine. Acetohydroxyacid synthase-inhibiting herbicides (AHAS herbicides) are five chemical families of herbicides that inhibit AHAS enzymes, including imidazolinones (IMI), sulfonylureas (SU), pyrimidinylthiobenzoates, triazolinones and triazolopyrimidines. Five AHAS genes have been identified in rapeseed, but little information is available regarding the role of miRNAs in response to AHAS herbicides. In this study, an AHAS herbicides tolerant genotype and a sensitive genotype were used for miRNA comparative analysis. A total of 20 small RNA libraries were obtained of these two genotypes at three time points (0h, 24 h and 48 h) after spraying SU and IMI herbicides with two replicates. We identified 940 conserved miRNAs and 1515 novel candidate miRNAs in Brassica napus using high-throughput sequencing methods combined with computing analysis. A total of 3284 genes were predicted to be targets of these miRNAs, and their functions were shown using GO, KOG and KEGG annotations. The differentiation expression results of miRNAs showed almost twice as many differentiated miRNAs were found in tolerant genotype M342 (309 miRNAs) after SU herbicide application than in sensitive genotype N131 (164 miRNAs). In additiond 177 and 296 miRNAs defined as differentiated in sensitive genotype and tolerant genotype in response to SU herbicides. The miR398 family was observed to be associated with AHAS herbicide tolerance because their expression increased in the tolerant genotype but decreased in the sensitive genotype. Moreover, 50 novel miRNAs from 39 precursors were predicted. There were 8 conserved miRNAs, 4 novel miRNAs and 3 target genes were validated by quantitative real-time PCR experiment. This study not only provides novel insights into the miRNA content of AHAS herbicides tolerant rapeseed in response to AHAS herbicides, but also demonstrates that miRNAs may be involved in AHAS herbicides tolerance. PMID:28950015

Novel Cadmium Responsive MicroRNAs in Daphnia pulex.

PubMed

Chen, Shuai; McKinney, Garrett J; Nichols, Krista M; Colbourne, John K; Sepúlveda, Maria S

2015-12-15

Daphnia pulex is a widely used toxicological model and is known for its sensitivity to cadmium (Cd). Recent research suggests that microRNAs (miRNAs) play a critical role in animal responses to heavy metals. To investigate the functions of D. pulex miRNAs under Cd exposure, we analyzed the miRNA profiles of D. pulex after 48 h using miRNA microarrays and validated our findings by q-PCR. miRNA dpu-let-7 was identified as a stably expressed gene and used as a reference. We identified 22 and 21 differentially expressed miRNAs under low (20 μg/L CdCl2) and high-exposure (40 μg/L CdCl2) concentrations compared to controls, respectively. Cellular functions of predicted miRNA target Cd-responsive genes included oxidative stress, ion transport, mitochondrial damage, and DNA repair. An insulin-related network was also identified in relation to several Cd-responsive miRNAs. The expression of three predicted target genes for miR-71 and miR-210 were evaluated, and expression of two of them (SCN2A and SLC31A1) was negatively correlated with the expression of their regulator miRNAs. We show miR-210 is hypoxia-responsive in D. pulex and propose Cd and hypoxia induce miR-210 via a same HIF1α modulated pathway. Collectively, this research advances our understanding on the role of miRNAs in response to heavy-metal exposure.

Mineral pitch induces apoptosis and inhibits proliferation via modulating reactive oxygen species in hepatic cancer cells.

PubMed

Pant, Kishor; Gupta, Parul; Damania, Preeti; Yadav, Ajay K; Gupta, Aanchal; Ashraf, Anam; Venugopal, Senthil K

2016-05-27

Mineral Pitch (MP) is a dark brown coloured humic matter originating from high altitude rocks. It is an Ayurvedic medicinal food, commonly used by the people of the Himalayan regions of Nepal and India for various body ailments. The Huh-7 cells were treated with different concentrations of MP for 24 h, and both apoptosis and proliferation was determined by the TUNEL and MTT assays respectively. The formation of ROS and nitric oxide was analysed by DCFH-DA and Griess reagent respectively. The expression of miRNA-21 and miRNA-22 were checked by the real time PCR. Effect of miRNA-22 on proliferation and c-myc was studied by over-expressing miRNA-22 premiRs in Huh-7 cells. We found that MP enhanced anti-cancer effects by inducing apoptosis and inhibiting proliferation. MP induced both ROS and NO, upon neutralizing them, there was a partial recovery of apoptosis and proliferation. MP also induced miRNA-22 expression, while miRNA-21 expression was inhibited. Over-expression of miRNA-22 resulted in a significant inhibition of proliferation. miRNA-22 directly targeted c-myc gene, thereby inhibited proliferation. These results clearly show that MP induces its anti-cancer activity by more than one pathway. The data clearly indicate that MP induced apoptosis via the production of ROS, and inhibited proliferation by inducing miRNA-22 and inhibiting miRNA-21 in Huh-7 cells.

Oligodendrocyte precursor cell transplantation promotes functional recovery following contusive spinal cord injury in rats and is associated with altered microRNA expression

PubMed Central

Yang, Jin; Xiong, Liu-Lin; Wang, You-Cui; He, Xiang; Jiang, Ling; Fu, Song-Jun; Han, Xue-Fei; Liu, Jia; Wang, Ting-Hua

2018-01-01

It has been reported that oligodendrocyte precursor cells (OPCs) may be used to treat contusive spinal cord injury (SCC), and may alter microRNA (miRNA/miR) expression following SCC in rats. However, the association between miRNA expression and the treatment of rats with SCC with OPC transplantation remain unclear. The present study transplanted OPCs into the spinal cord of rats with SCC and subsequently used the Basso, Beattie and Bresnahan (BBB) score to assess the functional recovery and pain scores. An miRNA assay was performed to detect differentially expressed miRNAs in the spinal cord of SCC rats transplanted with OPCs, compared with SCC rats transplanted with medium. Quantitative polymerase chain reaction was used to verify significantly altered miRNA expression levels. The results demonstrated that OPC transplantation was able to improve motor recovery and relieve mechanical allodynia in rats with SCC. In addition, through a miRNA assay, 45 differentially expressed miRNAs (40 upregulated miRNAs and 5 downregulated miRNAs) were detected in the spinal cord of rats in the OPC group compared with in the Medium group. Differentially expressed miRNAs were identified according to the following criteria: Fold change >2 and P<0.05. Furthermore, quantitative polymerase chain reaction was used to verify the most highly upregulated (miR-375-3p and miR-1-3p) and downregulated (miR-363-3p, miR-449a-5p and miR-3074) spinal cord miRNAs that were identified in the miRNA assay. In addition, a bioinformatics analysis of these miRNAs indicated that miR-375 and miR-1 may act primarily to inhibit cell proliferation and apoptosis via transcriptional and translational regulation, whereas miR-363, miR-449a and miR-3074 may act primarily to inhibit cell proliferation and neuronal differentiation through transcriptional regulation. These results suggested that OPC transplantation may promote functional recovery of rats with SCC, which may be associated with the expression of various miRNAs in the spinal cord, including miR-375-3p, miR-1-3p, miR-363-3p, miR-449a-5p and miR-3074. PMID:29115639

Elucidation of miRNAs-Mediated Responses to Low Nitrogen Stress by Deep Sequencing of Two Soybean Genotypes

PubMed Central

Wang, Yejian; Zhang, Chanjuan; Hao, Qinnan; Sha, Aihua; Zhou, Rong; Zhou, Xinan; Yuan, Longping

2013-01-01

Nitrogen (N) is a major limiting factor in crop production, and plant adaptive responses to low N are involved in many post-transcriptional regulation. Recent studies indicate that miRNAs play important roles in adaptive responses. However, miRNAs in soybean adaptive responses to N limitation have been not reported. We constructed sixteen libraries to identify low N-responsive miRNAs on a genome-wide scale using samples from 2 different genotypes (low N sensitive and low N tolerant) subjected to various periods of low nitrogen stress. Using high-throughput sequencing technology (Illumina-Solexa), we identified 362 known miRNAs variants belonging to 158 families and 90 new miRNAs belonging to 55 families. Among these known miRNAs variants, almost 50% were not different from annotated miRNAs in miRBase. Analyses of their expression patterns showed 150 known miRNAs variants as well as 2 novel miRNAs with differential expressions. These differentially expressed miRNAs between the two soybean genotypes were compared and classified into three groups based on their expression patterns. Predicted targets of these miRNAs were involved in various metabolic and regulatory pathways such as protein degradation, carbohydrate metabolism, hormone signaling pathway, and cellular transport. These findings suggest that miRNAs play important roles in soybean response to low N and contribute to the understanding of the genetic basis of differences in adaptive responses to N limitation between the two soybean genotypes. Our study provides basis for expounding the complex gene regulatory network of these miRNAs. PMID:23861762

Small RNA Sequencing Reveals Differential miRNA Expression in the Early Development of Broccoli (Brassica oleracea var. italica) Pollen

PubMed Central

Li, Hui; Wang, Yu; Wu, Mei; Li, Lihong; Jin, Chuan; Zhang, Qingli; Chen, Chengbin; Song, Wenqin; Wang, Chunguo

2017-01-01

Pollen development is an important and complex biological process in the sexual reproduction of flowering plants. Although the cytological characteristics of pollen development are well defined, the regulation of its early stages remains largely unknown. In the present study, miRNAs were explored in the early development of broccoli (Brassica oleracea var. italica) pollen. A total of 333 known miRNAs that originated from 235 miRNA families were detected. Fifty-five novel miRNA candidates were identified. Sixty of the 333 known miRNAs and 49 of the 55 predicted novel miRNAs exhibited significantly differential expression profiling in the three distinct developmental stages of broccoli pollen. Among these differentially expressed miRNAs, miRNAs that would be involved in the developmental phase transition from uninucleate microspores to binucleate pollen grains or from binucleate to trinucleate pollen grains were identified. miRNAs that showed significantly enriched expression in a specific early stage of broccoli pollen development were also observed. In addition, 552 targets for 127 known miRNAs and 69 targets for 40 predicted novel miRNAs were bioinformatically identified. Functional annotation and GO (Gene Ontology) analysis indicated that the putative miRNA targets showed significant enrichment in GO terms that were related to plant organ formation and morphogenesis. Some of enriched GO terms were detected for the targets directly involved in plant male reproduction development. These findings provided new insights into the functions of miRNA-mediated regulatory networks in broccoli pollen development. PMID:28392797

Small RNA Sequencing Reveals Differential miRNA Expression in the Early Development of Broccoli (Brassica oleracea var. italica) Pollen.

PubMed

Li, Hui; Wang, Yu; Wu, Mei; Li, Lihong; Jin, Chuan; Zhang, Qingli; Chen, Chengbin; Song, Wenqin; Wang, Chunguo

2017-01-01

Pollen development is an important and complex biological process in the sexual reproduction of flowering plants. Although the cytological characteristics of pollen development are well defined, the regulation of its early stages remains largely unknown. In the present study, miRNAs were explored in the early development of broccoli ( Brassica oleracea var. italica ) pollen. A total of 333 known miRNAs that originated from 235 miRNA families were detected. Fifty-five novel miRNA candidates were identified. Sixty of the 333 known miRNAs and 49 of the 55 predicted novel miRNAs exhibited significantly differential expression profiling in the three distinct developmental stages of broccoli pollen. Among these differentially expressed miRNAs, miRNAs that would be involved in the developmental phase transition from uninucleate microspores to binucleate pollen grains or from binucleate to trinucleate pollen grains were identified. miRNAs that showed significantly enriched expression in a specific early stage of broccoli pollen development were also observed. In addition, 552 targets for 127 known miRNAs and 69 targets for 40 predicted novel miRNAs were bioinformatically identified. Functional annotation and GO (Gene Ontology) analysis indicated that the putative miRNA targets showed significant enrichment in GO terms that were related to plant organ formation and morphogenesis. Some of enriched GO terms were detected for the targets directly involved in plant male reproduction development. These findings provided new insights into the functions of miRNA-mediated regulatory networks in broccoli pollen development.

MicroRNAome of Spodoptera frugiperda cells (Sf9) and its alteration following baculovirus infection.

PubMed

Mehrabadi, Mohammad; Hussain, Mazhar; Asgari, Sassan

2013-06-01

MicroRNAs (miRNAs) as small non-coding RNAs play important roles in many biological processes such as development, cell signalling and immune response. Studies also suggest that miRNAs are important in host-virus interactions where the host limits virus infection by differentially expressing miRNAs that target essential viral genes. Here, we identified conserved and new miRNAs from Spodoptera frugiperda cells (Sf9) using a combination of deep sequencing and bioinformatics as well as experimental approaches. S. frugiperda miRNAs share common features of miRNAs in other organisms, such as uracil (U) at the 5' end of miRNA. The 5' ends of the miRNAs were more conserved than the 3' ends, revealing evolutionary protection of the seed region in miRNAs. The predominant miRNAs were found to be conserved among arthropods. The majority of homologous miRNAs were found in Bombyx mori, with 76 of the 90 identified miRNAs. We found that seed shifting and arm switching have happened in this insect's miRNAs. Expression levels of the majority of miRNAs changed following baculovirus infection. Results revealed that baculovirus infection mainly led to an overall suppression of cellular miRNAs. We found four different genes being regulated by sfr-miR-184 at the post-transcriptional level. The data presented here further support conservation of miRNAs in insects and other organisms. In addition, the results reveal a differential expression of host miRNAs upon baculovirus infection, suggesting their potential roles in host-virus interactions. Seed shifting and arm switching happened during evolution of miRNAs in different insects and caused miRNA diversification, which led to changes in the target repository of miRNAs.

Identification and Functional Analysis of Flowering Related microRNAs in Common Wild Rice (Oryza rufipogon Griff.)

PubMed Central

Dong, Yibo; Yuan, Qianhua; Wang, Feng; Li, Weimin; Jiang, Ying; Jia, Shirong; Pei, XinWu

2013-01-01

Background MicroRNAs (miRNAs) is a class of non-coding RNAs involved in post- transcriptional control of gene expression, via degradation and/or translational inhibition. Six-hundred sixty-one rice miRNAs are known that are important in plant development. However, flowering-related miRNAs have not been characterized in Oryza rufipogon Griff. It was approved by supervision department of Guangdong wild rice protection. We analyzed flowering-related miRNAs in O. rufipogon using high-throughput sequencing (deep sequencing) to understand the changes that occurred during rice domestication, and to elucidate their functions in flowering. Results Three O. rufipogon sRNA libraries, two vegetative stage (CWR-V1 and CWR-V2) and one flowering stage (CWR-F2) were sequenced using Illumina deep sequencing. A total of 20,156,098, 21,531,511 and 20,995,942 high quality sRNA reads were obtained from CWR-V1, CWR-V2 and CWR-F2, respectively, of which 3,448,185, 4,265,048 and 2,833,527 reads matched known miRNAs. We identified 512 known rice miRNAs in 214 miRNA families and predicted 290 new miRNAs. Targeted functional annotation, GO and KEGG pathway analyses predicted that 187 miRNAs regulate expression of flowering-related genes. Differential expression analysis of flowering-related miRNAs showed that: expression of 95 miRNAs varied significantly between the libraries, 66 are flowering-related miRNAs, such as oru-miR97, oru-miR117, oru-miR135, oru-miR137, et al. 17 are early-flowering -related miRNAs, including osa-miR160f, osa-miR164d, osa-miR167d, osa-miR169a, osa-miR172b, oru-miR4, et al., induced during the floral transition. Real-time PCR revealed the same expression patterns as deep sequencing. miRNAs targets were confirmed for cleavage by 5′-RACE in vivo, and were negatively regulated by miRNAs. Conclusions This is the first investigation of flowering miRNAs in wild rice. The result indicates that variation in miRNAs occurred during rice domestication and lays a foundation for further study of phase change and flowering in O. rufipogon. Complicated regulatory networks mediated by multiple miRNAs regulate the expression of flowering genes that control the induction of flowering. PMID:24386120

Deep sequencing of small RNA libraries from human prostate epithelial and stromal cells reveal distinct pattern of microRNAs primarily predicted to target growth factors.

PubMed

Singh, Savita; Zheng, Yun; Jagadeeswaran, Guru; Ebron, Jey Sabith; Sikand, Kavleen; Gupta, Sanjay; Sunker, Ramanjulu; Shukla, Girish C

2016-02-28

Complex epithelial and stromal cell interactions are required during the development and progression of prostate cancer. Regulatory small non-coding microRNAs (miRNAs) participate in the spatiotemporal regulation of messenger RNA (mRNA) and regulation of translation affecting a large number of genes involved in prostate carcinogenesis. In this study, through deep-sequencing of size fractionated small RNA libraries we profiled the miRNAs of prostate epithelial (PrEC) and stromal (PrSC) cells. Over 50 million reads were obtained for PrEC in which 860,468 were unique sequences. Similarly, nearly 76 million reads for PrSC were obtained in which over 1 million were unique reads. Expression of many miRNAs of broadly conserved and poorly conserved miRNA families were identified. Sixteen highly expressed miRNAs with significant change in expression in PrSC than PrEC were further analyzed in silico. ConsensusPathDB showed the target genes of these miRNAs were significantly involved in adherence junction, cell adhesion, EGRF, TGF-β and androgen signaling. Let-7 family of tumor-suppressor miRNAs expression was highly pervasive in both, PrEC and PrSC cells. In addition, we have also identified several miRNAs that are unique to PrEC or PrSC cells and their predicted putative targets are a group of transcription factors. This study provides perspective on the miRNA expression in PrEC and PrSC, and reveals a global trend in miRNA interactome. We conclude that the most abundant miRNAs are potential regulators of development and differentiation of the prostate gland by targeting a set of growth factors. Additionally, high level expression of the most members of let-7 family miRNAs suggests their role in the fine tuning of the growth and proliferation of prostate epithelial and stromal cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Expressed microRNA associated with high rate of egg production in chicken ovarian follicles.

PubMed

Wu, N; Gaur, U; Zhu, Q; Chen, B; Xu, Z; Zhao, X; Yang, M; Li, D

2017-04-01

MicroRNA (miRNA) is a highly conserved class of small noncoding RNA about 19-24 nucleotides in length that function in a specific manner to post-transcriptionally regulate gene expression in organisms. Tissue miRNA expression studies have discovered a myriad of functions for miRNAs in various aspects, but a role for miRNAs in chicken ovarian tissue at 300 days of age has not hitherto been reported. In this study, we performed the first miRNA analysis of ovarian tissues in chickens with low and high rates of egg production using high-throughput sequencing. By comparing low rate of egg production chickens with high rate of egg production chickens, 17 significantly differentially expressed miRNAs were found (P < 0.05), including 11 known and six novel miRNAs. We found that all 11 known miRNAs were involved mainly in pathways of reproduction regulation, such as steroid hormone biosynthesis and dopaminergic synapse. Additionally, expression profiling of six randomly selected differentially regulated miRNAs were validated by quantitative real-time polymerase chain reaction (RT-qPCR). Some miRNAs, such as gga-miR-34b, gga-miR-34c and gga-miR-216b, were reported to regulate processes such as proliferation, cell cycle, apoptosis and metastasis and were expressed differentially in ovaries of chickens with high rates of egg production, suggesting that these miRNAs have an important role in ovary development and reproductive management of chicken. Furthermore, we uncovered that a significantly up-regulated miRNA-gga-miR-200a-3p-is ubiquitous in reproduction-regulation-related pathways. This miRNA may play a special central role in the reproductive management of chicken, and needs to be further studied for confirmation. © 2016 Stichting International Foundation for Animal Genetics.

microRNA-10b Is Overexpressed and Critical for Cell Survival and Proliferation in Medulloblastoma

PubMed Central

Pal, Rekha; Greene, Stephanie

2015-01-01

This study demonstrates the effects of miRNA-10b on medulloblastoma proliferation through transcriptional induction of the anti-apoptotic protein BCL2. Using a cancer specific miRNA-array, high expression of miRNA-10b in medulloblastoma cell lines compared to a normal cerebellar control was shown, and this was confirmed with real time PCR (RT-PCR). Two medulloblastoma cell lines (DAOY and UW228) were transiently transfected with control miRNA, miRNA-10b inhibitor or miRNA-10b mimic and subjected to RT-PCR, MTT, apoptosis, clonogenic assay and western blot analysis. Transfection of miRNA-10b inhibitor induced a significant down-regulation of miRNA-10b expression, inhibited proliferation, and induced apoptosis, while miRNA-10b mimic exerted an opposite effect. Inhibition of miRNA-10b abrogated the colony-forming capability of medulloblastoma cells, and markedly down-regulated the expression of BCL2. Down-regulation of BCL2 by antisense oligonucleotides or siRNA also significantly down-regulated miRNA-10b, suggesting that BCL2 is a major mediator of the effects of miRNA-10b. ABT-737 and ABT-199, potent inhibitors of BCL2, downregulated the expression of miRNA-10b and increased apoptosis. Analysis of miRNA-10b levels in 13 primary medulloblastoma samples revealed that the 2 patients with the highest levels of miRNA-10b had multiple recurrences (4.5) and died within 8 years of diagnosis, compared with the 11 patients with low levels of miRNA-10b who had a mean of 1.2 recurrences and nearly 40% long-term survival. The data presented here indicate that miRNA-10b may act as an oncomir in medulloblastoma tumorigenesis, and reveal a previously unreported mechanism with Bcl-2 as a mediator of the effects of miRNA-10b upon medulloblastoma cell survival. PMID:26394044

Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs.

PubMed

Khan, Aly A; Betel, Doron; Miller, Martin L; Sander, Chris; Leslie, Christina S; Marks, Debora S

2009-06-01

Transfection of small RNAs (such as small interfering RNAs (siRNAs) and microRNAs (miRNAs)) into cells typically lowers expression of many genes. Unexpectedly, increased expression of genes also occurs. We investigated whether this upregulation results from a saturation effect--that is, competition among the transfected small RNAs and the endogenous pool of miRNAs for the intracellular machinery that processes small RNAs. To test this hypothesis, we analyzed genome-wide transcript responses from 151 published transfection experiments in seven different human cell types. We show that targets of endogenous miRNAs are expressed at significantly higher levels after transfection, consistent with impaired effectiveness of endogenous miRNA repression. This effect exhibited concentration and temporal dependence. Notably, the profile of endogenous miRNAs can be largely inferred by correlating miRNA sites with gene expression changes after transfections. The competition and saturation effects have practical implications for miRNA target prediction, the design of siRNA and short hairpin RNA (shRNA) genomic screens and siRNA therapeutics.

Circulating microRNA profiles and the identification of miR-593 and miR-511 which directly target the PROP1 gene in children with combined pituitary hormone deficiency.

PubMed

Hu, Yanyan; Wang, Qian; Wang, Zengmin; Wang, Fengxue; Guo, Xiaobo; Li, Guimei

2015-02-01

Since the tissue of children with combined pituitary hormone deficiency (CPHD) is not readily accessible, a new focus in children with CPHD is the blood-based expression profiling of non-protein coding genes, such as microRNAs (miRNAs or miRs), which regulate gene expression by inhibiting the translation of mRNAs. In this study, to address this, we identified potential miRNA signatures for CPHD by comparing genome-wide miRNA expression profiles in the serum of children with CPHD vs. normal (healthy) controls. Human embryonic kidney 293T cells were transfected with miR-593 or miR-511 oligonucleotides. Potential target gene expression was validated by western blot analysis for proteins and by miR-593 or miR-511 reporter assay using PROP1 gene 3'-untranslated region (3'-UTR) reporter. The miR-593 and miR-511 levels in the serum of 103 children with CPHD were assessed using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method. We found 23 upregulated and 19 downregulated miRNAs with abnormal expression in children with CPHD compared with the normal controls using miRNA microarray analysis and RT-qPCR. miR-593 and miR-511 targeted the 3'-UTR of the PROP1 gene and attenuated the expression of PROP1. The levels of miR-593 and miR-511 in the serum of children with CPHD were increased compared with those in the control subjects. According to Youden's index, the sensitivity was 82.54 and 84.86%, and the specificity was 98.15 and 91.36% for miR-593 and miR-511, respectively. The various levels of specific miRNAs, particularly miR-593 and miR-511 whose direct target is the PROP1 gene, may serve as a non-invasive diagnostic biomarkers for children with CPHD.

The Effect of microRNAs in the Regulation of Human CYP3A4: a Systematic Study using a Mathematical Model

NASA Astrophysics Data System (ADS)

Wei, Zhiyun; Jiang, Songshan; Zhang, Yiting; Wang, Xiaofei; Peng, Xueling; Meng, Chunjie; Liu, Yichen; Wang, Honglian; Guo, Luo; Qin, Shengying; He, Lin; Shao, Fengmin; Zhang, Lirong; Xing, Qinghe

2014-03-01

CYP3A4 metabolizes more than 50% of the drugs on the market. The large inter-individual differences of CYP3A4 expression may contribute to the variability of human drug responses. Post-transcriptional regulation of CYP3A4 is poorly understood, whereas transcriptional regulation has been studied much more thoroughly. In this study, we used multiple software programs to predict miRNAs that might bind to CYP3A4 and identified 112 potentially functional miRNAs. Then a luciferase reporter system was used to assess the effect of the overexpression of each potentially functional miRNA in HEK 293T cells. Fourteen miRNAs that significantly decreased reporter activity were measured in human liver samples (N = 27) as candidate miRNAs. To establish a more effective way to analyze in vivo data for miRNA candidates, the relationship between functional miRNA and target mRNA was modeled mathematically. Taking advantage of this model, we found that hsa-miR-577, hsa-miR-1, hsa-miR-532-3p and hsa-miR-627 could significantly downregulate the translation efficiency of CYP3A4 mRNA in liver. This study used in silico, in vitro and in vivo methods to progressively screen functional miRNAs for CYP3A4 and to enhance our understanding of molecular events underlying the large inter-individual differences of CYP3A4 expression in human populations.

The miRNA and mRNA Signatures of Peripheral Blood Cells in Humans Infected with Trypanosoma brucei gambiense.

PubMed

Lueong, Smiths; Leong, Smiths; Simo, Gustave; Camara, Mamadou; Jamonneau, Vincent; Kabore, Jacques; Ilboudo, Hamidou; Bucheton, Bruno; Hoheisel, Jörg D; Clayton, Christine

2013-01-01

Simple, reliable tools for diagnosis of human African Trypanosomiases could ease field surveillance and enhance patient care. In particular, current methods to distinguish patients with (stage II) and without (stage I) brain involvement require samples of cerebrospinal fluid. We describe here an exploratory study to find out whether miRNAs from peripheral blood leukocytes might be useful in diagnosis of human trypanosomiasis, or for determining the stage of the disease. Using microarrays, we measured miRNAs in samples from Trypanosoma brucei gambiense-infected patients (9 stage I, 10 stage II), 8 seronegative parasite-negative controls and 12 seropositive, but parasite-negative subjects. 8 miRNAs (out of 1205 tested) showed significantly lower expression in patients than in seronegative, parasite-negative controls, and 1 showed increased expression. There were no clear differences in miRNAs between patients in different disease stages. The miRNA profiles could not distinguish seropositive, but parasitologically negative samples from controls and results within this group did not correlate with those from the trypanolysis test. Some of the regulated miRNAs, or their predicted mRNA targets, were previously reported changed during other infectious diseases or cancer. We conclude that the changes in miRNA profiles of peripheral blood lymphocytes in human African trypanosomiasis are related to immune activation or inflammation, are probably disease-non-specific, and cannot be used to determine the disease stage. The approach has little promise for diagnostics but might yield information about disease pathology.

Analysis of circulating miRNAs in patients with familial hypercholesterolaemia treated by LDL/Lp(a) apheresis.

PubMed

Dlouha, Dana; Blaha, Milan; Blaha, Vladimir; Fatorova, Ilona; Hubacek, Jaroslav A; Stavek, Petr; Lanska, Vera; Parikova, Alena; Pitha, Jan

2017-11-01

LDL/Lp(a) apheresis therapy is a well-established method of aggressively lowering LDL and Lp(a). Recently, miRNAs have been discussed as markers of vascular status including atherosclerosis. MiRNAs inhibit post-transcriptional processes through RNA duplex formation resulting in gene silencing or regulation of gene expression. We measured a profile of 175 plasma-circulating miRNAs using pre-defined Serum/Plasma Focus Human microRNA PCR Panels in pooled samples of 11 subjects with familial hypercholesterolaemia under long-term apheresis treatment. Subsequently we analysed expressions of ten pre-selected miRNAs potentially involved in lipid homeostasis in the same group of subjects. We compared plasma-circulating miRNA levels isolated from peripheral blood collected immediately before and after apheresis. The greatest differences in plasma levels were found in miR-451a, miR-16, miR-19a/b, miR-223 and miR-185. In subsequent individual miRNA assay we detected a significant increase in miR-33b levels after apheresis (P < 0.05). Additionally, correlations between plasma lipids and miR-33a (P < 0.04) and miR-122 (P < 0.01) have been determined. Moreover, miR-122 levels in LDLR homozygotes were higher compared to heterozygotes after, but not before, apheresis treatment (P < 0.04). LDL/Lp(a) apheresis has an impact on miRNAs associated with lipid homeostasis and vascular status. Copyright © 2017 Elsevier B.V. All rights reserved.

Altered expression of miRNAs in the uterus from a letrozole-induced rat PCOS model.

PubMed

Li, Chunjin; Chen, Lu; Zhao, Yun; Chen, Shuxiong; Fu, Lulu; Jiang, Yanwen; Gao, Shan; Liu, Zhuo; Wang, Fengge; Zhu, Xiaoling; Rao, Jiahui; Zhang, Jing; Zhou, Xu

2017-01-20

Polycystic ovary syndrome (PCOS) causes female subfertility with ovarian disorders and may be associated with increased rate of early-pregnancy failure. Rat PCOS models were established using letrozole to understand the uterine pathogenesis of PCOS. The differential expression of microRNAs (miRNAs) was observed in rat uterus with PCOS. After estrous cycles were disrupted, significantly abnormal ovarian morphology and hormone level were observed in rats with PCOS. A total of 148 miRNAs differentially expressed were identified in the uterus from the letrozole-induced rat model compared with the control. These miRNAs included 111 upregulated miRNAs and 37 downregulated miRNAs. The differential expression of miR-484, miR-375-3p, miR-324-5p, and miR-223-3p was further confirmed by quantitative reverse transcription polymerase chain reaction. Bioinformatic analysis showed that these four miRNAs were predicted to regulate a large number of genes with different functions. Pathway analysis supported that target genes of miRNAs were involved in insulin secretion and signaling pathways, such as wnt, AMPK, PI3K-Akt, and Ras. These data indicated that miRNAs differentially expressed in rat uterus with PCOS may be associated with PCOS pathogenesis in the uterus. Our findings can help clarify the mechanism of uterine defects in PCOS. Copyright © 2016. Published by Elsevier B.V.

Endothelial and circulating C19MC microRNAs are biomarkers of infantile hemangioma

PubMed Central

Strub, Graham M.; Kirsh, Andrew L.; Whipple, Mark E.; Kuo, Winston P.; Keller, Rachel B.; Kapur, Raj P.; Majesky, Mark W.; Perkins, Jonathan A.

2016-01-01

Infantile hemangioma (IH) is the most common vascular tumor of infancy, and it uniquely regresses in response to oral propranolol. MicroRNAs (miRNAs) have emerged as key regulators of vascular development and are dysregulated in many disease processes, but the role of miRNAs in IH growth has not been investigated. We report expression of C19MC, a primate-specific megacluster of miRNAs expressed in placenta with rare expression in postnatal tissues, in glucose transporter 1–expressing (GLUT-1–expressing) IH endothelial cells and in the plasma of children with IH. Tissue or circulating C19MC miRNAs were not detectable in patients having 9 other types of vascular anomalies or unaffected children, identifying C19MC miRNAs as the first circulating biomarkers of IH. Levels of circulating C19MC miRNAs correlated with IH tumor size and propranolol treatment response, and IH tissue from children treated with propranolol or from children with partially involuted tumors contained lower levels of C19MC miRNAs than untreated, proliferative tumors, implicating C19MC miRNAs as potential drivers of IH pathogenesis. Detection of C19MC miRNAs in the circulation of infants with IH may provide a specific and noninvasive means of IH diagnosis and identification of candidates for propranolol therapy as well as a means to monitor treatment response. PMID:27660822

Identification and Analysis of Expression of Novel MicroRNAs of Murine Gammaherpesvirus 68▿ †

PubMed Central

Zhu, Jia Yun; Strehle, Martin; Frohn, Anne; Kremmer, Elisabeth; Höfig, Kai P.; Meister, Gunter; Adler, Heiko

2010-01-01

Murine gammaherpesvirus 68 (MHV-68) is closely related to Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) and provides a small-animal model with which to study the pathogenesis of gammaherpesvirus (γHV) infections. To completely explore the potential of the MHV-68 system for the investigation of γHV microRNAs (miRNAs), it would be desirable to know the number and expression patterns of all miRNAs encoded by MHV-68. By deep sequencing of small RNAs, we systematically investigated the expression profiles of MHV-68 miRNAs in both lytically and persistently infected cells. In addition to the nine known MHV-68 miRNAs, we identified six novel MHV-68 miRNA genes and analyzed the expression levels of all MHV-68 miRNAs. Furthermore, we also characterized the cellular miRNA expression signatures in MHV-68-infected versus noninfected NIH 3T3 fibroblasts and in 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-treated versus nontreated S11 cells. We found that mmu-mir-15b and mmu-mir-16 are highly upregulated upon MHV-68 infection of NIH 3T3 cells, indicating a potential role for cellular miRNAs during MHV-68 infection. Our data will aid in the full exploration of the functions of γHV miRNAs. PMID:20668074

Non-targeted profiling of circulating microRNAs in women with polycystic ovary syndrome (PCOS): effects of obesity and sex hormones.

PubMed

Murri, Mora; Insenser, María; Fernández-Durán, Elena; San-Millán, José L; Luque-Ramírez, Manuel; Escobar-Morreale, Héctor F

2018-02-02

Circulating micro-ribonucleic acids (miRNAs) are small noncoding RNA molecules that influence gene transcription. We conducted the present profiling study to characterize the expression of circulating miRNAs in lean and obese patients with polycystic ovary syndrome (PCOS), the most common endocrine and metabolic disorder in premenopausal women. We selected 11 control women, 12 patients with PCOS and 12 men so that they were similar in terms of body mass index. Five control women, 6 men and 6 patients with PCOS had normal weight whereas 6 subjects per group were obese. We used miRCURY LNA™ Universal RT microRNA PCR for miRNA profiling. The expression of 38 miRNAs and was different between subjects with PCOS and male and female controls. The differences in 15 miRNAs followed a pattern suggestive of androgenization characterized by expression levels that were similar in patients with PCOS and men but were different compared with those of control women. The expression of 13 miRNAs in women with PCOS was similar to that of control women and different compared with the expression observed in men, suggesting sexual dimorphism and, lastly, we observed 5 miRNAs that were expressed differently in women with PCOS compared with both men and control women, suggesting a specific abnormality in expression associated with the syndrome. Obesity interacted with the differences in several of these miRNAs, and the expression levels of many of them correlated with the hirsutism score, sex hormones and/or indexes of obesity, adiposity and metabolic dysfunction. The present results suggest that several serum miRNAs are influenced by PCOS, sex hormones and obesity. Our findings may guide the targeted search of miRNAs as clinically relevant markers for PCOS and its association with obesity and metabolic dysfunction in future studies. Copyright © 2018. Published by Elsevier Inc.

Expression of microRNAs in bovine and human pre-implantation embryo culture media

PubMed Central

Kropp, Jenna; Salih, Sana M.; Khatib, Hasan

2014-01-01

MicroRNAs (miRNA) are short non-coding RNAs which act to regulate expression of genes driving numerous cellular processes. These RNAs are secreted within exosomes from cells into the extracellular environment where they may act as signaling molecules. In addition, they are relatively stable and are specifically expressed in association to certain cancers making them strong candidates as biological markers. Moreover, miRNAs have been detected in body fluids including urine, milk, saliva, semen, and blood plasma. However, it is unknown whether they are secreted by embryonic cells into the culture media. Given that miRNAs are expressed throughout embryonic cellular divisions and embryonic genome activation, we hypothesized that they are secreted from the embryo into the extracellular environment and may play a role in the developmental competence of bovine embryos. To test this hypothesis, bovine embryos were cultured individually from day 5 to day 8 of development in an in vitro fertilization system and gene expression of 5 miRNAs was analyzed in both embryos and culture media. Differential miRNA gene expression was observed between embryos that developed to the blastocyst stage and those that failed to develop from the morula to blastocyst stage, deemed degenerate embryos. MiR-25, miR-302c, miR-196a2, and miR-181a expression was found to be higher in degenerate embryos compared to blastocyst embryos. Interestingly, these miRNAs were also found to be expressed in the culture media of both bovine and human pre-implantation embryos. Overall, our results show for the first time that miRNAs are secreted from pre-implantation embryos into culture media and that miRNA expression may correlate with developmental competence of the embryo. Expression of miRNAs in in vitro culture media could allow for the development of biological markers for selection of better quality embryos and for subsequent successful pregnancy. PMID:24795753

A Rapid Screen for Host-Encoded miRNAs with Inhibitory Effects against Ebola Virus Using a Transcription- and Replication-Competent Virus-Like Particle System.

PubMed

Wang, Zhongyi; Li, Jiaming; Fu, Yingying; Zhao, Zongzheng; Zhang, Chunmao; Li, Nan; Li, Jingjing; Cheng, Hongliang; Jin, Xiaojun; Lu, Bing; Guo, Zhendong; Qian, Jun; Liu, Linna

2018-05-16

MicroRNAs (miRNAs) may become efficient antiviral agents against the Ebola virus (EBOV) targeting viral genomic RNAs or transcripts. We previously conducted a genome-wide search for differentially expressed miRNAs during viral replication and transcription. In this study, we established a rapid screen for miRNAs with inhibitory effects against EBOV using a tetracistronic transcription- and replication-competent virus-like particle (trVLP) system. This system uses a minigenome comprising an EBOV leader region, luciferase reporter, VP40, GP, VP24, EBOV trailer region, and three noncoding regions from the EBOV genome and can be used to model the life cycle of EBOV under biosafety level (BSL) 2 conditions. Informatic analysis was performed to select up-regulated miRNAs targeting the coding regions of the minigenome with the highest binding energy to perform inhibitory effect screening. Among these miRNAs, miR-150-3p had the most significant inhibitory effect. Reverse transcription polymerase chain reaction (RT-PCR), Western blot, and double fluorescence reporter experiments demonstrated that miR-150-3p inhibited the reproduction of trVLPs via the regulation of GP and VP40 expression by directly targeting the coding regions of GP and VP40. This novel, rapid, and convenient screening method will efficiently facilitate the exploration of miRNAs against EBOV under BSL-2 conditions.

Impaired expression of DICER and some microRNAs in HBZ expressing cells from acute adult T-cell leukemia patients

PubMed Central

Gazon, Hélène; Belrose, Gildas; Terol, Marie; Meniane, Jean-Come; Mesnard, Jean-Michel; Césaire, Raymond; Peloponese, Jean-Marie

2016-01-01

Global dysregulation of microRNAs (miRNAs), a class of non-coding RNAs that regulate genes expression, is a common feature of human tumors. Profiling of cellular miRNAs on Adult T cell Leukemia (ATL) cells by Yamagishi et al. showed a strong decrease in expression for 96.7% of cellular miRNAs in ATL cells. However, the mechanisms that regulate the expression of miRNAs in ATL cells are still largely unknown. In this study, we compared the expression of 12 miRs previously described for being overexpress by Tax and the expression of several key components of the miRNAs biogenesis pathways in different HBZ expressing cell lines as well as in primary CD4 (+) cells from acute ATL patients. We showed that the expression of miRNAs and Dicer1 were downregulated in cells lines expressing HBZ as well as in fresh CD4 (+) cells from acute ATL patients. Using qRT-PCR, western blotting analysis and Chromatin Immunoprecipitation, we showed that dicer transcription was regulated by c-Jun and JunD, two AP-1 transcription factors. We also demonstrated that HBZ affects the expression of Dicer by removing JunD from the proximal promoter. Furthermore, we showed that at therapeutic concentration of 1mM, Valproate (VPA) an HDAC inhibitors often used in cancer treatment, rescue Dicer expression and miRNAs maturation. These results might offer a rationale for clinical studies of new combined therapy in an effort to improve the outcome of patients with acute ATL. PMID:26849145

Identification of miRNA-mRNA regulatory modules by exploring collective group relationships.

PubMed

Masud Karim, S M; Liu, Lin; Le, Thuc Duy; Li, Jiuyong

2016-01-11

microRNAs (miRNAs) play an essential role in the post-transcriptional gene regulation in plants and animals. They regulate a wide range of biological processes by targeting messenger RNAs (mRNAs). Evidence suggests that miRNAs and mRNAs interact collectively in gene regulatory networks. The collective relationships between groups of miRNAs and groups of mRNAs may be more readily interpreted than those between individual miRNAs and mRNAs, and thus are useful for gaining insight into gene regulation and cell functions. Several computational approaches have been developed to discover miRNA-mRNA regulatory modules (MMRMs) with a common aim to elucidate miRNA-mRNA regulatory relationships. However, most existing methods do not consider the collective relationships between a group of miRNAs and the group of targeted mRNAs in the process of discovering MMRMs. Our aim is to develop a framework to discover MMRMs and reveal miRNA-mRNA regulatory relationships from the heterogeneous expression data based on the collective relationships. We propose DIscovering COllective group RElationships (DICORE), an effective computational framework for revealing miRNA-mRNA regulatory relationships. We utilize the notation of collective group relationships to build the computational framework. The method computes the collaboration scores of the miRNAs and mRNAs on the basis of their interactions with mRNAs and miRNAs, respectively. Then it determines the groups of miRNAs and groups of mRNAs separately based on their respective collaboration scores. Next, it calculates the strength of the collective relationship between each pair of miRNA group and mRNA group using canonical correlation analysis, and the group pairs with significant canonical correlations are considered as the MMRMs. We applied this method to three gene expression datasets, and validated the computational discoveries. Analysis of the results demonstrates that a large portion of the regulatory relationships discovered by DICORE is consistent with the experimentally confirmed databases. Furthermore, it is observed that the top mRNAs that are regulated by the miRNAs in the identified MMRMs are highly relevant to the biological conditions of the given datasets. It is also shown that the MMRMs identified by DICORE are more biologically significant and functionally enriched.

Circulating Organ-Specific MicroRNAs Serve as Biomarkers in Organ-Specific Diseases: Implications for Organ Allo- and Xeno-Transplantation

PubMed Central

Zhou, Ming; Hara, Hidetaka; Dai, Yifan; Mou, Lisha; Cooper, David K. C.; Wu, Changyou; Cai, Zhiming

2016-01-01

Different cell types possess different miRNA expression profiles, and cell/tissue/organ-specific miRNAs (or profiles) indicate different diseases. Circulating miRNA is either actively secreted by living cells or passively released during cell death. Circulating cell/tissue/organ-specific miRNA may serve as a non-invasive biomarker for allo- or xeno-transplantation to monitor organ survival and immune rejection. In this review, we summarize the proof of concept that circulating organ-specific miRNAs serve as non-invasive biomarkers for a wide spectrum of clinical organ-specific manifestations such as liver-related disease, heart-related disease, kidney-related disease, and lung-related disease. Furthermore, we summarize how circulating organ-specific miRNAs may have advantages over conventional methods for monitoring immune rejection in organ transplantation. Finally, we discuss the implications and challenges of applying miRNA to monitor organ survival and immune rejection in allo- or xeno-transplantation. PMID:27490531

Changes in microRNA (miRNA) expression during pancreatic cancer development and progression in a genetically engineered KrasG12D;Pdx1-Cre mouse (KC) model.

PubMed

Rachagani, Satyanarayana; Macha, Muzafar A; Menning, Melanie S; Dey, Parama; Pai, Priya; Smith, Lynette M; Mo, Yin-Yuan; Batra, Surinder K

2015-11-24

Differential expression of microRNAs (miRNAs) has been demonstrated in various cancers, including pancreatic cancer (PC). Due to the lack of tissue samples from early-stages of PC, the stage-specific alteration of miRNAs during PC initiation and progression is largely unknown. In this study, we investigated the global miRNA expression profile and their processing machinery during PC progression using the KrasG12D;Pdx1-Cre (KC) mouse model. At 25 weeks, the miRNA microarray analysis revealed significant downregulation of miR-150, miR-494, miR-138, miR-148a, miR-216a, and miR-217 and upregulation of miR-146b, miR-205, miR-31, miR-192, and miR-21 in KC mice compared to controls. Further, expression of miRNA biosynthetic machinery including Dicer, Exportin-5, TRKRA, and TARBP2 were downregulated, while DGCR8 and Ago2 were upregulated in KC mice. In addition, from 10 to 50 weeks of age, stage-specific expression profiling of miRNA in KC mice revealed downregulation of miR-216, miR-217, miR-100, miR-345, miR-141, miR-483-3p, miR-26b, miR-150, miR-195, Let-7b and Let-96 and upregulation of miR-21, miR-205, miR-146b, miR-34c, miR-1273, miR-223 and miR-195 compared to control mice. Interestingly, the differential expression of miRNA in mice also corroborated with the miRNA expression in human PC cell lines and tissue samples; ectopic expression of Let-7b in CD18/HPAF and Capan1 cells resulted in the downregulation of KRAS and MSST1 expression. Overall, the present study aids an understanding of miRNA expression patterns during PC pathogenesis and helps to facilitate the identification of promising and novel early diagnostic/prognostic markers and therapeutic targets.

Changes in microRNA (miRNA) expression during pancreatic cancer development and progression in a genetically engineered KrasG12D;Pdx1-Cre mouse (KC) model

PubMed Central

Rachagani, Satyanarayana; Dey, Parama; Pai, Priya; Smith, Lynette M.; Mo, Yin-Yuan; Batra, Surinder K.

2015-01-01

Differential expression of microRNAs (miRNAs) has been demonstrated in various cancers, including pancreatic cancer (PC). Due to the lack of tissue samples from early-stages of PC, the stage-specific alteration of miRNAs during PC initiation and progression is largely unknown. In this study, we investigated the global miRNA expression profile and their processing machinery during PC progression using the KrasG12D;Pdx1-Cre (KC) mouse model. At 25 weeks, the miRNA microarray analysis revealed significant downregulation of miR-150, miR-494, miR-138, miR-148a, miR-216a, and miR-217 and upregulation of miR-146b, miR-205, miR-31, miR-192, and miR-21 in KC mice compared to controls. Further, expression of miRNA biosynthetic machinery including Dicer, Exportin-5, TRKRA, and TARBP2 were downregulated, while DGCR8 and Ago2 were upregulated in KC mice. In addition, from 10 to 50 weeks of age, stage-specific expression profiling of miRNA in KC mice revealed downregulation of miR-216, miR-217, miR-100, miR-345, miR-141, miR-483-3p, miR-26b, miR-150, miR-195, Let-7b and Let-96 and upregulation of miR-21, miR-205, miR-146b, miR-34c, miR-1273, miR-223 and miR-195 compared to control mice. Interestingly, the differential expression of miRNA in mice also corroborated with the miRNA expression in human PC cell lines and tissue samples; ectopic expression of Let-7b in CD18/HPAF and Capan1 cells resulted in downregulation of KRAS and MSST1 expression. Overall, the present study aids an understanding of miRNA expression patterns during PC pathogenesis and helps to facilitate the identification of promising and novel early diagnostic/prognostic markers and therapeutic targets. PMID:26516699

Molecular mechanisms of repeated social defeat-induced glucocorticoid resistance: Role of microRNA.

PubMed

Jung, Seung Ho; Wang, Yufen; Kim, Taewan; Tarr, Andrew; Reader, Brenda; Powell, Nicole; Sheridan, John F

2015-02-01

Glucocorticoid (GC) resistance is a severe problem associated with various inflammatory diseases. Previous studies have shown that repeated social stress induces GC resistance in innate immune cells, but the underlying molecular mechanisms have not been fully elucidated. Therefore, the purpose of this study was to examine potential underlying molecular mechanism(s) of repeated social defeat (RSD) stress on GC resistance in splenic macrophages. It was hypothesized that mRNA expression of receptors for GC and nuclear translocating-associated regulators in splenic macrophages would be affected by RSD, and that these changes would be associated with epigenetic modification. The data showed that the mRNA expression of GC and mineralocorticoid receptors were significantly decreased in splenic macrophages by RSD. RSD also induced a significantly decreased mRNA expression in FK506-binding protein 52 (FKBP52), consequently resulting in a significantly increased ratio of FKBP51 to FKBP52. Moreover, DNA methyltransferases 3a and 3b showed a significant decrease in their mRNA expression in the RSD group as did mRNA expression of histone deacetyltransferase 2. The RSD group also showed a significantly reduced quantity of methylated DNA in splenic macrophages. Based on microRNA (miRNA) profiling data, it was determined that RSD induced significantly increased expression of 9 different miRNAs that were predicted to interact with mRNAs of the GC receptor (6 miRNAs), mineralocorticoid receptor (3 miRNAs) and FKBP52 (2 miRNAs). Spearman correlation analysis revealed significantly strong correlations between the expression of 2 miRNAs and their target mRNA expression for GC receptors. Among these miRNAs, we verified direct effects of miRNA-29b and -340 overexpression on mRNA expression of GC receptors in L929 cells. The overexpression of miRNA-29b or -340 in L929 cells significantly reduced LPS-induced overexpression of GC receptors. In conclusion, this study provides evidence that epigenetic regulation, such as DNA methylation and miRNA expression, may play a role in the RSD-induced GC resistance that we have observed in splenic macrophages. Copyright © 2014 Elsevier Inc. All rights reserved.

Molecular Mechanisms of Repeated Social Defeat-Induced Glucocorticoid Resistance: Role of microRNA

PubMed Central

Jung, Seung Ho; Wang, Yufen; Kim, Taewan; Tarr, Andrew; Reader, Brenda; Powell, Nicole; Sheridan, John F.

2014-01-01

Glucocorticoid (GC) resistance is a severe problem associated with various inflammatory diseases. Previous studies have shown that repeated social stress induces GC resistance in innate immune cells, but the underlying molecular mechanisms have not been fully elucidated. Therefore, the purpose of this study was to examine potential underlying molecular mechanism(s) of repeated social defeat (RSD) stress on GC resistance in splenic macrophages. It was hypothesized that mRNA expression of receptors for GC and nuclear translocating-associated regulators in splenic macrophages would be affected by RSD, and that these changes would be associated with epigenetic modification. The data showed that the mRNA expression of GC and mineralocorticoid receptors were significantly decreased in splenic macrophages by RSD. RSD also induced a significantly decreased mRNA expression in FK506-binding protein 52 (FKBP52), consequently resulting in a significantly increased ratio of FKBP51 to FKBP52. Moreover, DNA methyltransferases 3a and 3b showed a significant decrease in their mRNA expression in the RSD group as did mRNA expression of histone deacetyltransferase 2. The RSD group also showed a significantly reduced quantity of methylated DNA in splenic macrophages. Based on microRNA (miRNA) profiling data, it was determined that RSD induced significantly increased expression of 9 different miRNAs that were predicted to interact with mRNAs of the GC receptor (6 miRNAs), mineralocorticoid receptor (3 miRNAs) and FKBP52 (2 miRNAs). Spearman correlation analysis revealed significantly strong correlations between the expression of 2 miRNAs and their target mRNA expression for GC receptors. Among these miRNAs, we verified direct effects of miRNA-29b and -340 overexpression on mRNA expression of GC receptors in L929 cells. The overexpression of miRNA-29b or -340 in L929 cells significantly reduced LPS-induced overexpression of GC receptors. In conclusion, this study provides evidence that epigenetic regulation, such as DNA methylation and miRNA expression, may play a role in the RSD-induced GC resistance that we have observed in splenic macrophages. PMID:25317829

Using corngrass1 to engineer poplar as a bioenergy crop

DOEpatents

Meilan, Richard; Rubinelli, Peter Marius; Chuck, George

2016-05-10

Embodiments of the present invention relate generally to new bioenergy crops and methods of creating new bioenergy crops. For example, genes encoding microRNAs (miRNAs) are used to create transgenic crops. In some embodiments, over-expression of miRNA is used to produce transgenic perennials, such as trees, with altered lignin content or composition. In some embodiments, the transgenic perennials are Populus spp. In some embodiments, the miRNA is a member of the miR156 family. In some embodiments, the gene is Zea mays Cg1.

A genome-wide survey of sexually dimorphic expression of Drosophila miRNAs identifies the steroid hormone-induced miRNA let-7 as a regulator of sexual identity.

PubMed

Fagegaltier, Delphine; König, Annekatrin; Gordon, Assaf; Lai, Eric C; Gingeras, Thomas R; Hannon, Gregory J; Shcherbata, Halyna R

2014-10-01

MiRNAs bear an increasing number of functions throughout development and in the aging adult. Here we address their role in establishing sexually dimorphic traits and sexual identity in male and female Drosophila. Our survey of miRNA populations in each sex identifies sets of miRNAs differentially expressed in male and female tissues across various stages of development. The pervasive sex-biased expression of miRNAs generally increases with the complexity and sexual dimorphism of tissues, gonads revealing the most striking biases. We find that the male-specific regulation of the X chromosome is relevant to miRNA expression on two levels. First, in the male gonad, testis-biased miRNAs tend to reside on the X chromosome. Second, in the soma, X-linked miRNAs do not systematically rely on dosage compensation. We set out to address the importance of a sex-biased expression of miRNAs in establishing sexually dimorphic traits. Our study of the conserved let-7-C miRNA cluster controlled by the sex-biased hormone ecdysone places let-7 as a primary modulator of the sex-determination hierarchy. Flies with modified let-7 levels present doublesex-related phenotypes and express sex-determination genes normally restricted to the opposite sex. In testes and ovaries, alterations of the ecdysone-induced let-7 result in aberrant gonadal somatic cell behavior and non-cell-autonomous defects in early germline differentiation. Gonadal defects as well as aberrant expression of sex-determination genes persist in aging adults under hormonal control. Together, our findings place ecdysone and let-7 as modulators of a somatic systemic signal that helps establish and sustain sexual identity in males and females and differentiation in gonads. This work establishes the foundation for a role of miRNAs in sexual dimorphism and demonstrates that similar to vertebrate hormonal control of cellular sexual identity exists in Drosophila. Copyright © 2014 by the Genetics Society of America.

Bioinformatic identification and expression analysis of banana microRNAs and their targets.

PubMed

Chai, Juan; Feng, Renjun; Shi, Hourui; Ren, Mengyun; Zhang, Yindong; Wang, Jingyi

2015-01-01

MicroRNAs (miRNAs) represent a class of endogenous non-coding small RNAs that play important roles in multiple biological processes by degrading targeted mRNAs or repressing mRNA translation. Thousands of miRNAs have been identified in many plant species, whereas only a limited number of miRNAs have been predicted in M. acuminata (A genome) and M. balbisiana (B genome). Here, previously known plant miRNAs were BLASTed against the Expressed Sequence Tag (EST) and Genomic Survey Sequence (GSS), a database of banana genes. A total of 32 potential miRNAs belonging to 13 miRNAs families were detected using a range of filtering criteria. 244 miRNA:target pairs were subsequently predicted, most of which encode transcription factors or enzymes that participate in the regulation of development, growth, metabolism, and other physiological processes. In order to validate the predicted miRNAs and the mutual relationship between miRNAs and their target genes, qRT-PCR was applied to detect the tissue-specific expression levels of 12 putative miRNAs and 6 target genes in roots, leaves, flowers, and fruits. This study provides some important information about banana pre-miRNAs, mature miRNAs, and miRNA target genes and these findings can be applied to future research of miRNA functions.

Bioinformatic Identification and Expression Analysis of Banana MicroRNAs and Their Targets

PubMed Central

Shi, Hourui; Ren, Mengyun; Zhang, Yindong; Wang, Jingyi

2015-01-01

MicroRNAs (miRNAs) represent a class of endogenous non-coding small RNAs that play important roles in multiple biological processes by degrading targeted mRNAs or repressing mRNA translation. Thousands of miRNAs have been identified in many plant species, whereas only a limited number of miRNAs have been predicted in M. acuminata (A genome) and M. balbisiana (B genome). Here, previously known plant miRNAs were BLASTed against the Expressed Sequence Tag (EST) and Genomic Survey Sequence (GSS), a database of banana genes. A total of 32 potential miRNAs belonging to 13 miRNAs families were detected using a range of filtering criteria. 244 miRNA:target pairs were subsequently predicted, most of which encode transcription factors or enzymes that participate in the regulation of development, growth, metabolism, and other physiological processes. In order to validate the predicted miRNAs and the mutual relationship between miRNAs and their target genes, qRT-PCR was applied to detect the tissue-specific expression levels of 12 putative miRNAs and 6 target genes in roots, leaves, flowers, and fruits. This study provides some important information about banana pre-miRNAs, mature miRNAs, and miRNA target genes and these findings can be applied to future research of miRNA functions. PMID:25856313

A comparison of microRNA expression profiles from splenic hemangiosarcoma, splenic nodular hyperplasia, and normal spleens of dogs.

PubMed

Grimes, Janet A; Prasad, Nripesh; Levy, Shawn; Cattley, Russell; Lindley, Stephanie; Boothe, Harry W; Henderson, Ralph A; Smith, Bruce F

2016-12-03

Splenic masses are common in older dogs; yet diagnosis preceding splenectomy and histopathology remains elusive. MicroRNAs (miRNAs) are short, non-coding RNAs that play a role in post-transcriptional regulation, and differential expression of miRNAs between normal and tumor tissue has been used to diagnose neoplastic diseases. The objective of this study was to determine differential expression of miRNAs by use of RNA-sequencing in canine spleens that were histologically confirmed as hemangiosarcoma, nodular hyperplasia, or normal. Twenty-two miRNAs were found to be differentially expressed in hemangiosarcoma samples (4 between hemangiosarcoma and both nodular hyperplasia and normal spleen and 18 between hemangiosarcoma and normal spleen only). In particular, mir-26a, mir-126, mir-139, mir-140, mir-150, mir-203, mir-424, mir-503, mir-505, mir-542, mir-30e, mir-33b, mir-365, mir-758, mir-22, and mir-452 are of interest in the pathogenesis of hemangiosarcoma. Findings of this study confirm the hypothesis that miRNA expression profiles are different between canine splenic hemangiosarcoma, nodular hyperplasia, and normal spleens. A large portion of the differentially expressed miRNAs have roles in angiogenesis, with an additional group of miRNAs being dysregulated in vascular disease processes. Two other miRNAs have been implicated in cancer pathways such as PTEN and cell cycle checkpoints. The finding of multiple miRNAs with roles in angiogenesis and vascular disease is important, as hemangiosarcoma is a tumor of endothelial cells, which are driven by angiogenic stimuli. This study shows that miRNA dysregulation is a potential player in the pathogenesis of canine splenic hemangiosarcoma.

Onco-GPCR signaling and dysregulated expression of microRNAs in human cancer.

PubMed

Nohata, Nijiro; Goto, Yusuke; Gutkind, J Silvio

2017-01-01

The G-protein-coupled receptor (GPCR) family is the largest family of cell-surface receptors involved in signal transduction. Aberrant expression of GPCRs and G proteins are frequently associated with prevalent human diseases, including cancer. In fact, GPCRs represent the therapeutic targets of more than a quarter of the clinical drugs currently on the market. MiRNAs (miRNAs) are also aberrantly expressed in many human cancers, and they have significant roles in the initiation, development and metastasis of human malignancies. Recent studies have revealed that dysregulation of miRNAs and their target genes expression are associated with cancer progression. The emerging information suggests that miRNAs play an important role in the fine tuning of many signaling pathways, including GPCR signaling. We summarize our current knowledge of the individual functions of miRNAs regulated by GPCRs and GPCR signaling-associated molecules, and miRNAs that regulate the expression and activity of GPCRs, their endogenous ligands and their coupled heterotrimeric G proteins in human cancer.

Analysis of TP53 gene expression and p53 level of human hypopharyngeal FaDu (HTB-43) head and neck cancer cell line after microRNA-181a inhibition.

PubMed

Cheah, Y K; Cheng, R W; Yeap, S K; Khoo, C H; See, H S

2014-03-17

The identification of new biomarkers for early detection of highly recurrent head and neck cancer is urgently needed. MicroRNAs (miRNAs) are small and non-coding RNAs that regulate cancer-related gene expression, such as tumor protein 53 (TP53) gene expression. This study was carried out to analyze TP53 gene expression using real-time PCR and to determine changes in intracellular p53 level by flow cytometry after downregulation of miRNA-181a miRNA inhibitor in the FaDu cell line. TP53 gene expression showed a 3-fold increment and the p53 protein level was also increased in the miRNA-181a-treated cells. In conclusion, miRNA-181a binds to the TP53 gene and inhibits its expression, decreasing the synthesis of p53.

Novel miRNA-31 and miRNA-200a-Mediated Regulation of Retinoblastoma Proliferation.

PubMed

Montoya, Vanessa; Fan, Hanli; Bryar, Paul J; Weinstein, Joanna L; Mets, Marilyn B; Feng, Gang; Martin, Joshua; Martin, Alissa; Jiang, Hongmei; Laurie, Nikia A

2015-01-01

Retinoblastoma is the most common intraocular tumor in children. Current management includes broad-based treatments such as chemotherapy, enucleation, laser therapy, or cryotherapy. However, therapies that target specific pathways important for retinoblastoma progression could provide valuable alternatives for treatment. MicroRNAs are short, noncoding RNA transcripts that can regulate the expression of target genes, and their aberrant expression often facilitates disease. The identification of post-transcriptional events that occur after the initiating genetic lesions could further define the rapidly aggressive growth displayed by retinoblastoma tumors. In this study, we used two phenotypically different retinoblastoma cell lines to elucidate the roles of miRNA-31 and miRNA-200a in tumor proliferation. Our approach confirmed that miRNAs-31 and -200a expression is significantly reduced in human retinoblastomas. Moreover, overexpression of these two miRNAs restricts the expansion of a highly proliferative cell line (Y79), but does not restrict the growth rate of a less aggressive cell line (Weri1). Gene expression profiling of miRNA-31 and/or miRNA-200a-overexpressing cells identified differentially expressed mRNAs associated with the divergent response of the two cell lines. This work has the potential to enhance the development of targeted therapeutic approaches for retinoblastoma and improve the efficacy of treatment.

ARNetMiT R Package: association rules based gene co-expression networks of miRNA targets.

PubMed

Özgür Cingiz, M; Biricik, G; Diri, B

2017-03-31

miRNAs are key regulators that bind to target genes to suppress their gene expression level. The relations between miRNA-target genes enable users to derive co-expressed genes that may be involved in similar biological processes and functions in cells. We hypothesize that target genes of miRNAs are co-expressed, when they are regulated by multiple miRNAs. With the usage of these co-expressed genes, we can theoretically construct co-expression networks (GCNs) related to 152 diseases. In this study, we introduce ARNetMiT that utilize a hash based association rule algorithm in a novel way to infer the GCNs on miRNA-target genes data. We also present R package of ARNetMiT, which infers and visualizes GCNs of diseases that are selected by users. Our approach assumes miRNAs as transactions and target genes as their items. Support and confidence values are used to prune association rules on miRNA-target genes data to construct support based GCNs (sGCNs) along with support and confidence based GCNs (scGCNs). We use overlap analysis and the topological features for the performance analysis of GCNs. We also infer GCNs with popular GNI algorithms for comparison with the GCNs of ARNetMiT. Overlap analysis results show that ARNetMiT outperforms the compared GNI algorithms. We see that using high confidence values in scGCNs increase the ratio of the overlapped gene-gene interactions between the compared methods. According to the evaluation of the topological features of ARNetMiT based GCNs, the degrees of nodes have power-law distribution. The hub genes discovered by ARNetMiT based GCNs are consistent with the literature.

Increased Expression of Herpes Virus-Encoded hsv1-miR-H18 and hsv2-miR-H9-5p in Cancer-Containing Prostate Tissue Compared to That in Benign Prostate Hyperplasia Tissue

PubMed Central

Shinn, Helen Ki; Yan, Chunri; Kim, Tae-Hwan; Kim, Sang Tae; Kim, Won Tae; Lee, Ok-Jun; Moon, Sung-Kwon; Kim, Nam-Hyung; Kim, Jayoung; Cha, Eun-Jong

2016-01-01

Purpose: Previously, we reported the presence of virus-encoded microRNAs (miRNAs) in the urine of prostate cancer (CaP) patients. In this study, we investigated the expression of two herpes virus-encoded miRNAs in prostate tissue. Methods: A total of 175 tissue samples from noncancerous benign prostatic hyperplasia (BPH), 248 tissue samples from patients with CaP and BPH, and 50 samples from noncancerous surrounding tissues from these same patients were analyzed for the expression of two herpes virus-encoded miRNAs by real-time polymerase chain reaction (PCR) and immunocytochemistry using nanoparticles as molecular beacons. Results: Real-time reverse transcription-PCR results revealed significantly higher expression of hsv1-miR-H18 and hsv2-miRH9- 5p in surrounding noncancerous and CaP tissues than that in BPH tissue (each comparison, P<0.001). Of note, these miRNA were expressed equivalently in the CaP tissues and surrounding noncancerous tissues. Moreover, immunocytochemistry clearly demonstrated a significant enrichment of both hsv1-miR-H18 and hsv2-miR-H9 beacon-labeled cells in CaP and surrounding noncancerous tissue compared to that in BPH tissue (each comparison, P<0.05 for hsv1-miR-H18 and hsv2- miR-H9). Conclusions: These results suggest that increased expression of hsv1-miR-H18 and hsv2-miR-H95p might be associated with tumorigenesis in the prostate. Further studies will be required to elucidate the role of these miRNAs with respect to CaP and herpes viral infections. PMID:27377944

MiR-203 involves in neuropathic pain development and represses Rap1a expression in nerve growth factor differentiated neuronal PC12 cells.

PubMed

Li, Haixia; Huang, Yuguang; Ma, Chao; Yu, Xuerong; Zhang, Zhiyong; Shen, Le

2015-01-01

Although microRNAs (miRNAs) have been shown to play a role in numerous biological processes, their function in neuropathic pain is not clear. The rat bilateral sciatic nerve chronic constriction injury (bCCI) is an established model of neuropathic pain, so we examined miRNA expression and function in the spinal dorsal horn in bCCI rats. Microarray and real-time polymerase chain reaction were used to examine the expression of miRNA in nerve system of bCCI rats, and the targets of miRNA were predicted by bioinformatic approaches. The function of specific miRNA was estimated through the methods of gene engineering. This study revealed substantially (∼10-fold) decreased miR-203 expression in the spinal dorsal horns but not the dorsal root ganglions, hippocampus, or anterior cingulate cortexes of bCCI rats. Rap1a protein expression was upregulated in bCCI rat spinal dorsal horns. We further verified that miR-203 directly targeted the 3'-untranslated region of the rap1a gene, thereby decreasing rap1a protein expression in neuron-like cells. Rap1a has diverse neuronal functions and their perturbation is responsible for several mental disorders. For example, Rap1a/MEK/ERK is involved in peripheral sensitization. These data suggest a potential role for miR-203 in regulating neuropathic pain development, and Rap1a is a validated target gene in vitro. Results from our study and others indicate the possibility that Rap1a may be involved in pain. We hope that these results can provide support for future research into miR-203 in gene therapy for neuropathic pain.

MicroRNA Expression Analysis in Serum of Patients with Congenital Hemochromatosis and Age-Related Macular Degeneration (AMD)

PubMed Central

Szemraj, Maciej; Oszajca, Katarzyna; Szemraj, Janusz; Jurowski, Piotr

2017-01-01

Background Congenital hemochromatosis is a disorder caused by mutations of genes involved in iron metabolism, leading to increased levels of iron concentration in tissues and serum. High concentrations of iron can lead to the development of AMD. The aim of this study was to analyze circulating miRNAs in the serum of congenital hemochromatosis patients with AMD and their correlation with the expression of genes involved in iron metabolism. Material/Methods Peripheral blood monolayer cells and serum were obtained from patients with congenital hemochromatosis, congenital hemochromatosis and AMD, AMD patients without congenital hemochromatosis, and healthy controls. Serum miRNAs expressions were analyzed by RT-PCR (qRT-PCR) using TaqMan MicroRNA probes, and proteins levels were measured by ELSA kits. Gene polymorphisms in TF and TFRC genes were determined using the TaqMan discrimination assay. Results Statistical analysis of the miRNAs expressions selected for further study the miR-31, miR-133a, miR-141, miR-145, miR-149, and miR-182, which are involved in the posttranscriptional expression of iron-related genes: TF, TFRI, DMT1, FTL, and FPN1. It was discovered that the observed changes in the expressions of the miRNAs was correlated with the level of protein in the serum of the analyzed genes. There were no statistically significant differences in the distribution of genotype and allele frequencies in TF and TFRC genes between analyzed groups of patients. Conclusions The differences studied in the miRNA serum profile, in conjunction with the changes in the analyzed protein levels, may be useful in the early detection of congenital hemochromatosis in patients who may develop AMD disease. PMID:28827515

Human Milk Cells and Lipids Conserve Numerous Known and Novel miRNAs, Some of Which Are Differentially Expressed during Lactation

PubMed Central

Alsaweed, Mohammed; Lai, Ching Tat; Hartmann, Peter E.; Geddes, Donna T.; Kakulas, Foteini

2016-01-01

Human milk (HM) is rich in miRNAs, which are thought to contribute to infant protection and development. We used deep sequencing to profile miRNAs in the cell and lipid fractions of HM obtained post-feeding from 10 lactating women in months 2, 4, and 6 postpartum. In both HM fractions, 1,195 mature known miRNAs were identified, which were positively associated with the cell (p = 0.048) and lipid (p = 0.010) content of HM. An additional 5,167 novel miRNA species were predicted, of which 235 were high-confidence miRNAs. HM cells contained more known miRNAs than HM lipids (1,136 and 835 respectively, p<0.001). Although the profile of the novel miRNAs was very different between cells and lipids, with the majority conserved in the cell fraction and being mother-specific, 2/3 of the known miRNAs common between cells and lipids were similarly expressed (p>0.05). Great similarities between the two HM fractions were also found in the profile of the top 20 known miRNAs. These were largely similar also between the three lactation stages examined, as were the total miRNA concentration, and the number and expression of the known miRNAs common between cells and lipids (p>0.05). Yet, approximately a third of all known miRNAs were differentially expressed during the first 6 months of lactation (p<0.05), with more pronounced miRNA upregulation seen in month 4. These findings indicate that although the total miRNA concentration of HM cells and lipids provided to the infant does not change in first 6 months of lactation, the miRNA composition is altered, particularly in month 4 compared to months 2 and 6. This may reflect the remodeling of the gland in response to infant feeding patterns, which usually change after exclusive breastfeeding, suggesting adaptation to the infant’s needs. PMID:27074017

Mobile microRNAs hit the target.

PubMed

Gursanscky, Nial R; Searle, Iain R; Carroll, Bernard J

2011-11-01

MicroRNAs (miRNAs) are negative regulators of gene expression in eukaryotic organisms, whereas small interfering RNAs (siRNAs) guide host-cell defence against viruses, transposons and transgenes. A key issue in plant biology is whether miRNAs act only in cells in which they are formed, or if, like siRNAs, they also function after passive diffusion or active transportation into other cells. Recent reports show that miRNAs are indeed able to move between plant cells to direct developmental programming of gene expression. In both leaf and root development, miRNAs establish intercellular gradients of gene expression that are essential for cell and tissue differentiation. Gradients in gene expression also play crucial roles in animal development, and there is strong evidence for intercellular movement of miRNAs in animals. Thus, intercellular movement of miRNAs may be crucial to animal developmental biology as well as plants. © 2011 John Wiley & Sons A/S.

Analysis of microRNA and gene expression profiling in triazole fungicide-treated HepG2 cell line.

PubMed

An, Yu Ri; Kim, Seung Jun; Oh, Moon-Ju; Kim, Hyun-Mi; Shim, Il-Seob; Kim, Pil-Je; Choi, Kyunghee; Hwang, Seung Yong

2013-01-07

MicroRNA (miRNA) plays an important role in various diseases and in cellular and molecular responses to toxicants. In the present study, we investigated differential expression of miRNAs in response to three triazole fungicides (myclobutanil, propiconazole, and triadimefon). The human hepatoma cell line (HepG2) was treated with the above triazoles for 3 h or 48 h. miRNA-based microarray experiments were carried out using the Agilent human miRNA v13 array. At early exposure (3h), six miRNAs were differentially expressed and at late exposure (48 h), three miRNAs were significantly expressed. Overall, this study provides an array of potential biomarkers for the above triazole fungicides. Furthermore, these miRNAs induced by triazoles could be the foundation for the development of a miRNA-based toxic biomarker library that can predict environmental toxicity. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

MicroRNAs – Important Molecules in Lung Cancer Research

PubMed Central

Leidinger, Petra; Keller, Andreas; Meese, Eckart

2011-01-01

MicroRNAs (miRNA) are important regulators of gene expression. They are involved in many physiological processes ensuring the cellular homeostasis of human cells. Alterations of the miRNA expression have increasingly been associated with pathophysiologic changes of cancer cells making miRNAs currently to one of the most analyzed molecules in cancer research. Here, we provide an overview of miRNAs in lung cancer. Specifically, we address biological functions of miRNAs in lung cancer cells, miRNA signatures generated from tumor tissue and from patients’ body fluids, the potential of miRNAs as diagnostic and prognostic biomarker for lung cancer, and its role as therapeutic target. PMID:22303398

Human miRNome profiling in colorectal cancer and liver metastasis development

PubMed Central

Perilli, Lisa; Pizzini, Silvia; Bisognin, Andrea; Mandruzzato, Susanna; Biasiolo, Marta; Facciolli, Arianna; Rossi, Elisabetta; Esposito, Giovanni; Rugge, Massimo; Pilati, Pierluigi; Mocellin, Simone; Nitti, Donato; Bortoluzzi, Stefania; Zanovello, Paola

2014-01-01

Qualitative alterations or abnormal expression of microRNAs (miRNAs) in colorectal cancer has mainly been demonstrated in primary tumors. The miRNA expression profiles in 78 samples from 46 patients were analyzed to identify changes in miRNA expression level among normal colon mucosa, primary tumor and liver metastasis samples. Using this dataset, we describe the interplay of miRNA groups in regulating pathways that are important for tumor development. Here we describe in details the contents and quality controls for the miRNA expression and clinical data associated with the study published by Pizzini and colleagues in the BMC Genomics in 2013 (Pizzini et al., 2013). Data are deposited in GEO database as GSE35834 series. PMID:26484092

Changes in mRNA expression precede changes in microRNA expression in lesional psoriatic skin during treatment with adalimumab.

PubMed

Raaby, L; Langkilde, A; Kjellerup, R B; Vinter, H; Khatib, S H; Hjuler, K F; Johansen, C; Iversen, L

2015-08-01

Tumour necrosis factor (TNF)-α inhibition is an effective treatment for moderate to severe plaque-type psoriasis. A change in the cytokine expression profile occurs in the skin after 4 days of treatment, preceding any clinical or histological improvements. MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression, but miRNA expression has never been studied in psoriatic skin during treatment. To investigate changes in miRNA expression in psoriatic skin during adalimumab treatment and to compare results with changes in miRNA expression in a mouse model of Aldara-induced psoriasis-like skin inflammation. Punch biopsies were obtained from nonlesional and lesional psoriatic skin during adalimumab treatment. In the mouse model of Aldara-induced skin inflammation, biopsies were obtained from TNF-α knockout (KO), IL-17A KO and wild-type mice. miRNA expression levels were analysed with microarray, reverse transcriptase quantitative polymerase chain reaction and in situ hybridization. In psoriatic skin, no changes in miRNA expression were seen 4 days after treatment initiation. After 14 days of treatment, the expression of several miRNAs was normalized towards the level seen in nonlesional skin before treatment. miR-23b expression increased after 14 days of treatment and remained high for 84 days, despite unaltered levels at baseline. In the mouse model of Aldara-induced skin inflammation, the level of miR-146a increased, whereas no regulation was seen for miR-203, miR-214-3p, miR-125a, miR-23b or let-7d-5p. This study demonstrates that the changes seen in the cytokine expression levels after 4 days of treatment with adalimumab are not facilitated by early changes in miRNA expression. © 2015 British Association of Dermatologists.

MiRNA-124 is a link between measles virus persistent infection and cell division of human neuroblastoma cells.

PubMed

Naaman, Hila; Rall, Glenn; Matullo, Christine; Veksler-Lublinsky, Isana; Shemer-Avni, Yonat; Gopas, Jacob

2017-01-01

Measles virus (MV) infects a variety of lymphoid and non-lymphoid peripheral organs. However, in rare cases, the virus can persistently infect cells within the central nervous system. Although some of the factors that allow MV to persist are known, the contribution of host cell-encoded microRNAs (miRNA) have not been described. MiRNAs are a class of noncoding RNAs transcribed from genomes of all multicellular organisms and some viruses, which regulate gene expression in a sequence-specific manner. We have studied the contribution of host cell-encoded miRNAs to the establishment of MV persistent infection in human neuroblastoma cells. Persistent MV infection was accompanied by differences in the expression profile and levels of several host cell-encoded microRNAs as compared to uninfected cells. MV persistence infection of a human neuroblastoma cell line (UKF-NB-MV), exhibit high miRNA-124 expression, and reduced expression of cyclin dependent kinase 6 (CDK6), a known target of miRNA-124, resulting in slower cell division but not cell death. By contrast, acute MV infection of UKF-NB cells did not result in increased miRNA-124 levels or CDK6 reduction. Ectopic overexpression of miRNA-124 affected cell viability only in UKF-NB-MV cells, causing cell death; implying that miRNA-124 over expression can sensitize cells to death only in the presence of MV persistent infection. To determine if miRNA-124 directly contributes to the establishment of MV persistence, UKF-NB cells overexpressing miRNA-124 were acutely infected, resulting in establishment of persistently infected colonies. We propose that miRNA-124 triggers a CDK6-dependent decrease in cell proliferation, which facilitates the establishment of MV persistence in neuroblastoma cells. To our knowledge, this is the first report to describe the role of a specific miRNA in MV persistence.

Dehydration-responsive miRNAs in foxtail millet: genome-wide identification, characterization and expression profiling.

PubMed

Yadav, Amita; Khan, Yusuf; Prasad, Manoj

2016-03-01

A set of novel and known dehydration-responsive miRNAs have been identified in foxtail millet. These findings provide new insights into understanding the functional role of miRNAs and their respective targets in regulating plant response to dehydration stress. MicroRNAs perform significant regulatory roles in growth, development and stress response of plants. Though the miRNA-mediated gene regulatory networks under dehydration stress remain largely unexplored in plant including foxtail millet (Setaria italica), which is a natural abiotic stress tolerant crop. To find out the dehydration-responsive miRNAs at the global level, four small RNA libraries were constructed from control and dehydration stress treated seedlings of two foxtail millet cultivars showing contrasting tolerance behavior towards dehydration stress. Using Illumina sequencing technology, 55 known and 136 novel miRNAs were identified, representing 22 and 48 miRNA families, respectively. Eighteen known and 33 novel miRNAs were differentially expressed during dehydration stress. After the stress treatment, 32 dehydration-responsive miRNAs were up-regulated in tolerant cultivar and 22 miRNAs were down-regulated in sensitive cultivar, suggesting that miRNA-mediated molecular regulation might play important roles in providing contrasting characteristics to these cultivars. Predicted targets of identified miRNAs were found to encode various transcription factors and functional enzymes, indicating their involvement in broad spectrum regulatory functions and biological processes. Further, differential expression patterns of seven known miRNAs were validated by northern blot and expression of ten novel dehydration-responsive miRNAs were confirmed by SL-qRT PCR. Differential expression behavior of five miRNA-target genes was verified under dehydration stress treatment and two of them also validated by RLM RACE. Overall, the present study highlights the importance of dehydration stress-associated post-transcriptional regulation governed by miRNAs and their targets in a naturally stress-tolerant model crop.

Identification of Viscum album L. miRNAs and prediction of their medicinal values

PubMed Central

Adolf, Jacob; Melzig, Matthias F.

2017-01-01

MicroRNAs (miRNAs) are a class of approximately 22 nucleotides single-stranded non-coding RNA molecules that play crucial roles in gene expression. It has been reported that the plant miRNAs might enter mammalian bloodstream and have a functional role in human metabolism, indicating that miRNAs might be one of the hidden bioactive ingredients in medicinal plants. Viscum album L. (Loranthaceae, European mistletoe) has been widely used for the treatment of cancer and cardiovascular diseases, but its functional compounds have not been well characterized. We considered that miRNAs might be involved in the pharmacological activities of V. album. High-throughput Illumina sequencing was performed to identify the novel and conserved miRNAs of V. album. The putative human targets were predicted. In total, 699 conserved miRNAs and 1373 novel miRNAs have been identified from V. album. Based on the combined use of TargetScan, miRanda, PITA, and RNAhybrid methods, the intersection of 30697 potential human genes have been predicted as putative targets of 29 novel miRNAs, while 14559 putative targets were highly enriched in 33 KEGG pathways. Interestingly, these highly enriched KEGG pathways were associated with some human diseases, especially cancer, cardiovascular diseases and neurological disorders, which might explain the clinical use as well as folk medicine use of mistletoe. However, further experimental validation is necessary to confirm these human targets of mistletoe miRNAs. Additionally, target genes involved in bioactive components synthesis in V. album were predicted as well. A total of 68 miRNAs were predicted to be involved in terpenoid biosynthesis, while two miRNAs including val-miR152 and miR9738 were predicted to target viscotoxins and lectins, respectively, which increased the knowledge regarding miRNA-based regulation of terpenoid biosynthesis, lectin and viscotoxin expressions in V. album. PMID:29112983

Identification of microRNAs in Wool Follicles during Anagen, Catagen, and Telogen Phases in Tibetan Sheep

PubMed Central

Liu, Guangbin; Liu, Ruize; Li, Qinqun; Tang, Xiaohui; Yu, Mei; Li, Xinyun; Cao, Jianhua; Zhao, Shuhong

2013-01-01

Background Wool quality is one of the most important economic traits in sheep. The wool fiber is derived from specialized skin cells that are referred to as wool follicles. To understand the roles of microRNAs (miRNAs) in wool fiber growth, we detected the expression patterns of miRNAs in wool follicles at the anagen, catagen, and telogen stages from Tibetan sheep through Solexa sequencing. Results A total of 244 mature miRNAs were identified. Of these, only five miRNAs are listed in the database of sheep miRNAs (miRBase Database V19), and the other 239 miRNAs have not been previously described in this species. Further analyses indicated that 204 miRNAs are evolutionarily conserved among mammal species, whereas 35 of the identified miRNAs were first found specifically in sheep. The expression pattern analyses showed that the expression levels of 39, 34, and 20 of the miRNAs significantly change between anagen and catagen, between anagen and telogen, and between catagen and telogen, respectively. The results of the bioinformatics analysis show that these differentially expressed miRNAs might regulate wool follicle development by targeting genes in many different pathways, such as the MAPK and Wnt pathways, as well as the pathways that regulate the actin cytoskeleton, focal adhesion, and tight junctions. Furthermore, we identified six differentially expressed miRNAs (oar-miR-103-3P, oar-miR-148b-3P, oar-miR-320-3P, oar-miR-31-5P, oar-novel-1-5P, and oar-novel-2-3P) that might target the key genes of the Wnt pathway. It has been reported that the Wnt pathway is critical for wool follicle development. Therefore, these miRNAs may regulate wool development through the Wnt pathway. Conclusions Our results provide new information on the identification and expression pattern of miRNAs in wool follicles. Our data might therefore aid in the understanding of the mechanisms of wool follicle development in sheep. PMID:24204975

Independent effects of sham laparotomy and anesthesia on hepatic microRNA expression in rats.

PubMed

Werner, Wiebke; Sallmon, Hannes; Leder, Annekatrin; Lippert, Steffen; Reutzel-Selke, Anja; Morgül, Mehmet Haluk; Jonas, Sven; Dame, Christof; Neuhaus, Peter; Iacomini, John; Tullius, Stefan G; Sauer, Igor M; Raschzok, Nathanael

2014-10-08

Studies on liver regeneration following partial hepatectomy (PH) have identified several microRNAs (miRNAs) that show a regulated expression pattern. These studies involve major surgery to access the liver, which is known to have intrinsic effects on hepatic gene expression and may also affect miRNA screening results. We performed two-third PH or sham laparotomy (SL) in Wistar rats to investigate the effect of both procedures on miRNA expression in liver tissue and corresponding plasma samples by microarray and qRT-PCR analyses. As control groups, non-treated rats and rats undergoing anesthesia only were used. We found that 49 out of 323 miRNAs (15%) were significantly deregulated after PH in liver tissue 12 to 48 hours postoperatively (>20% change), while 45 miRNAs (14%) were deregulated following SL. Out of these miRNAs, 10 miRNAs were similarly deregulated after PH and SL, while one miRNA showed opposite regulation. In plasma, miRNA upregulation was observed for miR-133a and miR-133b following PH and SL, whereas miR-100 and miR-466c were similarly downregulated following anesthesia and surgery. We show that miRNAs are indeed regulated by sham laparotomy and anesthesia in rats. These findings illustrate the critical need for finding appropriate control groups in experimental surgery.

Massive deregulation of miRNAs from nuclear reprogramming errors during trophoblast differentiation for placentogenesis in cloned pregnancy.

PubMed

Hossain, Md Munir; Tesfaye, Dawit; Salilew-Wondim, Dessie; Held, Eva; Pröll, Maren J; Rings, Franca; Kirfel, Gregor; Looft, Christian; Tholen, Ernst; Uddin, Jasim; Schellander, Karl; Hoelker, Michael

2014-01-18

Low efficiency of Somatic Cell Nuclear Transfer (NT) has been widely addressed with high incidence of placental abnormalities due to genetic and epigenetic modifications. MiRNAs are shown to be major regulators of such modifications. The present study has been carried out to identify the expression patterns of 377 miRNAs, their functional associations and mechanism of regulation in bovine placentas derived from artificial insemination (AI), in vitro production (IVP) and NT pregnancies. This study reveals a massive deregulation of miRNAs as chromosomal cluster or miRNA families without sex-linkage in NT and in-vitro derived IVP placentas. Cell specific localization miRNAs in blastocysts and expression profiling of embryos and placentas at different developmental stages identified that the major deregulation of miRNAs exhibited in placentas at day 50 of pregnancies is found to be less dependent on global DNA methylation, rather than on aberrant miRNA biogenesis molecules. Among them, aberrant AGO2 expression due to hypermethylation of its promoter was evident. Along with other factors, aberrant AGO2 expression was observed to be associated with multiple defects in trophoblast differentiation through deregulation of miRNAs mediated mechanisms. These aberrant miRNA activities might be associated with genetic and epigenetic modifications in abnormal placentogenesis due to maldifferentiation of early trophoblast cell lineage in NT and IVP pregnancies. This study provides the first insight into genome wide miRNA expression, their role in regulation of trophoblast differentiation as well as abnormal placental development in Somatic Cell Nuclear Transfer pregnancies to pave the way to improve the efficiency of cloning by nuclear transfer.

Massive deregulation of miRNAs from nuclear reprogramming errors during trophoblast differentiation for placentogenesis in cloned pregnancy

PubMed Central

2014-01-01

Background Low efficiency of Somatic Cell Nuclear Transfer (NT) has been widely addressed with high incidence of placental abnormalities due to genetic and epigenetic modifications. MiRNAs are shown to be major regulators of such modifications. The present study has been carried out to identify the expression patterns of 377 miRNAs, their functional associations and mechanism of regulation in bovine placentas derived from artificial insemination (AI), in vitro production (IVP) and NT pregnancies. Results This study reveals a massive deregulation of miRNAs as chromosomal cluster or miRNA families without sex-linkage in NT and in-vitro derived IVP placentas. Cell specific localization miRNAs in blastocysts and expression profiling of embryos and placentas at different developmental stages identified that the major deregulation of miRNAs exhibited in placentas at day 50 of pregnancies is found to be less dependent on global DNA methylation, rather than on aberrant miRNA biogenesis molecules. Among them, aberrant AGO2 expression due to hypermethylation of its promoter was evident. Along with other factors, aberrant AGO2 expression was observed to be associated with multiple defects in trophoblast differentiation through deregulation of miRNAs mediated mechanisms. Conclusion These aberrant miRNA activities might be associated with genetic and epigenetic modifications in abnormal placentogenesis due to maldifferentiation of early trophoblast cell lineage in NT and IVP pregnancies. This study provides the first insight into genome wide miRNA expression, their role in regulation of trophoblast differentiation as well as abnormal placental development in Somatic Cell Nuclear Transfer pregnancies to pave the way to improve the efficiency of cloning by nuclear transfer. PMID:24438674

Integrated analyses of microRNAs demonstrate their widespread influence on gene expression in high-grade serous ovarian carcinoma.

PubMed

Creighton, Chad J; Hernandez-Herrera, Anadulce; Jacobsen, Anders; Levine, Douglas A; Mankoo, Parminder; Schultz, Nikolaus; Du, Ying; Zhang, Yiqun; Larsson, Erik; Sheridan, Robert; Xiao, Weimin; Spellman, Paul T; Getz, Gad; Wheeler, David A; Perou, Charles M; Gibbs, Richard A; Sander, Chris; Hayes, D Neil; Gunaratne, Preethi H

2012-01-01

The Cancer Genome Atlas (TCGA) Network recently comprehensively catalogued the molecular aberrations in 487 high-grade serous ovarian cancers, with much remaining to be elucidated regarding the microRNAs (miRNAs). Here, using TCGA ovarian data, we surveyed the miRNAs, in the context of their predicted gene targets. Integration of miRNA and gene patterns yielded evidence that proximal pairs of miRNAs are processed from polycistronic primary transcripts, and that intronic miRNAs and their host gene mRNAs derive from common transcripts. Patterns of miRNA expression revealed multiple tumor subtypes and a set of 34 miRNAs predictive of overall patient survival. In a global analysis, miRNA:mRNA pairs anti-correlated in expression across tumors showed a higher frequency of in silico predicted target sites in the mRNA 3'-untranslated region (with less frequency observed for coding sequence and 5'-untranslated regions). The miR-29 family and predicted target genes were among the most strongly anti-correlated miRNA:mRNA pairs; over-expression of miR-29a in vitro repressed several anti-correlated genes (including DNMT3A and DNMT3B) and substantially decreased ovarian cancer cell viability. This study establishes miRNAs as having a widespread impact on gene expression programs in ovarian cancer, further strengthening our understanding of miRNA biology as it applies to human cancer. As with gene transcripts, miRNAs exhibit high diversity reflecting the genomic heterogeneity within a clinically homogeneous disease population. Putative miRNA:mRNA interactions, as identified using integrative analysis, can be validated. TCGA data are a valuable resource for the identification of novel tumor suppressive miRNAs in ovarian as well as other cancers.

Next-generation sequencing identifies the natural killer cell microRNA transcriptome

PubMed Central

Fehniger, Todd A.; Wylie, Todd; Germino, Elizabeth; Leong, Jeffrey W.; Magrini, Vincent J.; Koul, Sunita; Keppel, Catherine R.; Schneider, Stephanie E.; Koboldt, Daniel C.; Sullivan, Ryan P.; Heinz, Michael E.; Crosby, Seth D.; Nagarajan, Rakesh; Ramsingh, Giridharan; Link, Daniel C.; Ley, Timothy J.; Mardis, Elaine R.

2010-01-01

Natural killer (NK) cells are innate lymphocytes important for early host defense against infectious pathogens and surveillance against malignant transformation. Resting murine NK cells regulate the translation of effector molecule mRNAs (e.g., granzyme B, GzmB) through unclear molecular mechanisms. MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate the translation of their mRNA targets, and are therefore candidates for mediating this control process. While the expression and importance of miRNAs in T and B lymphocytes have been established, little is known about miRNAs in NK cells. Here, we used two next-generation sequencing (NGS) platforms to define the miRNA transcriptomes of resting and cytokine-activated primary murine NK cells, with confirmation by quantitative real-time PCR (qRT-PCR) and microarrays. We delineate a bioinformatics analysis pipeline that identified 302 known and 21 novel mature miRNAs from sequences obtained from NK cell small RNA libraries. These miRNAs are expressed over a broad range and exhibit isomiR complexity, and a subset is differentially expressed following cytokine activation. Using these miRNA NGS data, miR-223 was identified as a mature miRNA present in resting NK cells with decreased expression following cytokine activation. Furthermore, we demonstrate that miR-223 specifically targets the 3′ untranslated region of murine GzmB in vitro, indicating that this miRNA may contribute to control of GzmB translation in resting NK cells. Thus, the sequenced NK cell miRNA transcriptome provides a valuable framework for further elucidation of miRNA expression and function in NK cell biology. PMID:20935160

miRNA expression in control and FSHD fetal human muscle biopsies.

PubMed

Portilho, Débora Morueco; Alves, Marcelo Ribeiro; Kratassiouk, Gueorgui; Roche, Stéphane; Magdinier, Frédérique; de Santana, Eliane Corrêa; Polesskaya, Anna; Harel-Bellan, Annick; Mouly, Vincent; Savino, Wilson; Butler-Browne, Gillian; Dumonceaux, Julie

2015-01-01

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disorder and is one of the most common forms of muscular dystrophy. We have recently shown that some hallmarks of FSHD are already expressed in fetal FSHD biopsies, thus opening a new field of investigation for mechanisms leading to FSHD. As microRNAs (miRNAs) play an important role in myogenesis and muscle disorders, in this study we compared miRNAs expression levels during normal and FSHD muscle development. Muscle biopsies were obtained from quadriceps of both healthy control and FSHD1 fetuses with ages ranging from 14 to 33 weeks of development. miRNA expression profiles were analyzed using TaqMan Human MicroRNA Arrays. During human skeletal muscle development, in control muscle biopsies we observed changes for 4 miRNAs potentially involved in secondary muscle fiber formation and 5 miRNAs potentially involved in fiber maturation. When we compared the miRNA profiles obtained from control and FSHD biopsies, we did not observe any differences in the muscle specific miRNAs. However, we identified 8 miRNAs exclusively expressed in FSHD1 samples (miR-330, miR-331-5p, miR-34a, miR-380-3p, miR-516b, miR-582-5p, miR-517* and miR-625) which could represent new biomarkers for this disease. Their putative targets are mainly involved in muscle development and morphogenesis. Interestingly, these FSHD1 specific miRNAs do not target the genes previously described to be involved in FSHD. This work provides new candidate mechanisms potentially involved in the onset of FSHD pathology. Whether these FSHD specific miRNAs cause deregulations during fetal development, or protect against the appearance of the FSHD phenotype until the second decade of life still needs to be investigated.

Characteristics of microRNAs enriched in specific cell types and primary tissue types in solid organs.

PubMed

Kriegel, Alison J; Liu, Yong; Liu, Pengyuan; Baker, Maria Angeles; Hodges, Matthew R; Hua, Xing; Liang, Mingyu

2013-12-01

Knowledge of miRNA expression and function in specific cell types in solid organs is limited because of difficulty in obtaining appropriate specimens. We used laser capture microdissection to obtain nine tissue regions from rats, including the nucleus of the solitary tract, hypoglossal motor nucleus, ventral respiratory column/pre-Bötzinger complex, and midline raphe nucleus from the brain stem, myocardium and coronary artery from the heart, and glomerulus, proximal convoluted tubule, and medullary thick ascending limb from the kidney. Each tissue region consists of or is enriched for a specific cell type. Differential patterns of miRNA expression obtained by deep sequencing of minute amounts of laser-captured cells were highly consistent with data obtained from real-time PCR analysis. miRNA expression patterns correctly clustered the specimens by tissue regions and then by primary tissue types (neural, muscular, or epithelial). The aggregate difference in miRNA profiles between tissue regions that contained the same primary tissue type was as large as one-half of the aggregate difference between primary tissue types. miRNAs differentially expressed between primary tissue types are more likely to be abundant miRNAs, while miRNAs differentially expressed between tissue regions containing the same primary tissue type were distributed evenly across the abundance spectrum. The tissue type-enriched miRNAs were more likely to target genes enriched for specific functional categories compared with either cell type-enriched miRNAs or randomly selected miRNAs. These data indicate that the role of miRNAs in determining characteristics of primary tissue types may be different than their role in regulating cell type-specific functions in solid organs.

Altered miRNA expression in aniline-mediated cell cycle progression in rat spleen.

PubMed

Wang, Gangduo; Wang, Jianling; Khan, M Firoze

2017-09-01

Aniline exposure is associated with toxicity to the spleen, however, early molecular events in aniline-induced cell cycle progression in the spleen remain unknown. MicroRNAs (miRNAs) have been implicated in tumor development by modulating key cell cycle regulators and controlling cell proliferation. This study was, therefore, undertaken on the expression of miRNAs, regulation of cyclins and cyclin-dependent kinases (CDKs) in an experimental condition that precedes a tumorigenic response. Male SD rats were treated with aniline (1 mmol/kg/day by gavage) for 7 days, and expression of miRNAs, cyclins and CDKs in rat spleens were analyzed. Microarray and/or qPCR analyses showed that aniline exposure led to significantly decreased miRNA expression of let-7a, miR-24, miR-34c, miR-100, miR-125b, and greatly increased miR-181a. The aberrant expression of miRNAs was associated with significantly increased protein expression of cyclins A, B1, D3 and E. Furthermore, remarkably enhanced expression of CDKs like CDK1, CDK2, CDK4, CDK6, especially p-CDK1 and p-CDK2 as well as alternations in the expression of pRB, p27, and CDC25A in the spleens of aniline-treated rats was also observed. The data suggest that aniline exposure leads to aberrant expression of miRNAs in the spleen which could be important in the regulation of cell cycle proteins. Our findings, thus, provide new insight into the role of miRNAs in cell cycle progression, which may contribute to aniline-induced tumorigenic response in the spleen.

The MicroRNA-200 Family Is Upregulated in Endometrial Carcinoma

PubMed Central

Snowdon, Jaime; Zhang, Xiao; Childs, Tim; Tron, Victor A.; Feilotter, Harriet

2011-01-01

Background MicroRNAs (miRNAs, miRs) are small non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. MicroRNAs are dysregulated in cancer and may play essential roles in tumorigenesis. Additionally, miRNAs have been shown to have prognostic and diagnostic value in certain types of cancer. The objective of this study was to identify dysregulated miRNAs in endometrioid endometrial adenocarcinoma (EEC) and the precursor lesion, complex atypical hyperplasia (CAH). Methodology We compared the expression profiles of 723 human miRNAs from 14 cases of EEC, 10 cases of CAH, and 10 normal proliferative endometria controls using Agilent Human miRNA arrays following RNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues. The expression of 4 dysregulated miRNAs was validated using real time reverse transcription-PCR. Results Forty-three miRNAs were dysregulated in EEC and CAH compared to normal controls (p<0.05). The entire miR-200 family (miR-200a/b/c, miR-141, and miR-429) was up-regulated in cases of EEC. Conclusions This information contributes to the candidate miRNA expression profile that has been generated for EEC and shows that certain miRNAs are dysregulated in the precursor lesion, CAH. These miRNAs in particular may play important roles in tumorigenesis. Examination of miRNAs that are consistently dysregulated in various studies of EEC, like the miR-200 family, will aid in the understanding of the role that miRNAs play in tumorigenesis in this tumour type. PMID:21897839

Combined RT-qPCR of mRNA and microRNA Targets within One Fluidigm Integrated Fluidic Circuit.

PubMed

Baldwin, Don A; Horan, Annamarie D; Hesketh, Patrick J; Mehta, Samir

2016-07-01

The ability to profile expression levels of a large number of mRNAs and microRNAs (miRNAs) within the same sample, using a single assay method, would facilitate investigations of miRNA effects on mRNA abundance and streamline biomarker screening across multiple RNA classes. A protocol is described for reverse transcription of long RNA and miRNA targets, followed by preassay amplification of the pooled cDNAs and quantitative PCR (qPCR) detection for a mixed panel of candidate RNA biomarkers. The method provides flexibility for designing custom target panels, is robust over a range of input RNA amounts, and demonstrated a high assay success rate.

MicroRNA Detection by Whole-Mount In Situ Hybridization in C. elegans.

PubMed

Andachi, Yoshiki; Kohara, Yuji

2018-01-01

MicroRNAs (miRNAs) loaded on argonaute proteins guide RNA-induced silencing complexes to target mRNAs. An excellent method to decipher the spatiotemporal expression patterns of miRNAs is whole-mount in situ hybridization (WISH), which has been successfully used in vertebrate embryos but still remains unavailable for many animal species. Here, we describe a WISH method for miRNA detection in Caenorhabditis elegans at both embryonic and post-embryonic stages. Strategies devised for detection include fixation of animals with carbodiimide at a high temperature and subsequent partial digestion of the fixed animals with an extremely high concentration of proteinase. WISH signals are visualized by staining with a chromogenic substrate or a fluorescent dye.

Chapter 17. Extension of endogenous primers as a tool to detect micro-RNA targets.

PubMed

Vatolin, Sergei; Weil, Robert J

2008-01-01

Mammalian cells express a large number of small, noncoding RNAs, including micro-RNAs (miRNAs), that can regulate both the level of a target mRNA and the protein produced by the target mRNA. Recognition of miRNA targets is a complicated process, as a single target mRNA may be regulated by several miRNAs. The potential for combinatorial miRNA-mediated regulation of miRNA targets complicates diagnostic and therapeutic applications of miRNAs. Despite significant progress in understanding the biology of miRNAs and advances in computational predictions of miRNA targets, methods that permit direct physical identification of miRNA-mRNA complexes in eukaryotic cells are still required. Several groups have utilized coimmunoprecipitation of RNA associated with a protein(s) that is part of the RNA silencing macromolecular complex. This chapter describes a detailed but straightforward strategy that identifies miRNA targets based on the assumption that small RNAs base paired with a complementary target mRNA can be used as a primer to synthesize cDNA that may be used for cloning, identification, and functional analysis.

Identification of Differentially Expressed miRNAs in Colorado Potato Beetles (Leptinotarsa decemlineata (Say)) Exposed to Imidacloprid.

PubMed

Morin, Mathieu D; Lyons, Pierre J; Crapoulet, Nicolas; Boquel, Sébastien; Morin, Pier Jr

2017-12-16

The Colorado potato beetle ( Leptinotarsa decemlineata (Say)) is a significant pest of potato plants that has been controlled for more than two decades by neonicotinoid imidacloprid. L. decemlineata can develop resistance to this agent even though the molecular mechanisms underlying this resistance are not well characterized. MicroRNAs (miRNAs) are short ribonucleic acids that have been linked to response to various insecticides in several insect models. Unfortunately, the information is lacking regarding differentially expressed miRNAs following imidacloprid treatment in L. decemlineata . In this study, next-generation sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) were used to identify modulated miRNAs in imidacloprid-treated versus untreated L. decemlineata . This approach identified 33 differentially expressed miRNAs between the two experimental conditions. Of interest, miR-282 and miR-989, miRNAs previously shown to be modulated by imidacloprid in other insects, and miR-100, a miRNA associated with regulation of cytochrome P450 expression, were significantly modulated in imidacloprid-treated beetles. Overall, this work presents the first report of a miRNA signature associated with imidacloprid exposure in L. decemlineata using a high-throughput approach. It also reveals interesting miRNA candidates that potentially underly imidacloprid response in this insect pest.

MicroRNA repertoire for functional genome research in tilapia identified by deep sequencing.

PubMed

Yan, Biao; Wang, Zhen-Hua; Zhu, Chang-Dong; Guo, Jin-Tao; Zhao, Jin-Liang

2014-08-01

The Nile tilapia (Oreochromis niloticus; Cichlidae) is an economically important species in aquaculture and occupies a prominent position in the aquaculture industry. MicroRNAs (miRNAs) are a class of noncoding RNAs that post-transcriptionally regulate gene expression involved in diverse biological and metabolic processes. To increase the repertoire of miRNAs characterized in tilapia, we used the Illumina/Solexa sequencing technology to sequence a small RNA library using pooled RNA sample isolated from the different developmental stages of tilapia. Bioinformatic analyses suggest that 197 conserved and 27 novel miRNAs are expressed in tilapia. Sequence alignments indicate that all tested miRNAs and miRNAs* are highly conserved across many species. In addition, we characterized the tissue expression patterns of five miRNAs using real-time quantitative PCR. We found that miR-1/206, miR-7/9, and miR-122 is abundantly expressed in muscle, brain, and liver, respectively, implying a potential role in the regulation of tissue differentiation or the maintenance of tissue identity. Overall, our results expand the number of tilapia miRNAs, and the discovery of miRNAs in tilapia genome contributes to a better understanding the role of miRNAs in regulating diverse biological processes.

Timescales and bottlenecks in miRNA-dependent gene regulation.

PubMed

Hausser, Jean; Syed, Afzal Pasha; Selevsek, Nathalie; van Nimwegen, Erik; Jaskiewicz, Lukasz; Aebersold, Ruedi; Zavolan, Mihaela

2013-12-03

MiRNAs are post-transcriptional regulators that contribute to the establishment and maintenance of gene expression patterns. Although their biogenesis and decay appear to be under complex control, the implications of miRNA expression dynamics for the processes that they regulate are not well understood. We derived a mathematical model of miRNA-mediated gene regulation, inferred its parameters from experimental data sets, and found that the model describes well time-dependent changes in mRNA, protein and ribosome density levels measured upon miRNA transfection and induction. The inferred parameters indicate that the timescale of miRNA-dependent regulation is slower than initially thought. Delays in miRNA loading into Argonaute proteins and the slow decay of proteins relative to mRNAs can explain the typically small changes in protein levels observed upon miRNA transfection. For miRNAs to regulate protein expression on the timescale of a day, as miRNAs involved in cell-cycle regulation do, accelerated miRNA turnover is necessary.

Clinical value of integrated-signature miRNAs in esophageal cancer.

PubMed

Zhang, Heng-Chao; Tang, Kai-Fu

2017-08-01

MicroRNAs (miRNAs) are crucial regulators of gene expression in tumorigenesis and are of great interest to researchers, but miRNA profiles are often inconsistent between studies. The aim of this study was to confirm candidate miRNA biomarkers for esophageal cancer from integrated-miRNA expression profiling data and TCGA (The Cancer Genome Atlas) data in tissues. Here, we identify five significant miRNAs by a comprehensive analysis in esophageal cancer, and two of them (hsa-miR-100-5p and hsa-miR-133b) show better prognoses with significant difference for both 3-year and 5-year survival. Additionally, they participate in esophageal cancer occurrence and development according to KEGG and Panther enrichment analyses. Therefore, these five miRNAs may serve as miRNA biomarkers in esophageal cancer. Analysis of differential expression for target genes of these miRNAs may also provide new therapeutic alternatives in esophageal cancer. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Cigarette-Smoke-Induced Dysregulation of MicroRNA Expression and Its Role in Lung Carcinogenesis

PubMed Central

Russ, Rebecca; Slack, Frank J.

2012-01-01

Dysregulation of microRNAs (miRNAs), particularly their downregulation, has been widely shown to be associated with the development of lung cancer. Downregulation of miRNAs leads to the overactivation of their oncogene targets, while upregulation of some miRNAs leads to inhibition of important tumor suppressors. Research has implicated cigarette smoke in miRNA dysregulation, leading to carcinogenesis. Cigarette smoke may lead to genetic or epigenetic damage to miRNAs, many of which map to fragile sites and some of which contain single nucleotide polymorphisms. Cigarette smoke may also cause dysregulation by affecting regulatory mechanisms controlling miRNA expression. Researchers have shown a correlation between smoke-exposure-induced dysregulation of miRNAs and age. Furthermore, dysregulation seems to be associated with intensity and duration of smoke exposure and duration of cessation. Longer exposure at a threshold level is needed for irreversibility of changes in expression. Better understanding of miRNA dysregulation may allow for improved biomonitoring and treatment regimens for lung cancer. PMID:22191027

Involvement of microRNA-133 and -29 in cardiac disturbances in diabetic ovariectomized rats.

PubMed

Habibi, Parisa; Alihemmati, Alireza; Nasirzadeh, Mohammadreza; Yousefi, Hadi; Habibi, Mohammadrasoul; Ahmadiasl, Nasser

2016-11-01

Menopause and diabetes obviously increase the risk of cardiovascular disease in women. The aims of the present study were to evaluate the effects of ovariectomy in type 2 diabetes on the histology and expression of miRNA-29, miRNA-133, IGF-1 and Bcl-2 genes and Bcl-2 protein and caspase 3 activity in the hearts of female rats. Forty Female Wistar rats were divided into four groups: control, sham, ovariectomized (OVX), and ovariectomized with type 2 diabetes (OVX.D). After the 8-week experiment, the histological evaluation of the heart tissue was performed using H&E staining and PAS analysis, and cardiac expression of miRNA-29, miRNA-133, IGF-1, and Bcl-2 were evaluated using real-time PCR, and Bcl-2 protein and caspase 3 activity were evaluated using Western blot and ELISA. Ovariectomy significantly decreased miRNA-29, miRNA-133, IGF-1, and BCL-2 expression and Bcl-2 protein and increased caspase 3 activity in the heart compared to sham animals group (P<0.05). Type 2 diabetes in ovariectomized rats markedly decreased expression of miRNA-29, miRNA-133, IGF-1, BCL-2 genes, and Bcl-2 protein, and increased caspase 3 activity and reduced collagen and fibroblast tissue and glycogen granule deposition in relation to OVX group (P<0.05). Our findings suggest that type 2 diabetes and menopause synergically could enhance the cardiac fibrosis through dysregulation of miRNA-29, miRNA-133, IGF-1, and Bcl-2 genes expression and Bcl-2 protein and upregulation of caspase 3 activity.

In vivo delivery of miRNAs for cancer therapy: Challenges and strategies⋆

PubMed Central

Chen, Yunching; Gao, Dong-Yu; Huang, Leaf

2016-01-01

MicroRNAs (miRNAs), small non-coding RNAs, can regulate post-transcriptional gene expressions and silence a broad set of target genes. miRNAs, aberrantly expressed in cancer cells, play an important role in modulating gene expressions, thereby regulating downstream signaling pathways and affecting cancer formation and progression. Oncogenes or tumor suppressor genes regulated by miRNAs mediate cell cycle progression, metabolism, cell death, angiogenesis, metastasis and immunosuppression in cancer. Recently, miRNAs have emerged as therapeutic targets or tools and biomarkers for diagnosis and therapy monitoring in cancer. Since miRNAs can regulate multiple cancer-related genes simultaneously, using miRNAs as a therapeutic approach plays an important role in cancer therapy. However, one of the major challenges of miRNA-based cancer therapy is to achieve specific, efficient and safe systemic delivery of therapeutic miRNAs In vivo. This review discusses the key challenges to the development of the carriers for miRNA-based therapy and explores current strategies to systemically deliver miRNAs to cancer without induction of toxicity. PMID:24859533

microRNAs and Their Targets in Apple (Malus domestica cv. "Fuji") Involved in Response to Infection of Pathogen Valsa mali.

PubMed

Feng, Hao; Xu, Ming; Zheng, Xiang; Zhu, Tongyi; Gao, Xiaoning; Huang, Lili

2017-01-01

miRNAs are important regulators involving in plant-pathogen interactions. However, their roles in apple tree response to Valsa canker pathogen ( Valsa mali, Vm ) infection were poorly understood. In this study, we constructed two miRNA libraries using the twig bark tissues of apple tree ( Malus domestica Borkh. cv. "Fuji") inoculated with Vm (IVm) and PDA medium (control, BMd). Among all detected miRNAs, 23 miRNAs were specifically isolated from BMd and 39 miRNAs were specifically isolated from IVm. Meanwhile, the expression of 294 miRNAs decreased; and another 172 miRNAs showed an increased expression trend in IVm compared with that in BMd. Furthermore, two degradome sequencing libraries were also constructed to identify the target genes of these miRNAs. In total, 353 differentially expressed miRNAs between IVm and BMd were detected to be able to target 1,077 unigenes with 2,251 cleavage sites. Based on GO and KEGG analysis, these genes were found to be mainly related to transcription regulation and signal transduction. In addition, we selected 17 miRNAs and 22 corresponding target genes to screen the expression profiles when apple twigs were infected by Vm . The expression trends of most miRNAs/target genes were consist with the results of deep sequencing. Many of them may involve in the apple twig- Vm interaction by inducing/reducing their expression. What's more, miRNAs and their target genes regulate the apple twig- Vm interaction by forming many complicated regulation networks rather than one to one model. It is worth that a conserved miRNAs mdm-miR482b, which was down regulated in IVm compared with BMd, has 14 potential target genes, most of which are disease resistance related genes. This indicates that mdm-miR482b may play important roles in apple twig response to Vm . More important, the feedback regulation of sRNA pathway in apple twig is also very complex, and play critical role in the interaction between apple twig and Vm based on the results of expression analysis. In all, the results will provide insights into the crucial functions of miRNAs in the woody plant, apple tree- Vm interaction.

microRNAs and Their Targets in Apple (Malus domestica cv. “Fuji”) Involved in Response to Infection of Pathogen Valsa mali

PubMed Central

Feng, Hao; Xu, Ming; Zheng, Xiang; Zhu, Tongyi; Gao, Xiaoning; Huang, Lili

2017-01-01

miRNAs are important regulators involving in plant-pathogen interactions. However, their roles in apple tree response to Valsa canker pathogen (Valsa mali, Vm) infection were poorly understood. In this study, we constructed two miRNA libraries using the twig bark tissues of apple tree (Malus domestica Borkh. cv. “Fuji”) inoculated with Vm (IVm) and PDA medium (control, BMd). Among all detected miRNAs, 23 miRNAs were specifically isolated from BMd and 39 miRNAs were specifically isolated from IVm. Meanwhile, the expression of 294 miRNAs decreased; and another 172 miRNAs showed an increased expression trend in IVm compared with that in BMd. Furthermore, two degradome sequencing libraries were also constructed to identify the target genes of these miRNAs. In total, 353 differentially expressed miRNAs between IVm and BMd were detected to be able to target 1,077 unigenes with 2,251 cleavage sites. Based on GO and KEGG analysis, these genes were found to be mainly related to transcription regulation and signal transduction. In addition, we selected 17 miRNAs and 22 corresponding target genes to screen the expression profiles when apple twigs were infected by Vm. The expression trends of most miRNAs/target genes were consist with the results of deep sequencing. Many of them may involve in the apple twig-Vm interaction by inducing/reducing their expression. What's more, miRNAs and their target genes regulate the apple twig-Vm interaction by forming many complicated regulation networks rather than one to one model. It is worth that a conserved miRNAs mdm-miR482b, which was down regulated in IVm compared with BMd, has 14 potential target genes, most of which are disease resistance related genes. This indicates that mdm-miR482b may play important roles in apple twig response to Vm. More important, the feedback regulation of sRNA pathway in apple twig is also very complex, and play critical role in the interaction between apple twig and Vm based on the results of expression analysis. In all, the results will provide insights into the crucial functions of miRNAs in the woody plant, apple tree-Vm interaction. PMID:29270184

Identification of miRNAs during mouse postnatal ovarian development and superovulation.

PubMed

Khan, Hamid Ali; Zhao, Yi; Wang, Li; Li, Qian; Du, Yu-Ai; Dan, Yi; Huo, Li-Jun

2015-07-08

MicroRNAs are small noncoding RNAs that play critical roles in regulation of gene expression in wide array of tissues including the ovary through sequence complementarity at post-transcriptional level. Tight regulation of multitude of genes involved in ovarian development and folliculogenesis could be regulated at transcription level by these miRNAs. Therefore, tissue specific miRNAs identification is considered a key step towards understanding the role of miRNAs in biological processes. To investigate the role of microRNAs during ovarian development and folliculogenesis we sequenced eight different libraries using Illumina deep sequencing technology. Different developmental stages were selected to explore miRNAs expression pattern at different stages of gonadal maturation with/without treatment of PMSG/hCG for superovulation. From massive sequencing reads, clean reads of 16-26 bp were selected for further analysis of differential expression analysis and novel microRNA annotation. Expression analysis of all miRNAs at different developmental stages showed that some miRNAs were present ubiquitously while others were differentially expressed at different stages. Among differentially expressed miRNAs we reported 61 miRNAs with a fold change of more than 2 at different developmental stages among all libraries. Among the up-regulated miRNAs, mmu-mir-1298 had the highest fold change with 4.025 while mmu-mir-150 was down-regulated more than 3 fold. Furthermore, we found 2659 target genes for 20 differentially expressed microRNAs using seven different target predictions programs (DIANA-mT, miRanda, miRDB, miRWalk, RNAhybrid, PICTAR5, TargetScan). Analysis of the predicted targets showed certain ovary specific genes targeted by single or multiple microRNAs. Furthermore, pathway annotation and Gene ontology showed involvement of these microRNAs in basic cellular process. These results suggest the presence of different miRNAs at different stages of ovarian development and superovulation. Potential role of these microRNAs was elucidated using bioinformatics tools in regulation of different pathways, biological functions and cellular components underlying ovarian development and superovulation. These results provide a framework for extended analysis of miRNAs and their roles during ovarian development and superovulation. Furthermore, this study provides a base for characterization of individual miRNAs to discover their role in ovarian development and female fertility.

Global identification of microRNAs associated with chlorantraniliprole resistance in diamondback moth Plutella xylostella (L.)

PubMed Central

Zhu, Bin; Li, Xiuxia; Liu, Ying; Gao, Xiwu; Liang, Pei

2017-01-01

The diamondback moth (DBM), Plutella xylostella (L.), is one of the most serious cruciferous pests and has developed high resistance to most insecticides, including chlorantraniliprole. Previous studies have reported several protein-coding genes that involved in chlorantraniliprole resistance, but research on resistance mechanisms at the post-transcription level is still limited. In this study, a global screen of microRNAs (miRNAs) associated with chlorantraniliprole resistance in P. xylostella was performed. The small RNA libraries for a susceptible (CHS) and two chlorantraniliprole resistant strains (CHR, ZZ) were constructed and sequenced, and a total of 199 known and 30 novel miRNAs were identified. Among them, 23 miRNAs were differentially expressed between CHR and CHS, and 90 miRNAs were differentially expressed between ZZ and CHS, of which 11 differentially expressed miRNAs were identified in both CHR and ZZ. Using miRanda and RNAhybrid, a total of 1,411 target mRNAs from 102 differentially expressed miRNAs were predicted, including mRNAs in several groups of detoxification enzymes. The expression of several differentially expressed miRNAs and their potential targets was validated by qRT-PCR. The results may provide important clues for further study of the mechanisms of miRNA-mediated chlorantraniliprole resistance in DBM and other target insects. PMID:28098189

Induced miR-99a expression represses Mtor cooperatively with miR-150 to promote regulatory T-cell differentiation

PubMed Central

Warth, Sebastian C; Hoefig, Kai P; Hiekel, Anian; Schallenberg, Sonja; Jovanovic, Ksenija; Klein, Ludger; Kretschmer, Karsten; Ansel, K Mark; Heissmeyer, Vigo

2015-01-01

Peripheral induction of regulatory T (Treg) cells provides essential protection from inappropriate immune responses. CD4+ T cells that lack endogenous miRNAs are impaired to differentiate into Treg cells, but the relevant miRNAs are unknown. We performed an overexpression screen with T-cell-expressed miRNAs in naive mouse CD4+ T cells undergoing Treg differentiation. Among 130 candidates, the screen identified 29 miRNAs with a negative and 10 miRNAs with a positive effect. Testing reciprocal Th17 differentiation revealed specific functions for miR-100, miR-99a and miR-10b, since all of these promoted the Treg and inhibited the Th17 program without impacting on viability, proliferation and activation. miR-99a cooperated with miR-150 to repress the expression of the Th17-promoting factor mTOR. The comparably low expression of miR-99a was strongly increased by the Treg cell inducer “retinoic acid”, and the abundantly expressed miR-150 could only repress Mtor in the presence of miR-99a. Our data suggest that induction of Treg cell differentiation is regulated by a miRNA network, which involves cooperation of constitutively expressed as well as inducible miRNAs. PMID:25712478

MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias

PubMed Central

Calin, George Adrian; Liu, Chang-Gong; Sevignani, Cinzia; Ferracin, Manuela; Felli, Nadia; Dumitru, Calin Dan; Shimizu, Masayoshi; Cimmino, Amelia; Zupo, Simona; Dono, Mariella; Dell'Aquila, Marie L.; Alder, Hansjuerg; Rassenti, Laura; Kipps, Thomas J.; Bullrich, Florencia; Negrini, Massimo; Croce, Carlo M.

2004-01-01

Little is known about the expression levels or function of micro-RNAs (miRNAs) in normal and neoplastic cells, although it is becoming clear that miRNAs play important roles in the regulation of gene expression during development [Ambros, V. (2003) Cell 113, 673–676; McManus, M. T. (2003) Semin. Cancer Biol. 13, 253–258]. We now report the genomewide expression profiling of miRNAs in human B cell chronic lymphocytic leukemia (CLL) by using a microarray containing hundreds of human precursor and mature miRNA oligonucleotide probes. This approach allowed us to identify significant differences in miRNome expression between CLL samples and normal CD5+ B cells; data were confirmed by Northern blot analyses and real-time RT-PCR. At least two distinct clusters of CLL samples can be identified that were associated with the presence or absence of Zap-70 expression, a predictor of early disease progression. Two miRNA signatures were associated with the presence or absence of mutations in the expressed Ig variableregion genes or with deletions at 13q14, respectively. These data suggest that miRNA expression patterns have relevance to the biological and clinical behavior of this leukemia. PMID:15284443

MicroRNA Regulation of Glycolytic Metabolism in Glioblastoma

PubMed Central

McIntyre, Alan; Smith, Stuart

2017-01-01

Glioblastoma (GBM) is the most aggressive and common malignant brain tumour in adults. A well-known hallmark of GMB and many other tumours is aerobic glycolysis. MicroRNAs (miRNAs) are a class of short nonprotein coding sequences that exert posttranscriptional controls on gene expression and represent critical regulators of aerobic glycolysis in GBM. In GBM, miRNAs regulate the expression of glycolytic genes directly and via the regulation of metabolism-associated tumour suppressors and oncogenic signalling pathways. This review aims to establish links between miRNAs expression levels, the expression of GBM glycolytic regulatory genes, and the malignant progression and prognosis of GBM. In this review, the involvement of 25 miRNAs in the regulation of glycolytic metabolism of GBM is discussed. Seven of these miRNAs have been shown to regulate glycolytic metabolism in other tumour types. Further eight miRNAs, which are differentially expressed in GBM, have also been reported to regulate glycolytic metabolism in other cancer types. Thus, these miRNAs could serve as potential glycolytic regulators in GBM but will require functional validation. As such, the characterisation of these molecular and metabolic signatures in GBM can facilitate a better understanding of the molecular pathogenesis of this disease. PMID:28804724

Psmir: a database of potential associations between small molecules and miRNAs

PubMed Central

Meng, Fanlin; Wang, Jing; Dai, Enyu; Yang, Feng; Chen, Xiaowen; Wang, Shuyuan; Yu, Xuexin; Liu, Dianming; Jiang, Wei

2016-01-01

miRNAs are key post-transcriptional regulators of many essential biological processes, and their dysregulation has been validated in almost all human cancers. Restoring aberrantly expressed miRNAs might be a novel therapeutics. Recently, many studies have demonstrated that small molecular compounds can affect miRNA expression. Thus, prediction of associations between small molecules and miRNAs is important for investigation of miRNA-targeted drugs. Here, we analyzed 39 miRNA-perturbed gene expression profiles, and then calculated the similarity of transcription responses between miRNA perturbation and drug treatment to predict drug-miRNA associations. At the significance level of 0.05, we obtained 6501 candidate associations between 1295 small molecules and 25 miRNAs, which included 624 FDA approved drugs. Finally, we constructed the Psmir database to store all potential associations and the related materials. In a word, Psmir served as a valuable resource for dissecting the biological significance in small molecules’ effects on miRNA expression, which will facilitate developing novel potential therapeutic targets or treatments for human cancers. Psmir is supported by all major browsers, and is freely available at http://www.bio-bigdata.com/Psmir/. PMID:26759061

Psmir: a database of potential associations between small molecules and miRNAs.

PubMed

Meng, Fanlin; Wang, Jing; Dai, Enyu; Yang, Feng; Chen, Xiaowen; Wang, Shuyuan; Yu, Xuexin; Liu, Dianming; Jiang, Wei

2016-01-13

miRNAs are key post-transcriptional regulators of many essential biological processes, and their dysregulation has been validated in almost all human cancers. Restoring aberrantly expressed miRNAs might be a novel therapeutics. Recently, many studies have demonstrated that small molecular compounds can affect miRNA expression. Thus, prediction of associations between small molecules and miRNAs is important for investigation of miRNA-targeted drugs. Here, we analyzed 39 miRNA-perturbed gene expression profiles, and then calculated the similarity of transcription responses between miRNA perturbation and drug treatment to predict drug-miRNA associations. At the significance level of 0.05, we obtained 6501 candidate associations between 1295 small molecules and 25 miRNAs, which included 624 FDA approved drugs. Finally, we constructed the Psmir database to store all potential associations and the related materials. In a word, Psmir served as a valuable resource for dissecting the biological significance in small molecules' effects on miRNA expression, which will facilitate developing novel potential therapeutic targets or treatments for human cancers. Psmir is supported by all major browsers, and is freely available at http://www.bio-bigdata.com/Psmir/.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamrick, Mark W., E-mail: mhamrick@mail.mcg.edu; Department of Orthopaedic Surgery, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA; Herberg, Samuel

Research highlights: {yields} Aging is associated with muscle atrophy and loss of muscle mass, known as the sarcopenia of aging. {yields} We demonstrate that age-related muscle atrophy is associated with marked changes in miRNA expression in muscle. {yields} Treating aged mice with the adipokine leptin significantly increased muscle mass and the expression of miRNAs involved in muscle repair. {yields} Recombinant leptin therapy may therefore be a novel approach for treating age-related muscle atrophy. -- Abstract: Age-associated loss of muscle mass, or sarcopenia, contributes directly to frailty and an increased risk of falls and fractures among the elderly. Aged mice andmore » elderly adults both show decreased muscle mass as well as relatively low levels of the fat-derived hormone leptin. Here we demonstrate that loss of muscle mass and myofiber size with aging in mice is associated with significant changes in the expression of specific miRNAs. Aging altered the expression of 57 miRNAs in mouse skeletal muscle, and many of these miRNAs are now reported to be associated specifically with age-related muscle atrophy. These include miR-221, previously identified in studies of myogenesis and muscle development as playing a role in the proliferation and terminal differentiation of myogenic precursors. We also treated aged mice with recombinant leptin, to determine whether leptin therapy could improve muscle mass and alter the miRNA expression profile of aging skeletal muscle. Leptin treatment significantly increased hindlimb muscle mass and extensor digitorum longus fiber size in aged mice. Furthermore, the expression of 37 miRNAs was altered in muscles of leptin-treated mice. In particular, leptin treatment increased the expression of miR-31 and miR-223, miRNAs known to be elevated during muscle regeneration and repair. These findings suggest that aging in skeletal muscle is associated with marked changes in the expression of specific miRNAs, and that nutrient-related hormones such as leptin may be able to reverse muscle atrophy and alter the expression of atrophy-related miRNAs in aging skeletal muscle.« less

Identification of microRNAs in Response to Drought in Common Wild Rice (Oryza rufipogon Griff.) Shoots and Roots.

PubMed

Zhang, Jing-Wen; Long, Yan; Xue, Man-de; Xiao, Xing-Guo; Pei, Xin-Wu

2017-01-01

Drought is the most important factor that limits rice production in drought-prone environments. Plant microRNAs (miRNAs) are involved in biotic and abiotic stress responses. Common wild rice (Oryza rufipogon Griff.) contains abundant drought-resistant genes, which provide an opportunity to explore these excellent resources as contributors to improve rice resistance, productivity, and quality. In this study, we constructed four small RNA libraries, called CL and CR from PEG6000-free samples and DL and DR from PEG6000-treated samples, where 'R' indicates the root tissue and 'L' indicates the shoot tissue. A total of 200 miRNAs were identified to be differentially expressed under the drought-treated conditions (16% PEG6000 for 24 h), and the changes in the miRNA expression profile of the shoot were distinct from those of the root. At the miRNA level, 77 known miRNAs, which belong to 23 families, including 40 up-regulated and 37 down-regulated in the shoot, and 85 known miRNAs in 46 families, including 65 up-regulated and 20 down-regulated in the root, were identified as differentially expressed. In addition, we predicted 26 new miRNA candidates from the shoot and 43 from the root that were differentially expressed during the drought stress. The quantitative real-time PCR analysis results were consistent with high-throughput sequencing data. Moreover, 88 miRNAs that were differentially-expressed were predicted to match with 197 targets for drought-stress. Our results suggest that the miRNAs of O. rufipogon are responsive to drought stress. The differentially expressed miRNAs that are tissue-specific under drought conditions could play different roles in the regulation of the auxin pathway, the flowering pathway, the drought pathway, and lateral root formation. Thus, the present study provides an account of tissue-specific miRNAs that are involved in the drought adaption of O. rufipogon.

Identification of organ tissue types and skin from forensic samples by microRNA expression analysis.

PubMed

Sauer, Eva; Extra, Antje; Cachée, Philipp; Courts, Cornelius

2017-05-01

The identification of organ tissues in traces recovered from scenes and objects with regard to violent crimes involving serious injuries can be of considerable relevance in forensic investigations. Molecular genetic approaches are provably superior to histological and immunological assays in characterizing organ tissues, and micro-RNAs (miRNAs), due to their cell type specific expression patterns and stability against degradation, emerged as a promising molecular species for forensic analyses, with a range of tried and tested indicative markers. Thus, herein we present the first miRNA based approach for the forensic identification of organ tissues. Using quantitative PCR employing an empirically derived strategy for data normalization and unbiased statistical decision making, we assessed the differential expression of 15 preselected miRNAs in tissues of brain, kidney, lung, liver, heart muscle, skeletal muscle and skin. We show that not only can miRNA expression profiling be used to reliably differentiate between organ tissues but also that this method, which is compatible with and complementary to forensic DNA analysis, is applicable to realistic forensic samples e.g. mixtures, aged and degraded material as well as traces generated by mock stabbings and experimental shootings at ballistic models. Copyright © 2017 Elsevier B.V. All rights reserved.

Resistance Training Regulates Cardiac Function through Modulation of miRNA-214

PubMed Central

Melo, Stéphano Freitas Soares; Barauna, Valério Garrone; Júnior, Miguel Araújo Carneiro; Bozi, Luiz Henrique Marchesi; Drummond, Lucas Rios; Natali, Antônio José; de Oliveira, Edilamar Menezes

2015-01-01

Aims: To determine the effects of resistance training (RT) on the expression of microRNA (miRNA)-214 and its target in sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), and on the morphological and mechanical properties of isolated left ventricular myocytes. Main methods: Male Wistar rats were divided into two groups (n = 7/group): Control (CO) or trained (TR). The exercise-training protocol consisted of: 4 × 12 bouts, 5×/week during 8 weeks, with 80% of one repetition maximum. Key findings: RT increased the left ventricular myocyte width by 15% and volume by 12%, compared with control animals (p < 0.05). The time to half relaxation and time to peak were 8.4% and 4.4% lower, respectively, in cells from TR group as compared to CO group (p < 0.05). RT decreased miRNA-214 level by 18.5% while its target SERCA2a expression were 18.5% higher (p < 0.05). Significance: Our findings showed that RT increases single left ventricular myocyte dimensions and also leads to faster cell contraction and relaxation. These mechanical adaptations may be related to the augmented expression of SERCA2a which, in turn, may be associated with the epigenetic modification of decreased miRNA-214 expression. PMID:25822872

Bioinformatic Identification of Potential MicroRNAs and Their Targets in the Lingzhi or Reishi Medicinal Mushroom Ganoderma lucidum (Higher Basidiomycetes).

PubMed

Mu, Da-Shuai; Li, Chenyang; Shi, Liang; Zhang, Xuchen; Ren, Ang; Zhao, Ming-Wen

2015-01-01

MicroRNAs (miRNAs) are a class of small, endogenous, noncoding RNA molecules that negatively regulate gene expression at the transcriptional or the post-transcriptional level. Although a large number of miRNAs have been identified in many species, especially model plants and animals, miRNAs in fungi remain largely unknown. In this study, based on a database of expressed sequence tags in Ganoderma lucidum, 89 potential miRNAs were identified using computational methods. Real-time polymerase chain reaction analysis of miRNA-like samples prepared from G. lucidum at different development stages revealed that miRNA-like RNAs were differentially expressed in different stages. Furthermore, a total of 28 potential targets were found based on near-perfect or perfect complementarity between the randomly selected 9 miRNA-like RNAs and the target sequences, and potential targets for G. lucidum miRNA-like RNAs were predicted. Finally, we studied the expression pattern of 4 target genes in 3 different development stages of G. lucidum to further understand the mechanism of interaction between miRNA-like RNAs and their target genes. Our analysis paves the way toward identifying fungal miRNA-like RNAs that might be involved in various physiological and cellular differentiation processes.

MicroRNA-22 and microRNA-140 suppress NF-{kappa}B activity by regulating the expression of NF-{kappa}B coactivators

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takata, Akemi; Otsuka, Motoyuki, E-mail: otsukamo-tky@umin.ac.jp; Kojima, Kentaro

2011-08-12

Highlights: {yields} miRNAs were screened for their ability to regulate NF-{kappa}B activity. {yields} miRNA-22 and miRNA-140-3p suppress NF-{kappa}B activity by regulating coactivators. {yields} miRNA-22 targets nuclear receptor coactivator 1 (NCOA1). {yields} miRNA-140-3p targets nuclear receptor-interacting protein 1 (NRIP1). -- Abstract: Nuclear factor {kappa}B (NF-{kappa}B) is a transcription factor that regulates a set of genes that are critical to many biological phenomena, including liver tumorigenesis. To identify microRNAs (miRNAs) that regulate NF-{kappa}B activity in the liver, we screened 60 miRNAs expressed in hepatocytes for their ability to modulate NF-{kappa}B activity. We found that miRNA-22 and miRNA-140-3p significantly suppressed NF-{kappa}B activity bymore » regulating the expression of nuclear receptor coactivator 1 (NCOA1) and nuclear receptor-interacting protein 1 (NRIP1), both of which are NF-{kappa}B coactivators. Our results provide new information about the roles of miRNAs in the regulation of NF-{kappa}B activity.« less

Changes in miRNA expression profile of space-flown Caenorhabditis elegans during Shenzhou-8 mission

NASA Astrophysics Data System (ADS)

Xu, Dan; Gao, Ying; Huang, Lei; Sun, Yeqing

2014-04-01

Recent advances in the field of molecular biology have demonstrated that small non-coding microRNAs (miRNAs) have a broad effect on gene expression networks and play a key role in biological responses to environmental stressors. However, little is known about how space radiation exposure and altered gravity affect miRNA expression. The "International Space Biological Experiments" project was carried out in November 2011 by an international collaboration between China and Germany during the Shenzhou-8 (SZ-8) mission. To study the effects of spaceflight on Caenorhabditis elegans (C. elegans), we explored the expression profile miRNA changes in space-flown C. elegans. Dauer C. elegans larvae were taken by SZ-8 spacecraft and experienced the 16.5-day shuttle spaceflight. We performed miRNA microarray analysis, and the results showed that 23 miRNAs were altered in a complex space environment and different expression patterns were observed in the space synthetic and radiation environments. Most putative target genes of the altered miRNAs in the space synthetic environment were predicted to be involved in developmental processes instead of in the regulation of transcription, and the enrichment of these genes was due to space radiation. Furthermore, integration analysis of the miRNA and mRNA expression profiles confirmed that twelve genes were differently regulated by seven miRNAs. These genes may be involved in embryonic development, reproduction, transcription factor activity, oviposition in a space synthetic environment, positive regulation of growth and body morphogenesis in a space radiation environment. Specifically, we found that cel-miR-52, -55, and -56 of the miR-51 family were sensitive to space environmental stressors and could regulate biological behavioural responses and neprilysin activity through the different isoforms of T01C4.1 and F18A12.8. These findings suggest that C. elegans responded to spaceflight by altering the expression of miRNAs and some target genes that function in diverse regulatory pathways.

1α,25(OH)2D3 differentially regulates miRNA expression in human bladder cancer cells

PubMed Central

Ma, Yingyu; Hu, Qiang; Luo, Wei; Pratt, Rachel N.; Glenn, Sean T.; Liu, Song; Trump, Donald L.; Johnson, Candace S.

2014-01-01

Bladder cancer is the fourth most commonly diagnosed cancer in men and eighth leading cause of cancer-related death in the US. Epidemiological and experimental studies strongly suggest a role for 1α,25(OH)2D3 in cancer prevention and treatment. The antitumor activities of 1α,25(OH)2D3 are mediated by the induction of cell cycle arrest, apoptosis, differentiation and the inhibition of angiogenesis and metastasis. MiRNAs play important regulatory roles in cancer development and progression. However, the role of 1α,25(OH)2D3 in the regulation of miRNA expression and the potential impact in bladder cancer has not been investigated. Therefore, we studied 1α,25(OH)2D3-regulated miRNA expression profiles in human bladder cancer cell line 253J and the highly tumorigenic and metastatic derivative line 253J-BV by miRNA qPCR panels. 253 J and 253J-BV cells express endogenous vitamin D receptor (VDR) which can be further induced by 1α,25(OH)2D3. VDR target gene 24-hydroxylase was induced by 1α,25(OH)2D3 in both cell lines, indicating functional 1α,25(OH)2D3 signaling. The miRNA qPCR panel assay results showed that 253J and 253J-BV cells have distinct miRNA expression profiles. Further, 1α,25(OH)2D3 differentially regulated miRNA expression profiles in 253J and 253 J-BV cells in a dynamic manner. Pathway analysis of the miRNA target genes revealed distinct patterns of contribution to the molecular functions and biological processes in the two cell lines. In conclusion, 1α,25(OH)2D3 differentially regulates the expression of miRNAs, which may contribute to distinct biological functions, in human bladder 253J and 253J-BV cells. PMID:25263658

Curcumin inhibits the proliferation and invasion of human osteosarcoma cell line MG-63 by regulating miR-138.

PubMed

Yu, Dazhi; An, Fengmei; He, Xu; Cao, Xuecheng

2015-01-01

In this study, we screened the different human osteosarcoma cell line MG-63 miRNAs after the treatment of curcumin and explored the effects of curcumin on MG-63 cells and its mechanism. Affemitrix miRNA chip was used to detect the changes of miRNA expression profile in MG-63 cells before and after curcumin treatment, and screen different expression of miRNAs. The target gene of miRNA was analyzed by bioinformatics. The expression levels of miRNA-138 target genes Smad4, NFκB p65 and cyclin D3 were detected. MTT and Transwell Cell invasion assays were used to observe the effects of curcumin on MG-63 cells. Curcumin could significantly inhibit the proliferation of MG-63 cells and the expression levels of miRNA-138 target genes Smad4, NFκB p65 and cyclin D3 in MG-63 cells (P<0.05); overexpression of hsa-miR-138 down-regulated the expression levels of Smad4, NFκB p65 and cyclin D3 compared with the treatment of curcumin, while inhibition of hsa-miR-138 up-regulated the expression levels of Smad4, NFκB p65 and cyclin D3. Curcumin could increase the expression of hsa-miR-138, hsa-miR-138 inhibited cell proliferation and invasive ability by inhibition of its target genes.

An imprinted non-coding genomic cluster at 14q32 defines clinically relevant molecular subtypes in osteosarcoma across multiple independent datasets.

PubMed

Hill, Katherine E; Kelly, Andrew D; Kuijjer, Marieke L; Barry, William; Rattani, Ahmed; Garbutt, Cassandra C; Kissick, Haydn; Janeway, Katherine; Perez-Atayde, Antonio; Goldsmith, Jeffrey; Gebhardt, Mark C; Arredouani, Mohamed S; Cote, Greg; Hornicek, Francis; Choy, Edwin; Duan, Zhenfeng; Quackenbush, John; Haibe-Kains, Benjamin; Spentzos, Dimitrios

2017-05-15

A microRNA (miRNA) collection on the imprinted 14q32 MEG3 region has been associated with outcome in osteosarcoma. We assessed the clinical utility of this miRNA set and their association with methylation status. We integrated coding and non-coding RNA data from three independent annotated clinical osteosarcoma cohorts (n = 65, n = 27, and n = 25) and miRNA and methylation data from one in vitro (19 cell lines) and one clinical (NCI Therapeutically Applicable Research to Generate Effective Treatments (TARGET) osteosarcoma dataset, n = 80) dataset. We used time-dependent receiver operating characteristic (tdROC) analysis to evaluate the clinical value of candidate miRNA profiles and machine learning approaches to compare the coding and non-coding transcriptional programs of high- and low-risk osteosarcoma tumors and high- versus low-aggressiveness cell lines. In the cell line and TARGET datasets, we also studied the methylation patterns of the MEG3 imprinting control region on 14q32 and their association with miRNA expression and tumor aggressiveness. In the tdROC analysis, miRNA sets on 14q32 showed strong discriminatory power for recurrence and survival in the three clinical datasets. High- or low-risk tumor classification was robust to using different microRNA sets or classification methods. Machine learning approaches showed that genome-wide miRNA profiles and miRNA regulatory networks were quite different between the two outcome groups and mRNA profiles categorized the samples in a manner concordant with the miRNAs, suggesting potential molecular subtypes. Further, miRNA expression patterns were reproducible in comparing high-aggressiveness versus low-aggressiveness cell lines. Methylation patterns in the MEG3 differentially methylated region (DMR) also distinguished high-aggressiveness from low-aggressiveness cell lines and were associated with expression of several 14q32 miRNAs in both the cell lines and the large TARGET clinical dataset. Within the limits of available CpG array coverage, we observed a potential methylation-sensitive regulation of the non-coding RNA cluster by CTCF, a known enhancer-blocking factor. Loss of imprinting/methylation changes in the 14q32 non-coding region defines reproducible previously unrecognized osteosarcoma subtypes with distinct transcriptional programs and biologic and clinical behavior. Future studies will define the precise relationship between 14q32 imprinting, non-coding RNA expression, genomic enhancer binding, and tumor aggressiveness, with possible therapeutic implications for both early- and advanced-stage patients.

miRNA Expression Profile after Status Epilepticus and Hippocampal Neuroprotection by Targeting miR-132

PubMed Central

Jimenez-Mateos, Eva M.; Bray, Isabella; Sanz-Rodriguez, Amaya; Engel, Tobias; McKiernan, Ross C.; Mouri, Genshin; Tanaka, Katsuhiro; Sano, Takanori; Saugstad, Julie A.; Simon, Roger P.; Stallings, Raymond L.; Henshall, David C.

2011-01-01

When an otherwise harmful insult to the brain is preceded by a brief, noninjurious stimulus, the brain becomes tolerant, and the resulting damage is reduced. Epileptic tolerance develops when brief seizures precede an episode of prolonged seizures (status epilepticus). MicroRNAs (miRNAs) are small, noncoding RNAs that function as post-transcriptional regulators of gene expression. We investigated how prior seizure preconditioning affects the miRNA response to status epilepticus evoked by intra-amygdalar kainic acid in mice. The miRNA was extracted from the ipsilateral CA3 subfield 24 hours after focal-onset status epilepticus in animals that had previously received either seizure preconditioning (tolerance) or no preconditioning (injury), and mature miRNA levels were measured using TaqMan low-density arrays. Expression of 21 miRNAs was increased, relative to control, after status epilepticus alone, and expression of 12 miRNAs was decreased. Increased miR-132 levels were matched with increased binding to Argonaute-2, a constituent of the RNA-induced silencing complex. In tolerant animals, expression responses of >40% of the injury-group-detected miRNAs differed, being either unchanged relative to control or down-regulated, and this included miR-132. In vivo microinjection of locked nucleic acid-modified oligonucleotides (antagomirs) against miR-132 depleted hippocampal miR-132 levels and reduced seizure-induced neuronal death. Thus, our data strongly suggest that miRNAs are important regulators of seizure-induced neuronal death. PMID:21945804

MiRNA-21 has effects to protect kidney injury induced by sepsis.

PubMed

Fu, Dian; Dong, Jie; Li, Ping; Tang, Chaopeng; Cheng, Wen; Xu, Zhenyu; Zhou, Wenquan; Ge, Jingping; Xia, Chen; Zhang, Zhengyu

2017-10-01

To investigate the miRNA-21 over-expression in the acute kidney injury induced by sepsis, we developed a sepsis induced in vitro model by lip polysaccharide (LPS) and in vovo model by cecal ligation and puncture (CLP) surgery. LPS or CLP surgery induced kidney cell apoptosis increasing. However, the kidney injury indexes of miRNA groups which were transfected with miRNA-21 were significantly suppressed. In further study, the relative proteins expressions were evaluated to explain the miRNA-21 mechanism to improve sepsis induced kidney cell apoptosis. The results were shown that miRNA-21 over-expression had effects to protect kidney cell apoptosis induced by sepsis via PTEN/PI3K/AKT signaling pathway. Copyright © 2017. Published by Elsevier Masson SAS.

MicroRNAs and their roles in aging

PubMed Central

Smith-Vikos, Thalyana; Slack, Frank J.

2012-01-01

MicroRNAs (miRNAs) are a class of short non-coding RNAs that bind mRNAs through partial base-pair complementarity with their target genes, resulting in post-transcriptional repression of gene expression. The role of miRNAs in controlling aging processes has been uncovered recently with the discovery of miRNAs that regulate lifespan in the nematode Caenorhabditis elegans through insulin and insulin-like growth factor-1 signaling and DNA damage checkpoint factors. Furthermore, numerous miRNAs are differentially expressed during aging in C. elegans, but the specific functions of many of these miRNAs are still unknown. Recently, various miRNAs have been identified that are up- or down-regulated during mammalian aging by comparing their tissue-specific expression in younger and older mice. In addition, many miRNAs have been implicated in governing senescence in a variety of human cell lines, and the precise functions of some of these miRNAs in regulating cellular senescence have helped to elucidate mechanisms underlying aging. In this Commentary, we review the various regulatory roles of miRNAs during aging processes. We highlight how certain miRNAs can regulate aging on the level of organism lifespan, tissue aging or cellular senescence. Finally, we discuss future approaches that might be used to investigate the mechanisms by which miRNAs govern aging processes. PMID:22294612

Identification and characterization of microRNAs and their target genes from Nile tilapia (Oreochromis niloticus).

PubMed

Huang, Yong; Ma, Xiu Ying; Yang, You Bing; Ren, Hong Tao; Sun, Xi Hong; Wang, Li Rui

MicroRNAs (miRNAs) are a class of small single-stranded, endogenous 21-22 nt non-coding RNAs that regulate their target mRNA levels by causing either inactivation or degradation of the mRNAs. In recent years, miRNA genes have been identified from mammals, insects, worms, plants, and viruses. In this research, bioinformatics approaches were used to predict potential miRNAs and their targets in Nile tilapia from the expressed sequence tag (EST) and genomic survey sequence (GSS) database, respectively, based on the conservation of miRNAs in many animal species. A total of 19 potential miRNAs were detected following a range of strict filtering criteria. To test the validity of the bioinformatics method, seven predicted Nile tilapia miRNA genes were selected for further biological validation, and their mature miRNA transcripts were successfully detected by stem-loop RT-PCR experiments. Using these potential miRNAs, we found 56 potential targets in this species. Most of the target mRNAs appear to be involved in development, metabolism, signal transduction, transcription regulation and stress responses. Overall, our findings will provide an important foundation for further research on miRNAs function in the Nile tilapia.

Characterization of microRNA profile in mammary tissue of dairy and beef breed heifers.

PubMed

Wicik, Z; Gajewska, M; Majewska, A; Walkiewicz, D; Osińska, E; Motyl, T

2016-02-01

MicroRNAs (miRNAs) are small non-coding RNAs that participate in the regulation of gene expression. Their role during mammary gland development is still largely unknown. In this study, we performed a microarray analysis to identify miRNAs associated with high mammogenic potential of the bovine mammary gland. We identified 54 significantly differentially expressed miRNAs between the mammary tissue of dairy (Holstein-Friesian, HF) and beef (Limousin, LM) postpubertal heifers. Fifty-two miRNAs had higher expression in the mammary tissue of LM heifers. The expression of the top candidate miRNAs (bta-miR-10b, bta-miR-29b, bta-miR-101, bta-miR-375, bta-miR-2285t, bta-miR-146b, bta-let7b, bta-miR-107, bta-miR-1434-3p) identified in the microarray experiment was additionally evaluated by qPCR. Enrichment analyses for targeted genes revealed that the major differences between miRNA expression in the mammary gland of HF versus LM were associated with the regulation of signalling pathways that are crucial for mammary gland development, such as TGF-beta, insulin, WNT and inflammatory pathways. Moreover, a number of genes potentially targeted by significantly differentially expressed miRNAs were associated with the activity of mammary stem cells. These data indicate that the high developmental potential of the mammary gland in dairy cattle, leading to high milk productivity, depends also on a specific miRNA expression pattern. © 2015 Blackwell Verlag GmbH.

MicroRNAs as regulators of drug transporters, drug-metabolizing enzymes, and tight junctions: implication for intestinal barrier function.

PubMed

Ikemura, Kenji; Iwamoto, Takuya; Okuda, Masahiro

2014-08-01

Drug transporters, drug-metabolizing enzymes, and tight junctions in the small intestine function as an absorption barrier and sometimes as a facilitator of orally administered drugs. The expression of these proteins often fluctuates and thereby causes individual pharmacokinetic variability. MicroRNAs (miRNAs), which are small non-coding RNAs, have recently emerged as a new class of gene regulator. MiRNAs post-transcriptionally regulate gene expression by binding to target mRNA to suppress its translation or regulate its degradation. They have been shown to be key regulators of proteins associated with pharmacokinetics. Moreover, the role of miRNAs on the expression of some proteins expressed in the small intestine has recently been clarified. In this review, we summarize current knowledge regarding the role of miRNAs in the regulation of drug transporters, drug-metabolizing enzymes, and tight junctions as well as its implication for intestinal barrier function. MiRNAs play vital roles in the differentiation, architecture, and barrier function of intestinal epithelial cells, and directly and/or indirectly regulate the expression and function of proteins associated with drug absorption in intestinal epithelial cells. Moreover, the variation of miRNA expression caused by pathological and physiological conditions as well as genetic factors should affect the expression of these proteins. Therefore, miRNAs could be significant factors affecting inter- and intra-individual variations in the pharmacokinetics and intestinal absorption of drugs. Overall, miRNAs could be promising targets for personalized pharmacotherapy or other attractive therapies through intestinal absorption of drugs. Copyright © 2014 Elsevier Inc. All rights reserved.

Effects of MicroRNA-23a on Differentiation and Gene Expression Profiles in 3T3-L1 Adipocytes

PubMed Central

Huang, Yong; Huang, Jinxiu; Qi, Renli; Wang, Qi; Wu, Yongjiang; Wang, Jing

2016-01-01

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate growth, development, and programmed death of cells. A newly-published study has shown that miRNA-23a could regulate 3T3-L1 adipocyte differentiation. Here, we identified miRNA-23a as a negative regulator of 3T3-L1 adipocyte differentiation again. Over-expression of miRNA-23a inhibited differentiation and decreased lipogenesis as well as down-regulated mRNA and protein expression of both peroxisome proliferator-activated receptor (PPAR) γ and fatty acid binding protein (FABP) 4, whereas knock down of miRNA-23a showed the opposite effects on differentiation as well as increasing the number of apoptotic cells. Additionally, digital gene expression profiling sequencing (DGE-Seq) was used to assay changes in gene expression profiles following alterations in the level of miR-23a. In total, over-expression or knock down of miRNA-23a significantly changed the expression of 313 and 425 genes, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that these genes were mainly involved in the stress response, immune system, metabolism, cell cycle, among other pathways. Additionally, the signal transducer and activator of transcription 1 (Stat1) was shown to be a target of miRNA-23a by computational and dual-luciferase reporter assays that indicated Janus Kinase (Jak)-Stat signal pathway was implicated in regulating adipogenesis mediated by miRNA-23a in adipocytes. PMID:27783036

Effects of Modeled Microgravity on Expression Profiles of Micro RNA in Human Lymphoblastoid Cells

NASA Technical Reports Server (NTRS)

Mangala, Lingegowda S.; Emami, Kamal; Story, Michael; Ramesh, Govindarajan; Rohde, Larry; Wu, Honglu

2010-01-01

Among space radiation and other environmental factors, microgravity or an altered gravity is undoubtedly the most significant stress experienced by living organisms during flight. In comparison to the static 1g, microgravity has been shown to alter global gene expression patterns and protein levels in cultured cells or animals. Micro RNA (miRNA) has recently emerged as an important regulator of gene expression, possibly regulating as many as one-third of all human genes. miRNA represents a class of single-stranded noncoding regulatory RNA molecules ( 22 nt) that control gene expressions by inhibiting the translation of mRNA to proteins. However, very little is known on the effect of altered gravity on miRNA expression. We hypothesized that the miRNA expression profile will be altered in zero gravity resulting in regulation of the gene expression and functional changes of the cells. To test this hypothesis, we cultured TK6 human lymphoblastoid cells in Synthecon s Rotary cell culture system (bioreactors) for 72 h either in the rotating (10 rpm) to model the microgravity in space or in the static condition. The cell viability was determined before and after culturing the cells in the bioreactor using both trypan blue and guava via count. Expressions of a panel of 352 human miRNA were analyzed using the miRNA PCRarray. Out of 352 miRNAs, expressions of 75 were significantly altered by a change of greater than 1.5 folds and seven miRNAs were altered by a fold change greater than 2 under the rotating culture condition. Among these seven, miR-545 and miR-517a were down regulated by 2 folds, whereas miR-150, miR-302a, miR-139-3p, miR-515-3p and miR-564 were up regulated by 2 to 8 folds. To confirm whether this altered miRNA expression correlates with gene expression and functional changes of the cells, we performed DNA Illumina Microarray Analysis and validated the related genes using q-RT PCR.

microRNA-137 promotes apoptosis in ovarian cancer cells via the regulation of XIAP

PubMed Central

Li, Xiaodi; Chen, Wei; Zeng, Wenshu; Wan, Chunling; Duan, Shiwei; Jiang, Songshan

2017-01-01

Background: microRNAs (miRNAs) have regulatory roles in various cellular processes, including apoptosis. Recently, X-linked inhibitor of apoptosis protein (XIAP) has been reported to be dysregulated in epithelial ovarian cancer (EOC). However, the mechanism underlying this dysregulation is largely unknown. Methods: Using bioinformatics and a literature analysis, a panel of miRNAs dysregulated in EOC was chosen for further experimental confirmation from hundreds of miRNAs that were predicted to interact with the XIAP 3′UTR. A dual-luciferase reporter assay was employed to detect the interaction by cellular co-transfection of an miRNA expression vector and a reporter vector with the XIAP 3′UTR fused to a Renilla luciferase reporter. DAPI and TUNEL approaches were used to further determine the effects of an miR-137 mimic and inhibitor on cisplatin-induced apoptosis in ovarian cancer cells. Results: We identified eight miRNAs by screening a panel of dysregulated miRNAs that may target the XIAP 3′UTR. The strongest inhibitory miRNA, miR-137, suppressed the activity of a luciferase reporter gene fused with the XIAP 3′UTR and decreased the levels of XIAP protein in SKOV3 ovarian cancer cells. Furthermore, forced expression of miR-137 increased cisplatin-induced apoptosis, and the depressed expression of miR-137 decreased cisplatin-induced apoptosis in SKOV3 and primary EOC cells. Consistently, the disruption of miR-137 via CRISPR/Cas9 inhibited apoptosis and upregulated XIAP in A2780 cells. Furthermore, the effect of miR-137 on apoptosis could be rescued by XIAP in SKOV3 cells. In addition, miR-137 expression is inversely correlated with the level of XIAP protein in both ovarian cancer tissues and cell lines. Conclusions: Our data suggest that multiple miRNAs can regulate XIAP via its 3′UTR. miR-137 can sensitise ovarian cancer cells to cisplatin-induced apoptosis, providing new insight into overcoming drug resistance in ovarian cancer. PMID:27875524

Differential Sensitivity of Target Genes to Translational Repression by miR-17~92

PubMed Central

Jin, Hyun Yong; Oda, Hiroyo; Chen, Pengda; Kang, Seung Goo; Valentine, Elizabeth; Liao, Lujian; Zhang, Yaoyang; Gonzalez-Martin, Alicia; Shepherd, Jovan; Head, Steven R.; Kim, Pyeung-Hyeun; Fu, Guo; Liu, Wen-Hsien; Han, Jiahuai

2017-01-01

MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5’UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genes. PMID:28241004

Urinary Exosomal miRNA Signature in Type II Diabetic Nephropathy Patients

PubMed Central

Delić, Denis; Eisele, Claudia; Schmid, Ramona; Baum, Patrick; Wiech, Franziska; Gerl, Martin; Zimdahl, Heike; Pullen, Steven S.; Urquhart, Richard

2016-01-01

MicroRNAs (miRNAs) are short non-coding RNA species which are important post-transcriptional regulators of gene expression and play an important role in the pathogenesis of diabetic nephropathy. miRNAs are present in urine in a remarkably stable form packaged in extracellular vesicles, predominantly exosomes. In the present study, urinary exosomal miRNA profiling was conducted in urinary exosomes obtained from 8 healthy controls (C), 8 patients with type II diabetes (T2D) and 8 patients with type II diabetic nephropathy (DN) using Agilent´s miRNA microarrays. In total, the expression of 16 miRNA species was deregulated (>2-fold) in DN patients compared to healthy donors and T2D patients: the expression of 14 miRNAs (miR-320c, miR-6068, miR-1234-5p, miR-6133, miR-4270, miR-4739, miR-371b-5p, miR-638, miR-572, miR-1227-5p, miR-6126, miR-1915-5p, miR-4778-5p and miR-2861) was up-regulated whereas the expression of 2 miRNAs (miR-30d-5p and miR-30e-5p) was down-regulated. Most of the deregulated miRNAs are involved in progression of renal diseases. Deregulation of urinary exosomal miRNAs occurred in micro-albuminuric DN patients but not in normo-albuminuric DN patients. We used qRT-PCR based analysis of the most strongly up-regulated miRNAs in urinary exosomes from DN patients, miRNAs miR-320c and miR-6068. The correlation of miRNA expression and micro-albuminuria levels could be replicated in a confirmation cohort. In conclusion, urinary exosomal miRNA content is altered in type II diabetic patients with DN. Deregulated miR-320c, which might have an impact on the TGF-β-signaling pathway via targeting thrombospondin 1 (TSP-1) shows promise as a novel candidate marker for disease progression in type II DN that should be evaluated in future studies. PMID:26930277

Epidrug mediated re-expression of miRNA targeting the HMGA transcripts in pituitary cells.

PubMed

Kitchen, Mark O; Yacqub-Usman, Kiren; Emes, Richard D; Richardson, Alan; Clayton, Richard N; Farrell, William E

2015-10-01

Transgenic mice overexpressing the high mobility group A (HMGA) genes, Hmga1 or Hmga2 develop pituitary tumours and their overexpression is also a frequent finding in human pituitary adenomas. In some cases, increased expression of HMGA2 but not that of HMGA1 is consequent to genetic perturbations. However, recent studies show that down-regulation of microRNA (miRNA), that contemporaneously target the HMGA1 and HMGA2 transcripts, are associated with their overexpression. In a cohort of primary pituitary adenoma we determine the impact of epigenetic modifications on the expression of HMGA-targeting miRNA. For these miRNAs, chromatin immunoprecipitations showed that transcript down-regulation is correlated with histone tail modifications associated with condensed silenced genes. The functional impact of epigenetic modification on miRNA expression was determined in the rodent pituitary cell line, GH3. In these cells, histone tail, miRNA-associated, modifications were similar to those apparent in human adenoma and likely account for their repression. Indeed, challenge of GH3 cells with the epidrugs, zebularine and TSA, led to enrichment of the histone modification, H3K9Ac, associated with active genes, and depletion of the modification, H3K27me3, associated with silent genes and re-expression of HMGA-targeting miRNA. Moreover, epidrugs challenges were also associated with a concomitant decrease in hmga1 transcript and protein levels and concurrent increase in bmp-4 expression. These findings show that the inverse relationship between HMGA expression and targeting miRNA is reversible through epidrug interventions. In addition to showing a mechanistic link between epigenetic modifications and miRNA expression these findings underscore their potential as therapeutic targets in this and other diseases.

Expression profiling and bioinformatic analyses suggest new target genes and pathways for human hair follicle related microRNAs.

PubMed

Hochfeld, Lara M; Anhalt, Thomas; Reinbold, Céline S; Herrera-Rivero, Marisol; Fricker, Nadine; Nöthen, Markus M; Heilmann-Heimbach, Stefanie

2017-02-22

Human hair follicle (HF) cycling is characterised by the tight orchestration and regulation of signalling cascades. Research shows that micro(mi)RNAs are potent regulators of these pathways. However, knowledge of the expression of miRNAs and their target genes and pathways in the human HF is limited. The objective of this study was to improve understanding of the role of miRNAs and their regulatory interactions in the human HF. Expression levels of ten candidate miRNAs with reported functions in hair biology were assessed in HFs from 25 healthy male donors. MiRNA expression levels were correlated with mRNA-expression levels from the same samples. Identified target genes were tested for enrichment in biological pathways and accumulation in protein-protein interaction (PPI) networks. Expression in the human HF was confirmed for seven of the ten candidate miRNAs, and numerous target genes for miR-24, miR-31, and miR-106a were identified. While the latter include several genes with known functions in hair biology (e.g., ITGB1, SOX9), the majority have not been previously implicated (e.g., PHF1). Target genes were enriched in pathways of interest to hair biology, such as integrin and GnRH signalling, and the respective gene products showed accumulation in PPIs. Further investigation of miRNA expression in the human HF, and the identification of novel miRNA target genes and pathways via the systematic integration of miRNA and mRNA expression data, may facilitate the delineation of tissue-specific regulatory interactions, and improve our understanding of both normal hair growth and the pathobiology of hair loss disorders.

Discovery and profiling of novel and conserved microRNAs during flower development in Carya cathayensis via deep sequencing.

PubMed

Wang, Zheng Jia; Huang, Jian Qin; Huang, You Jun; Li, Zheng; Zheng, Bing Song

2012-08-01

Hickory (Carya cathayensis Sarg.) is an economically important woody plant in China, but its long juvenile phase delays yield. MicroRNAs (miRNAs) are critical regulators of genes and important for normal plant development and physiology, including flower development. We used Solexa technology to sequence two small RNA libraries from two floral differentiation stages in hickory to identify miRNAs related to flower development. We identified 39 conserved miRNA sequences from 114 loci belonging to 23 families as well as two novel and ten potential novel miRNAs belonging to nine families. Moreover, 35 conserved miRNA*s and two novel miRNA*s were detected. Twenty miRNA sequences from 49 loci belonging to 11 families were differentially expressed; all were up-regulated at the later stage of flower development in hickory. Quantitative real-time PCR of 12 conserved miRNA sequences, five novel miRNA families, and two novel miRNA*s validated that all were expressed during hickory flower development, and the expression patterns were similar to those detected with Solexa sequencing. Finally, a total of 146 targets of the novel and conserved miRNAs were predicted. This study identified a diverse set of miRNAs that were closely related to hickory flower development and that could help in plant floral induction.

Discovery and characterization of miRNA genes in atlantic salmon (Salmo salar) by use of a deep sequencing approach

PubMed Central

2013-01-01

Background MicroRNAs (miRNAs) are an abundant class of endogenous small RNA molecules that downregulate gene expression at the posttranscriptional level. They play important roles in multiple biological processes by regulating genes that control developmental timing, growth, stem cell division and apoptosis by binding to the mRNA of target genes. Despite the position Atlantic salmon (Salmo salar) has as an economically important domesticated animal, there has been little research on miRNAs in this species. Knowledge about miRNAs and their target genes may be used to control health and to improve performance of economically important traits. However, before their biological function can be unravelled they must be identified and annotated. The aims of this study were to identify and characterize miRNA genes in Atlantic salmon by deep sequencing analysis of small RNA libraries from nine different tissues. Results A total of 180 distinct mature miRNAs belonging to 106 families of evolutionary conserved miRNAs, and 13 distinct novel mature miRNAs were discovered and characterized. The mature miRNAs corresponded to 521 putative precursor sequences located at unique genome locations. About 40% of these precursors were part of gene clusters, and the majority of the Salmo salar gene clusters discovered were conserved across species. Comparison of expression levels in samples from different tissues applying DESeq indicated that there were tissue specific expression differences in three conserved and one novel miRNA. Ssa-miR 736 was detected in heart tissue only, while two other clustered miRNAs (ssa-miR 212 and132) seems to be at a higher expression level in brain tissue. These observations correlate well with their expected functions as regulators of signal pathways in cardiac and neuronal cells, respectively. Ssa-miR 8163 is one of the novel miRNAs discovered and its function remains unknown. However, differential expression analysis using DESeq suggests that this miRNA is enriched in liver tissue and the precursor was mapped to intron 7 of the transferrin gene. Conclusions The identification and annotation of evolutionary conserved and novel Salmo salar miRNAs as well as the characterization of miRNA gene clusters provide biological knowledge that will greatly facilitate further functional studies on miRNAs in this species. PMID:23865519

Epigenetic regulation of normal human mammary cell type-specific miRNAs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vrba, Lukas; Garbe, James C.; Stampfer, Martha R.

2011-08-26

Epigenetic mechanisms are important regulators of cell type–specific genes, including miRNAs. In order to identify cell type-specific miRNAs regulated by epigenetic mechanisms, we undertook a global analysis of miRNA expression and epigenetic states in three isogenic pairs of human mammary epithelial cells (HMEC) and human mammary fibroblasts (HMF), which represent two differentiated cell types typically present within a given organ, each with a distinct phenotype and a distinct epigenotype. While miRNA expression and epigenetic states showed strong interindividual concordance within a given cell type, almost 10% of the expressed miRNA showed a cell type–specific pattern of expression that was linkedmore » to the epigenetic state of their promoter. The tissue-specific miRNA genes were epigenetically repressed in nonexpressing cells by DNA methylation (38%) and H3K27me3 (58%), with only a small set of miRNAs (21%) showing a dual epigenetic repression where both DNA methylation and H3K27me3 were present at their promoters, such as MIR10A and MIR10B. Individual miRNA clusters of closely related miRNA gene families can each display cell type–specific repression by the same or complementary epigenetic mechanisms, such as the MIR200 family, and MIR205, where fibroblasts repress MIR200C/141 by DNA methylation, MIR200A/200B/429 by H3K27me3, and MIR205 by both DNA methylation and H3K27me3. Since deregulation of many of the epigenetically regulated miRNAs that we identified have been linked to disease processes such as cancer, it is predicted that compromise of the epigenetic control mechanisms is important for this process. Overall, these results highlight the importance of epigenetic regulation in the control of normal cell type–specific miRNA expression.« less

Genome-Wide Identification of miRNAs Responsive to Drought in Peach (Prunus persica) by High-Throughput Deep Sequencing

PubMed Central

Eldem, Vahap; Çelikkol Akçay, Ufuk; Ozhuner, Esma; Bakır, Yakup; Uranbey, Serkan; Unver, Turgay

2012-01-01

Peach (Prunus persica L.) is one of the most important worldwide fresh fruits. Since fruit growth largely depends on adequate water supply, drought stress is considered as the most important abiotic stress limiting fleshy fruit production and quality in peach. Plant responses to drought stress are regulated both at transcriptional and post-transcriptional level. As post-transcriptional gene regulators, miRNAs (miRNAs) are small (19–25 nucleotides in length), endogenous, non-coding RNAs. Recent studies indicate that miRNAs are involved in plant responses to drought. Therefore, Illumina deep sequencing technology was used for genome-wide identification of miRNAs and their expression profile in response to drought in peach. In this study, four sRNA libraries were constructed from leaf control (LC), leaf stress (LS), root control (RC) and root stress (RS) samples. We identified a total of 531, 471, 535 and 487 known mature miRNAs in LC, LS, RC and RS libraries, respectively. The expression level of 262 (104 up-regulated, 158 down-regulated) of the 453 miRNAs changed significantly in leaf tissue, whereas 368 (221 up-regulated, 147 down-regulated) of the 465 miRNAs had expression levels that changed significantly in root tissue upon drought stress. Additionally, a total of 197, 221, 238 and 265 novel miRNA precursor candidates were identified from LC, LS, RC and RS libraries, respectively. Target transcripts (137 for LC, 133 for LS, 148 for RC and 153 for RS) generated significant Gene Ontology (GO) terms related to DNA binding and catalytic activites. Genome-wide miRNA expression analysis of peach by deep sequencing approach helped to expand our understanding of miRNA function in response to drought stress in peach and Rosaceae. A set of differentially expressed miRNAs could pave the way for developing new strategies to alleviate the adverse effects of drought stress on plant growth and development. PMID:23227166

Response of microRNAs to cold treatment in the young spikes of common wheat.

PubMed

Song, Guoqi; Zhang, Rongzhi; Zhang, Shujuan; Li, Yulian; Gao, Jie; Han, Xiaodong; Chen, Mingli; Wang, Jiao; Li, Wei; Li, Genying

2017-02-28

MicroRNAs (miRNAs) are a class of small non-coding RNAs that play important roles in biotic and abiotic stresses by regulating their target genes. For common wheat, spring frost damage frequently occurs, especially when low temperature coincides with plants at early floral organ differentiation, which may result in significant yield loss. Up to date, the role of miRNAs in wheat response to frost stress is not well understood. We report here the sequencing of small RNA transcriptomes from the young spikes that were treated with cold stress and the comparative analysis with those of the control. A total of 192 conserved miRNAs from 105 families and nine novel miRNAs were identified. Among them, 34 conserved and five novel miRNAs were differentially expressed between the cold-stressed samples and the controls. The expression patterns of 18 miRNAs were further validated by quantitative real time polymerase chain reaction (qRT-PCR). Moreover, nearly half of the miRNAs were cross inducible by biotic and abiotic stresses when compared with previously published work. Target genes were predicted and validated by degradome sequencing. Gene Ontology (GO) enrichment analysis showed that the target genes of differentially expressed miRNAs were enriched for response to the stimulus, regulation of transcription, and ion transport functions. Since many targets of differentially expressed miRNAs were transcription factors that are associated with floral development such as ARF, SPB (Squamosa Promoter Binding like protein), MADS-box (MCM1, AG, DEFA and SRF), MYB, SPX (SYG1, Pho81 and XPR1), TCP (TEOSINTE BRANCHED, Cycloidea and PCF), and PPR (PentatricoPeptide Repeat) genes, cold-altered miRNA expression may cause abnormal reproductive organ development. Analysis of small RNA transcriptomes and their target genes provide new insight into miRNA regulation in developing wheat inflorescences under cold stress. MiRNAs provide another layer of gene regulation in cold stress response that can be genetically manipulated to reduce yield loss in wheat.

The PTTG1-targeting miRNAs miR-329, miR-300, miR-381, and miR-655 inhibit pituitary tumor cell tumorigenesis and are involved in a p53/PTTG1 regulation feedback loop

PubMed Central

Diao, Cai-feng; Li, Jian-wei; Su, Jing-liang; Zhang, Sai

2015-01-01

Deregulation of the pituitary tumor transforming gene (PTTG1), a newly discovered oncogene, is a hallmark of various malignancies, including pituitary tumors. However, the mechanisms regulating PTTG1 expression are still needed to be explored. MicroRNAs (miRNAs) are a novel class of small RNA molecules that act as posttranscriptional regulators of gene expression and can play a significant role in tumor development. Here, we identified a series of miRNAs, namely, miR-329, miR-300, miR-381 and miR-655, which could target PTTG1 messenger RNA and inhibit its expression. Interestingly, all four miRNAs significantly that are downregulated in pituitary tumors were mapped to the 14q32.31 locus, which acts as a tumor suppressor in several cancers. Functional studies show that the PTTG1-targeting miRNAs inhibit proliferation, migration and invasion but induce apoptosis in GH3 and MMQ cells. Furthermore, overexpression of a PTTG1 expression vector lacking the 3′UTR partially reverses the tumor suppressive effects of these miRNAs. Next, we identified the promoter region of PTTG1-targeting miRNAs with binding sites for p53. In our hands, p53 transcriptionally activated the expression of these miRNAs in pituitary tumor cells. Finally, we found that PTTG1 could inhibit p53 transcriptional activity to the four miRNAs. These data indicate the existence of a feedback loop between PTTG1 targeting miRNAs, PTTG1 and p53 that promotes pituitary tumorigenesis. Together, these findings suggest that these PTTG1-targeting miRNAs are important players in the regulation of pituitary tumorigenesis and that these miRNAs may serve as valuable therapeutic targets for cancer treatment. PMID:26320179

Salivary extracellular vesicle-associated miRNAs as potential biomarkers in oral squamous cell carcinoma.

PubMed

Gai, Chiara; Camussi, Francesco; Broccoletti, Roberto; Gambino, Alessio; Cabras, Marco; Molinaro, Luca; Carossa, Stefano; Camussi, Giovanni; Arduino, Paolo G

2018-04-18

Several studies in the past have investigated the expression of micro RNAs (miRNAs) in saliva as potential biomarkers. Since miRNAs associated with extracellular vesicles (EVs) are known to be protected from enzymatic degradation, we evaluated whether salivary EVs from patients with oral squamous cell carcinoma (OSCC) were enriched with specific subsets of miRNAs. OSCC patients and controls were matched with regards to age, gender and risk factors. Total RNA was extracted from salivary EVs and the differential expression of miRNAs was evaluated by qRT-PCR array and qRT-PCR. The discrimination power of up-regulated miRNAs as biomarkers in OSCC patients versus controls was evaluated by the Receiver Operating Characteristic (ROC) curves. A preliminary qRT-PCR array was performed on samples from 5 OSCC patients and 5 healthy controls whereby a subset of miRNAs were identified that were differentially expressed. On the basis of these results, a cohort of additional 16 patients and 6 controls were analyzed to further confirm the miRNAs that were up-regulated or selectively expressed in the previous pilot study. The following miRNAs: miR-302b-3p and miR-517b-3p were expressed only in EVs from OSCC patients and miR-512-3p and miR-412-3p were up-regulated in salivary EVs from OSCC patients compared to controls with the ROC curve showing a good discrimination power for OSCC diagnosis. The Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analysis suggested the possible involvement of the miRNAs identified in pathways activated in OSCC. In this work, we suggest that salivary EVs isolated by a simple charge-based precipitation technique can be exploited as a non-invasive source of miRNAs for OSCC diagnosis. Moreover, we have identified a subset of miRNAs selectively enriched in EVs of OSCC patients that could be potential biomarkers.

Evolution of Fish Let-7 MicroRNAs and Their Expression Correlated to Growth Development in Blunt Snout Bream

PubMed Central

Zhao, Bo-Wen; Zhou, Lai-Fang; Liu, Yu-Long; Wan, Shi-Ming; Gao, Ze-Xia

2017-01-01

The lethal-7 (let-7) miRNA, known as one of the first founding miRNAs, is present in multiple copies in a genome and has diverse functions in animals. In this study, comparative genomic analysis of let-7 miRNAs members in fish species indicated that let-7 miRNA is a sequence conserved family in fish, while different species have the variable gene copy numbers. Among the ten members including let-7a/b/c/d/e/f/g/h/i/j, the let-7a precursor sequence was more similar to ancestral sequences, whereas other let-7 miRNA members were separate from the late differentiation of let-7a. The mostly predicted target genes of let-7 miRNAs are involved in biological process, especially developmental process and growth through Gene Ontology (GO) enrichment analysis. In order to identify the possible different functions of these ten miRNAs in fish growth development, their expression levels were quantified in adult males and females of Megalobrama amblycephala, as well as in 3-, 6-, and 12-months-old individuals with relatively slow- and fast-growth rates. These ten miRNAs had similar tissue expression patterns between males and females, with higher expression levels in the brain and pituitary than that in other tissues (p < 0.05). Among these miRNAs, the relative expression level of let-7a was the highest among almost all the tested tissues, followed by let-7b, let-7d and let-7c/e/f/g/h/i/j. As to the groups with different growth rates, the expression levels of let-7 miRNAs in pituitary and brain from the slow-growth group were always significantly higher than that in the fast-growth group (p < 0.05). These results suggest that let-7 miRNA members could play an important role in the regulation of growth development in M. amblycephala through negatively regulating expression of their target genes. PMID:28300776

Arsenic exposure triggers a shift in microRNA expression.

PubMed

Sturchio, Elena; Colombo, Teresa; Boccia, Priscilla; Carucci, Nicoletta; Meconi, Claudia; Minoia, Claudio; Macino, Giuseppe

2014-02-15

Exposure to inorganic Arsenic (iAs) through drinking water is a major public health problem affecting most countries. iAs has been classified by the International Agency for Research on Cancer as Group 1: "Carcinogenic to humans". Although numerous studies have shown the related adverse effects of iAs, sensitive appropriate biomarkers for studies of environmental epidemiology are still required. The present work aims at investigate the role of microRNAs (miRNAs), powerful negative regulators of gene expression, playing a key role in many physiological and pathological cellular processes, in iAs exposure. To this end, we analyzed miRNA changes in expression profile triggered by iAs exposure in Jurkat cell line. We used microarray technology to profile the expression of miRNAs following 2 μmol/L sodium arsenite treatment at different time points. Moreover, we performed phenotypic analysis of iAs treated cells. Real Time Polymerase Chain Reaction (RT-PCR) was used to validate miRNA microarray data and to assay expression modulation of selected relevant mRNAs. Finally, bioinformatics techniques were applied to reconstruct iAs-relevant molecular pathways and miRNA regulatory networks from the expression data. We report miRNAs modulated after iAs treatment in Jurkat cells. In particular, we highlight 36 miRNAs exhibiting consistent dysregulation and particularly a panel of 8 miRNAs which we also validated by RT-PCR analysis. Computational analysis of lists of putative target genes for these 8 miRNAs points to an involvement in arsenic-response pathways, for a subset of them, that were analyzed by RT-PCR. Furthermore, iAs exposure reveals induction of cell cycle progression and the failure of apoptosis, supporting the idea of iAs carcinogenic activity. Our study provides a list of miRNAs whose expression levels are affected by iAs treatment, corroborating the importance of proceeding with the hunt for specific subset of miRNAs, which can serve as potential biomarkers of iAs effects with useful diagnostic value. Copyright © 2013 Elsevier B.V. All rights reserved.

Signs of embryo-maternal communication: miRNAs in the maternal serum of pregnant pigs.

PubMed

Reliszko, Z P; Gajewski, Z; Kaczmarek, M M

2017-09-01

Circulating miRNAs were proposed to be indicators of normal or complicated pregnancies. Based on this knowledge and our recent transcriptomic approach showing expression of miRNAs in the porcine endometrium, conceptuses and uterine extracellular vesicles during pregnancy, we have hypothesized that signs of ongoing local embryo-maternal crosstalk involving miRNAs can be detected in the circulation of pregnant gilts as early as a few days after maternal recognition of pregnancy. By applying several molecular biology techniques that differ in dynamic range and precision in maternal serum of Day 16 pregnant pigs, we were able to show for the first time increased levels of several miRNAs, previously reported to be expressed in either conceptuses and extracellular vesicles (miR-26a and miR-125b) or pregnant endometrium (miR-23b). Our results clearly showed that real-time RT-PCR and digital PCR are the most reliable methods, being able to detect small-fold changes of low-abundant circulating miRNAs. Further validation in a separate group of gilts confirmed an increase in miR-23b and miR-125b levels. In silico analyses identified pregnancy-related biological processes and pathways affected by these miRNAs. Target prediction analysis revealed hundreds of porcine transcripts with conserved sites for these miRNAs, which were classified into signaling pathways relevant to pregnancy. We conclude that a unique set of miRNAs can already be observed in the circulation of pigs during the first weeks of pregnancy, as a result of the initiation of embryo-maternal communication. © 2017 Society for Reproduction and Fertility.

Expression of Versican 3′-Untranslated Region Modulates Endogenous MicroRNA Functions

PubMed Central

Lee, Daniel Y.; Jeyapalan, Zina; Fang, Ling; Yang, Jennifer; Zhang, Yaou; Yee, Albert Y.; Li, Minhui; Du, William W.; Shatseva, Tatiana; Yang, Burton B.

2010-01-01

Background Mature microRNAs (miRNAs) are single-stranded RNAs that regulate post-transcriptional gene expression. In our previous study, we have shown that versican 3′UTR, a fragment of non-coding transcript, has the ability to antagonize miR-199a-3p function thereby regulating expression of the matrix proteins versican and fibronectin, and thus resulting in enhanced cell-cell adhesion and organ adhesion. However, the impact of this non-coding fragment on tumorigenesis is yet to be determined. Methods and Findings Using computational prediction confirmed with in vitro and in vivo experiments, we report that the expression of versican 3′UTR not only antagonizes miR-199a-3p but can also lower its steady state expression. We found that expression of versican 3′UTR in a mouse breast carcinoma cell line, 4T1, decreased miR-199a-3p levels. The decrease in miRNA activity consequently translated into differences in tumor growth. Computational analysis indicated that both miR-199a-3p and miR-144 targeted a cell cycle regulator, Rb1. In addition, miR-144 and miR-136, which have also been shown to interact with versican 3′UTR, was found to target PTEN. Expression of Rb1 and PTEN were up-regulated synergistically in vitro and in vivo, suggesting that the 3′UTR binds and modulates miRNA activities, freeing Rb1 and PTEN mRNAs for translation. In tumor formation assays, cells transfected with the 3′UTR formed smaller tumors compared with cells transfected with a control vector. Conclusion Our results demonstrated that a 3′UTR fragment can be used to modulate miRNA functions. Our study also suggests that miRNAs in the cancer cells are more susceptible to degradation, due to its interaction with a non-coding 3′UTR. This non-coding component of mRNA may be used retrospectively to modulate miRNA activities. PMID:21049042

Mirnacle: machine learning with SMOTE and random forest for improving selectivity in pre-miRNA ab initio prediction.

PubMed

Marques, Yuri Bento; de Paiva Oliveira, Alcione; Ribeiro Vasconcelos, Ana Tereza; Cerqueira, Fabio Ribeiro

2016-12-15

MicroRNAs (miRNAs) are key gene expression regulators in plants and animals. Therefore, miRNAs are involved in several biological processes, making the study of these molecules one of the most relevant topics of molecular biology nowadays. However, characterizing miRNAs in vivo is still a complex task. As a consequence, in silico methods have been developed to predict miRNA loci. A common ab initio strategy to find miRNAs in genomic data is to search for sequences that can fold into the typical hairpin structure of miRNA precursors (pre-miRNAs). The current ab initio approaches, however, have selectivity issues, i.e., a high number of false positives is reported, which can lead to laborious and costly attempts to provide biological validation. This study presents an extension of the ab initio method miRNAFold, with the aim of improving selectivity through machine learning techniques, namely, random forest combined with the SMOTE procedure that copes with imbalance datasets. By comparing our method, termed Mirnacle, with other important approaches in the literature, we demonstrate that Mirnacle substantially improves selectivity without compromising sensitivity. For the three datasets used in our experiments, our method achieved at least 97% of sensitivity and could deliver a two-fold, 20-fold, and 6-fold increase in selectivity, respectively, compared with the best results of current computational tools. The extension of miRNAFold by the introduction of machine learning techniques, significantly increases selectivity in pre-miRNA ab initio prediction, which optimally contributes to advanced studies on miRNAs, as the need of biological validations is diminished. Hopefully, new research, such as studies of severe diseases caused by miRNA malfunction, will benefit from the proposed computational tool.

Testing the efficiency of plant artificial microRNAs by transient expression in Nicotiana benthamiana reveals additional action at the translational level

PubMed Central

Yu, Shi; Pilot, Guillaume

2014-01-01

Artificial microRNAs (amiRNAs) have become an important tool to assess gene functions due to their high efficiency and specificity to decrease target gene expression. Based on the observed degree of complementarity between microRNAs (miRNAs) and their targets, it was widely accepted that plant miRNAs act at the mRNA stability level, while the animal miRNAs act at the translational level. Contrary to these canonical dogmas, recent evidence suggests that both plant and animal miRNAs act at both levels. Nevertheless, it is still impossible to predict the effect of an artificial miRNA on the stability or translation of the target mRNA in plants. Consequently, identifying and discarding inefficient amiRNAs prior to stable plant transformation would help getting suppressed mutants faster and at reduced cost. We designed and tested a method using transient expression of amiRNAs and the corresponding target genes in Nicotiana benthamiana leaves to test the efficacy of amiRNAs for suppression of the target protein accumulation. The ability of the amiRNAs to suppress the target gene expression in N. benthamiana was then compared to that in stably transformed Arabidopsis. It was found that the efficacy of 16 amiRNAs, targeting a total of four genes, varied greatly. The effects of amiRNAs on target mRNA accumulation did not always correlate with target protein accumulation or the corresponding phenotypes, while a similar trend of the silencing efficacy of amiRNAs could be observed between N. benthamiana and stably transformed Arabidopsis. Our results showed that, similar to endogenous plant miRNAs, plant amiRNAs could act at the translational level, a property needed to be taken into account when testing the efficacy of individual amiRNAs. Preliminary tests in N. benthamiana can help determine which amiRNA would be the most likely to suppress target gene expression in stably transformed plants. PMID:25477887

Effects of MRP8, LPS, and lenalidomide on the expressions of TNF-α , brain-enriched, and inflammation-related microRNAs in the primary astrocyte culture.

PubMed

Omran, Ahmed; Ashhab, Muhammad Usman; Gan, Na; Kong, Huimin; Peng, Jing; Yin, Fei

2013-01-01

Astrocytes are now recognized as a heterogeneous class of cells with many important and diverse functions in healthy and diseased central nervous system (CNS). MicroRNAs (miRNAs) are small, noncoding RNAs which may have key roles in astrocytes activation in response to various stimuli. We performed quantitative real-time PCR (qPCR) to detect changes in the expressions of brain-enriched miRNAs (124, 134, 9, 132, and 138), inflammation-related miRNAs (146a, 21, 181a, 221, and 222), and tumor necrosis factor alpha (TNF- α ) in the rat primary astrocyte cultures after stimulation with myeloid-related protein 8 (MRP8) and lipopolysaccharides (LPS). Further, we inhibited the expression of TNF- α in the astrocytes by using TNF- α inhibitor (lenalidomide) and tested for the first time the effect of this inhibition on the expressions of the same tested miRNAs. Stimulation of the astrocytes with MRP8 or LPS leads to significant upregulation of miRNAs (124, 134, 9, 132, 146a, 21, 181a, 221, and 222), while miRNA-138 was downregulated. TNF- α inhibition with lenalidomide leads to opposite expressions of the tested miRNAs. These miRNAs may play an important role in activation of the astrocytes and may be a novel target for cell-specific therapeutic interventions in multiple CNS diseases.

Oxidized Low-density Lipoprotein (ox-LDL) Cholesterol Induces the Expression of miRNA-223 and L-type Calcium Channel Protein in Atrial Fibrillation

PubMed Central

He, Fengping; Xu, Xin; Yuan, Shuguo; Tan, Liangqiu; Gao, Lingjun; Ma, Shaochun; Zhang, Shebin; Ma, Zhanzhong; Jiang, Wei; Liu, Fenglian; Chen, Baofeng; Zhang, Beibei; Pang, Jungang; Huang, Xiuyan; Weng, Jiaqiang

2016-01-01

Atrial fibrillation (AF) is the most common sustained arrhythmia causing high morbidity and mortality. While changing of the cellular calcium homeostasis plays a critical role in AF, the L-type calcium channel α1c protein has suggested as an important regulator of reentrant spiral dynamics and is a major component of AF-related electrical remodeling. Our computational modeling predicted that miRNA-223 may regulate the CACNA1C gene which encodes the cardiac L-type calcium channel α1c subunit. We found that oxidized low-density lipoprotein (ox-LDL) cholesterol significantly up-regulates both the expression of miRNA-223 and L-type calcium channel protein. In contrast, knockdown of miRNA-223 reduced L-type calcium channel protein expression, while genetic knockdown of endogenous miRNA-223 dampened AF vulnerability. Transfection of miRNA-223 by adenovirus-mediated expression enhanced L-type calcium currents and promoted AF in mice while co-injection of a CACNA1C-specific miR-mimic counteracted the effect. Taken together, ox-LDL, as a known factor in AF-associated remodeling, positively regulates miRNA-223 transcription and L-type calcium channel protein expression. Our results implicate a new molecular mechanism for AF in which miRNA-223 can be used as an biomarker of AF rheumatic heart disease. PMID:27488468

Oxidized Low-density Lipoprotein (ox-LDL) Cholesterol Induces the Expression of miRNA-223 and L-type Calcium Channel Protein in Atrial Fibrillation

NASA Astrophysics Data System (ADS)

He, Fengping; Xu, Xin; Yuan, Shuguo; Tan, Liangqiu; Gao, Lingjun; Ma, Shaochun; Zhang, Shebin; Ma, Zhanzhong; Jiang, Wei; Liu, Fenglian; Chen, Baofeng; Zhang, Beibei; Pang, Jungang; Huang, Xiuyan; Weng, Jiaqiang

2016-08-01

Atrial fibrillation (AF) is the most common sustained arrhythmia causing high morbidity and mortality. While changing of the cellular calcium homeostasis plays a critical role in AF, the L-type calcium channel α1c protein has suggested as an important regulator of reentrant spiral dynamics and is a major component of AF-related electrical remodeling. Our computational modeling predicted that miRNA-223 may regulate the CACNA1C gene which encodes the cardiac L-type calcium channel α1c subunit. We found that oxidized low-density lipoprotein (ox-LDL) cholesterol significantly up-regulates both the expression of miRNA-223 and L-type calcium channel protein. In contrast, knockdown of miRNA-223 reduced L-type calcium channel protein expression, while genetic knockdown of endogenous miRNA-223 dampened AF vulnerability. Transfection of miRNA-223 by adenovirus-mediated expression enhanced L-type calcium currents and promoted AF in mice while co-injection of a CACNA1C-specific miR-mimic counteracted the effect. Taken together, ox-LDL, as a known factor in AF-associated remodeling, positively regulates miRNA-223 transcription and L-type calcium channel protein expression. Our results implicate a new molecular mechanism for AF in which miRNA-223 can be used as an biomarker of AF rheumatic heart disease.

In silico analysis and expression profiling of miRNAs targeting genes of steviol glycosides biosynthetic pathway and their relationship with steviol glycosides content in different tissues of Stevia rebaudiana.

PubMed

Saifi, Monica; Nasrullah, Nazima; Ahmad, Malik Mobeen; Ali, Athar; Khan, Jawaid A; Abdin, M Z

2015-09-01

miRNAs are emerging as potential regulators of the gene expression. Their proven promising role in regulating biosynthetic pathways related gene networks may hold the key to understand the genetic regulation of these pathways which may assist in selection and manipulation to get high performing plant genotypes with better secondary metabolites yields and increased biomass. miRNAs associated with genes of steviol glycosides biosynthetic pathway, however, have not been identified so far. In this study miRNAs targeting genes of steviol glycosides biosynthetic pathway were identified for the first time whose precursors were potentially generated from ESTs and nucleotide sequences of Stevia rebaudiana. Thereafter, stem-loop coupled real time PCR based expressions of these miRNAs in different tissues of Stevia rebaudiana were investigated and their relationship pattern was analysed with the expression levels of their target mRNAs as well as steviol glycoside contents. All the miRNAs investigated showed differential expressions in all the three tissues studied, viz. leaves, flowers and stems. Out of the eleven miRNAs validated, the expression levels of nine miRNAs (miR319a, miR319b, miR319c, miR319d, miR319e, miR319f, miR319h, miRstv_7, miRstv_9) were found to be inversely related, while expression levels of the two, i.e. miR319g and miRstv_11 on the contrary, showed direct relation with the expression levels of their target mRNAs and steviol glycoside contents in the leaves, flowers and stems. This study provides a platform for better understanding of the steviol glycosides biosynthetic pathway and these miRNAs can further be employed to manipulate the biosynthesis of these metabolites to enhance their contents and yield in S. rebaudiana. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Expression level of miRNAs on chromosome 14q32.31 region correlates with tumor aggressiveness and survival of glioblastoma patients.

PubMed

Shahar, Tal; Granit, Avital; Zrihan, Daniel; Canello, Tamar; Charbit, Hanna; Einstein, Ofira; Rozovski, Uri; Elgavish, Sharona; Ram, Zvi; Siegal, Tali; Lavon, Iris

2016-12-01

The 54 microRNAs (miRNAs) within the DLK-DIO3 genomic region on chromosome 14q32.31 (cluster-14-miRNAs) are organized into sub-clusters 14A and 14B. These miRNAs are downregulated in glioblastomas and might have a tumor suppressive role. Any association between the expression levels of cluster-14-miRNAs with overall survival (OS) is undetermined. We randomly selected miR-433, belonging to sub-cluster 14A and miR-323a-3p and miR-369-3p, belonging to sub-cluster 14B, and assessed their role in glioblastomas in vitro and in vivo. We also determined the expression level of cluster-14-miRNAs in 27 patients with newly diagnosed glioblastoma, and analyzed the association between their level of expression and OS. Overexpression of miR-323a-3p and miR-369-3p, but not miR-433, in glioblastoma cells inhibited their proliferation and migration in vitro. Mice implanted with glioblastoma cells overexpressing miR323a-3p and miR369-3p, but not miR433, exhibited prolonged survival compared to controls (P = .003). Bioinformatics analysis identified 13 putative target genes of cluster-14-miRNAs, and real-time RT-PCR validated these findings. Pathway analysis of the putative target genes identified neuregulin as the most enriched pathway. The expression level of cluster-14-miRNAs correlated with patients' OS. The median OS was 8.5 months for patients with low expression levels and 52.7 months for patients with high expression levels (HR 0.34; 95 % CI 0.12-0.59, P = .003). The expression level of cluster-14-miRNAs correlates directly with OS, suggesting a role for this cluster in promoting aggressive behavior of glioblastoma, possibly through ErBb/neuregulin signaling.

MicroRNA Profiling of Neurons Generated Using Induced Pluripotent Stem Cells Derived from Patients with Schizophrenia and Schizoaffective Disorder, and 22q11.2 Del

PubMed Central

Zhao, Dejian; Lin, Mingyan; Chen, Jian; Pedrosa, Erika; Hrabovsky, Anastasia; Fourcade, H. Matthew; Zheng, Deyou; Lachman, Herbert M.

2015-01-01

We are using induced pluripotent stem cell (iPSC) technology to study neuropsychiatric disorders associated with 22q11.2 microdeletions (del), the most common known schizophrenia (SZ)-associated genetic factor. Several genes in the region have been implicated; a promising candidate is DGCR8, which codes for a protein involved in microRNA (miRNA) biogenesis. We carried out miRNA expression profiling (miRNA-seq) on neurons generated from iPSCs derived from controls and SZ patients with 22q11.2 del. Using thresholds of p<0.01 for nominal significance and 1.5-fold differences in expression, 45 differentially expressed miRNAs were detected (13 lower in SZ and 32 higher). Of these, 6 were significantly down-regulated in patients after correcting for genome wide significance (FDR<0.05), including 4 miRNAs that map to the 22q11.2 del region. In addition, a nominally significant increase in the expression of several miRNAs was found in the 22q11.2 neurons that were previously found to be differentially expressed in autopsy samples and peripheral blood in SZ and autism spectrum disorders (e.g., miR-34, miR-4449, miR-146b-3p, and miR-23a-5p). Pathway and function analysis of predicted mRNA targets of the differentially expressed miRNAs showed enrichment for genes involved in neurological disease and psychological disorders for both up and down regulated miRNAs. Our findings suggest that: i. neurons with 22q11.2 del recapitulate the miRNA expression patterns expected of 22q11.2 haploinsufficiency, ii. differentially expressed miRNAs previously identified using autopsy samples and peripheral cells, both of which have significant methodological problems, are indeed disrupted in neuropsychiatric disorders and likely have an underlying genetic basis. PMID:26173148

Microarray‑based bioinformatics analysis of the prospective target gene network of key miRNAs influenced by long non‑coding RNA PVT1 in HCC.

PubMed

Zhang, Yu; Mo, Wei-Jia; Wang, Xiao; Zhang, Tong-Tong; Qin, Yuan; Wang, Han-Lin; Chen, Gang; Wei, Dan-Ming; Dang, Yi-Wu

2018-05-02

The long non‑coding RNA (lncRNA) PVT1 plays vital roles in the tumorigenesis and development of various types of cancer. However, the potential expression profiling, functions and pathways of PVT1 in HCC remain unknown. PVT1 was knocked down in SMMC‑7721 cells, and a miRNA microarray analysis was performed to detect the differentially expressed miRNAs. Twelve target prediction algorithms were used to predict the underlying targets of these differentially expressed miRNAs. Bioinformatics analysis was performed to explore the underlying functions, pathways and networks of the targeted genes. Furthermore, the relationship between PVT1 and the clinical parameters in HCC was confirmed based on the original data in the TCGA database. Among the differentially expressed miRNAs, the top two upregulated and downregulated miRNAs were selected for further analysis based on the false discovery rate (FDR), fold‑change (FC) and P‑values. Based on the TCGA database, PVT1 was obviously highly expressed in HCC, and a statistically higher PVT1 expression was found for sex (male), ethnicity (Asian) and pathological grade (G3+G4) compared to the control groups (P<0.05). Furthermore, Gene Ontology (GO) analysis revealed that the target genes were involved in complex cellular pathways, such as the macromolecule biosynthetic process, compound metabolic process, and transcription. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the MAPK and Wnt signaling pathways may be correlated with the regulation of the four candidate miRNAs. The results therefore provide significant information on the differentially expressed miRNAs associated with PVT1 in HCC, and we hypothesized that PVT1 may play vital roles in HCC by regulating different miRNAs or target gene expression (particularly MAPK8) via the MAPK or Wnt signaling pathways. Thus, further investigation of the molecular mechanism of PVT1 in HCC is needed.

Integrated analysis of mRNA and miRNA expression profiling in rice backcrossed progenies (BC2F12) with different plant height

PubMed Central

Cao, Aqin; Jin, Jie; Li, Shaoqing

2017-01-01

Inter-specific hybridization and backcrossing commonly occur in plants. The use of progeny generated from inter-specific hybridization and backcrossing has been developed as a novel model system to explore gene expression divergence. The present study investigated the analysis of gene expression and miRNA regulation in backcrossed introgression lines constructed from cultivated and wild rice. High-throughput sequencing was used to compare gene and miRNA expression profiles in three progeny lines (L1710, L1817 and L1730), with different plant heights resulting from the backcrossing of introgression lines (BC2F12) and their parents (O. sativa and O. longistaminata). A total of 25,387 to 26,139 mRNAs and 379 to 419 miRNAs were obtained in these rice lines. More differentially expressed genes and miRNAs were detected in progeny/O. longistaminata comparison groups than in progeny/O. sativa comparison groups. Approximately 80% of the genes and miRNAs showed expression level dominance to O. sativa, indicating that three progeny lines were closer to the recurrent parent, which might be influenced by their parental genome dosage. Approximately 16% to 64% of the differentially expressed miRNAs possessing coherent target genes were predicted, and many of these miRNAs regulated multiple target genes. Most genes were up-regulated in progeny lines compared with their parents, but down-regulated in the higher plant height line in the comparison groups among the three progeny lines. Moreover, certain genes related to cell walls and plant hormones might play crucial roles in the plant height variations of the three progeny lines. Taken together, these results provided valuable information on the molecular mechanisms of hybrid backcrossing and plant height variations based on the gene and miRNA expression levels in the three progeny lines. PMID:28859136

The use of high-throughput small RNA sequencing reveals differentially expressed microRNAs in response to aster yellows phytoplasma-infection in Vitis vinifera cv. ‘Chardonnay’

PubMed Central

Solofoharivelo, Marie-Chrystine; Souza-Richards, Rose; Stephan, Dirk; Murray, Shane; Burger, Johan T.

2017-01-01

Phytoplasmas are cell wall-less plant pathogenic bacteria responsible for major crop losses throughout the world. In grapevine they cause grapevine yellows, a detrimental disease associated with a variety of symptoms. The high economic impact of this disease has sparked considerable interest among researchers to understand molecular mechanisms related to pathogenesis. Increasing evidence exist that a class of small non-coding endogenous RNAs, known as microRNAs (miRNAs), play an important role in post-transcriptional gene regulation during plant development and responses to biotic and abiotic stresses. Thus, we aimed to dissect complex high-throughput small RNA sequencing data for the genome-wide identification of known and novel differentially expressed miRNAs, using read libraries constructed from healthy and phytoplasma-infected Chardonnay leaf material. Furthermore, we utilised computational resources to predict putative miRNA targets to explore the involvement of possible pathogen response pathways. We identified multiple known miRNA sequence variants (isomiRs), likely generated through post-transcriptional modifications. Sequences of 13 known, canonical miRNAs were shown to be differentially expressed. A total of 175 novel miRNA precursor sequences, each derived from a unique genomic location, were predicted, of which 23 were differentially expressed. A homology search revealed that some of these novel miRNAs shared high sequence similarity with conserved miRNAs from other plant species, as well as known grapevine miRNAs. The relative expression of randomly selected known and novel miRNAs was determined with real-time RT-qPCR analysis, thereby validating the trend of expression seen in the normalised small RNA sequencing read count data. Among the putative miRNA targets, we identified genes involved in plant morphology, hormone signalling, nutrient homeostasis, as well as plant stress. Our results may assist in understanding the role that miRNA pathways play during plant pathogenesis, and may be crucial in understanding disease symptom development in aster yellows phytoplasma-infected grapevines. PMID:28813447

The Effect of microRNAs in the Regulation of Human CYP3A4: a Systematic Study using a Mathematical Model

PubMed Central

Wei, Zhiyun; Jiang, Songshan; Zhang, Yiting; Wang, Xiaofei; Peng, Xueling; Meng, Chunjie; Liu, Yichen; Wang, Honglian; Guo, Luo; Qin, Shengying; He, Lin; Shao, Fengmin; Zhang, Lirong; Xing, Qinghe

2014-01-01

CYP3A4 metabolizes more than 50% of the drugs on the market. The large inter-individual differences of CYP3A4 expression may contribute to the variability of human drug responses. Post-transcriptional regulation of CYP3A4 is poorly understood, whereas transcriptional regulation has been studied much more thoroughly. In this study, we used multiple software programs to predict miRNAs that might bind to CYP3A4 and identified 112 potentially functional miRNAs. Then a luciferase reporter system was used to assess the effect of the overexpression of each potentially functional miRNA in HEK 293T cells. Fourteen miRNAs that significantly decreased reporter activity were measured in human liver samples (N = 27) as candidate miRNAs. To establish a more effective way to analyze in vivo data for miRNA candidates, the relationship between functional miRNA and target mRNA was modeled mathematically. Taking advantage of this model, we found that hsa-miR-577, hsa-miR-1, hsa-miR-532-3p and hsa-miR-627 could significantly downregulate the translation efficiency of CYP3A4 mRNA in liver. This study used in silico, in vitro and in vivo methods to progressively screen functional miRNAs for CYP3A4 and to enhance our understanding of molecular events underlying the large inter-individual differences of CYP3A4 expression in human populations. PMID:24594634

Tissue engineering and microRNAs: future perspectives in regenerative medicine.

PubMed

Gori, Manuele; Trombetta, Marcella; Santini, Daniele; Rainer, Alberto

2015-01-01

Tissue engineering is a growing area of biomedical research, holding great promise for a broad range of potential applications in the field of regenerative medicine. In recent decades, multiple tissue engineering strategies have been adopted to mimic and improve specific biological functions of tissues and organs, including biomimetic materials, drug-releasing scaffolds, stem cells, and dynamic culture systems. MicroRNAs (miRNAs), noncoding small RNAs that negatively regulate the expression of downstream target mRNAs, are considered a novel class of molecular targets and therapeutics that may play an important role in tissue engineering. Herein, we highlight the latest achievements in regenerative medicine, focusing on the role of miRNAs as key modulators of gene expression, stem cell self-renewal, proliferation and differentiation, and eventually in driving cell fate decisions. Finally, we will discuss the contribution of miRNAs in regulating the rearrangement of the tissue microenvironment and angiogenesis, and the range of strategies for miRNA delivery into target cells and tissues. Manipulation of miRNAs is an alternative approach and an attractive strategy for controlling several aspects of tissue engineering, although some issues concerning their in vivo effects and optimal delivery methods still remain uncovered.

Gene silencing efficiency and INF-β induction effects of splicing miRNA 155-based artificial miRNA with pre-miRNA stem-loop structures.

PubMed

Sin, Onsam; Mabiala, Prudence; Liu, Ye; Sun, Ying; Hu, Tao; Liu, Qingzhen; Guo, Deyin

2012-02-01

Artificial microRNA (miRNA) expression vectors have been developed and used for RNA interference. The secondary structure of artificial miRNA is important for RNA interference efficacy. We designed two groups of six artificial splicing miRNA 155-based miRNAs (SM155-based miRNAs) with the same target in the coding region or 3' UTR of a target gene and studied their RNA silencing efficiency and interferon β (IFN-β) induction effects. SM155-based miRNA with a mismatch at the +1 position and a bulge at the +11, +12 positions in a miRNA precursor stem-loop structure showed the highest gene silencing efficiency and lowest IFN-β induction effect (increased IFN-β mRNA level by 10% in both target cases), regardless of the specificity of the target sequence, suggesting that pSM155-based miRNA with this design could be a valuable miRNA expression vector.

Targeted gene deletion of miRNAs in mice by TALEN system.

PubMed

Takada, Shuji; Sato, Tempei; Ito, Yoshiaki; Yamashita, Satoshi; Kato, Tomoko; Kawasumi, Miyuri; Kanai-Azuma, Masami; Igarashi, Arisa; Kato, Tomomi; Tamano, Moe; Asahara, Hiroshi

2013-01-01

Mice are among the most valuable model animal species with an enormous amount of heritage in genetic modification studies. However, targeting genes in mice is sometimes difficult, especially for small genes, such as microRNAs (miRNAs) and targeting genes in repeat sequences. Here we optimized the application of TALEN system for mice and successfully obtained gene targeting technique in mice for intergenic region and series of microRNAs. Microinjection of synthesized RNA of TALEN targeting each gene in one cell stage of embryo was carried out and injected oocytes were transferred into pseudopregnant ICR female mice, producing a high success rate of the targeted deletion of miRNA genes. In our condition, TALEN RNA without poly(A) tail worked better than that of with poly(A) tail. This mutated allele in miRNA was transmitted to the next generation, suggesting the successful germ line transmission of this targeting method. Consistent with our notion of miRNAs maturation mechanism, in homozygous mutant mice of miR-10a, the non- mutated strand of miRNAs expression was completely diminished. This method will lead us to expand and accelerate our genetic research using mice in a high throughput way.

Novel Modeling of Combinatorial miRNA Targeting Identifies SNP with Potential Role in Bone Density

PubMed Central

Coronnello, Claudia; Hartmaier, Ryan; Arora, Arshi; Huleihel, Luai; Pandit, Kusum V.; Bais, Abha S.; Butterworth, Michael; Kaminski, Naftali; Stormo, Gary D.; Oesterreich, Steffi; Benos, Panayiotis V.

2012-01-01

MicroRNAs (miRNAs) are post-transcriptional regulators that bind to their target mRNAs through base complementarity. Predicting miRNA targets is a challenging task and various studies showed that existing algorithms suffer from high number of false predictions and low to moderate overlap in their predictions. Until recently, very few algorithms considered the dynamic nature of the interactions, including the effect of less specific interactions, the miRNA expression level, and the effect of combinatorial miRNA binding. Addressing these issues can result in a more accurate miRNA:mRNA modeling with many applications, including efficient miRNA-related SNP evaluation. We present a novel thermodynamic model based on the Fermi-Dirac equation that incorporates miRNA expression in the prediction of target occupancy and we show that it improves the performance of two popular single miRNA target finders. Modeling combinatorial miRNA targeting is a natural extension of this model. Two other algorithms show improved prediction efficiency when combinatorial binding models were considered. ComiR (Combinatorial miRNA targeting), a novel algorithm we developed, incorporates the improved predictions of the four target finders into a single probabilistic score using ensemble learning. Combining target scores of multiple miRNAs using ComiR improves predictions over the naïve method for target combination. ComiR scoring scheme can be used for identification of SNPs affecting miRNA binding. As proof of principle, ComiR identified rs17737058 as disruptive to the miR-488-5p:NCOA1 interaction, which we confirmed in vitro. We also found rs17737058 to be significantly associated with decreased bone mineral density (BMD) in two independent cohorts indicating that the miR-488-5p/NCOA1 regulatory axis is likely critical in maintaining BMD in women. With increasing availability of comprehensive high-throughput datasets from patients ComiR is expected to become an essential tool for miRNA-related studies. PMID:23284279

MicroRNA genes are frequently located near mouse cancer susceptibility loci

PubMed Central

Sevignani, Cinzia; Calin, George A.; Nnadi, Stephanie C.; Shimizu, Masayoshi; Davuluri, Ramana V.; Hyslop, Terry; Demant, Peter; Croce, Carlo M.; Siracusa, Linda D.

2007-01-01

MicroRNAs (miRNAs) are short 19- to 24-nt RNA molecules that have been shown to regulate the expression of other genes in a variety of eukaryotic systems. Abnormal expression of miRNAs has been observed in several human cancers, and furthermore, germ-line and somatic mutations in human miRNAs were recently identified in patients with chronic lymphocytic leukemia. Thus, human miRNAs can act as tumor suppressor genes or oncogenes, where mutations, deletions, or amplifications can underlie the development of certain types of leukemia. In addition, previous studies have shown that miRNA expression profiles can distinguish among human solid tumors from different organs. Because a single miRNA can simultaneously influence the expression of two or more protein-coding genes, we hypothesized that miRNAs could be candidate genes for cancer risk. Research in complex trait genetics has demonstrated that genetic background determines cancer susceptibility or resistance in various tissues, such as colon and lung, of different inbred mouse strains. We compared the genome positions of mouse tumor susceptibility loci with those of mouse miRNAs. Here, we report a statistically significant association between the chromosomal location of miRNAs and those of mouse cancer susceptibility loci that influence the development of solid tumors. Furthermore, we identified distinct patterns of flanking DNA sequences for several miRNAs located at or near susceptibility loci in inbred strains with different tumor susceptibilities. These data provide a catalog of miRNA genes in inbred strains that could represent genes involved in the development and penetrance of solid tumors. PMID:17470785

Sexual Dimorphism Floral MicroRNA Profiling and Target Gene Expression in Andromonoecious Poplar (Populus tomentosa)

PubMed Central

Song, Yuepeng; Ma, Kaifeng; Ci, Dong; Zhang, Zhiyi; Zhang, Deqiang

2013-01-01

Although the molecular basis of poplar sex-specific flower development remains largely unknown, increasing evidence indicates an essential role for microRNAs (miRNAs). The specific miRNA types and precise miRNA expression patterns in dioecious plant flower development remain unclear. Here, we used andromonoecious poplar, an exceptional model system, to eliminate the confounding effects of genetic background of dioecious plants. This system, combined with high-throughput sequencing and computational analysis, allowed us to characterize sex-specific miRNAomes from female and male flowers. Comparative miRNAome analysis combined with quantitative real-time PCR revealed the expression patterns of 27 miRNAs in poplar flower and showed that the targets of these miRNAs are involved in flower organogenesis, Ca2+ transport, phytohormone synthesis and metabolism, and DNA methylation. This paper describes a complex regulatory network consisting of these miRNAs expressed in sex-specific flower development in a dioecious plant. The conserved and novel miRNA locations were annotated in the Populus trichocarpa genome. Among these, miRNA Pto-F70 and 4 targets are located in the sex-determination regions of chromosome XIX. Furthermore, two novel miRNAs, Pto-F47 and Pto-F68, were shown for the first time to be regulatory factors in phytohormone interactions. To our knowledge, this report is the first systematic investigation of sex-specific flower-related miRNAs and their targets in poplar, and it deepens our understanding of the important regulatory functions of miRNAs in female and male flower development in this dioecious plant. PMID:23667507

GSK3 inhibitors CHIR99021 and 6-bromoindirubin-3′-oxime inhibit microRNA maturation in mouse embryonic stem cells

PubMed Central

Wu, Yongyan; Liu, Fayang; Liu, Yingying; Liu, Xiaolei; Ai, Zhiying; Guo, Zekun; Zhang, Yong

2015-01-01

Wnt/β-catenin signalling plays a prominent role in maintaining self-renewal and pluripotency of mouse embryonic stem cells (mESCs). microRNAs (miRNAs) have critical roles in maintaining pluripotency and directing reprogramming. To investigate the effect of GSK3 inhibitors on miRNA expression, we analysed the miRNA expression profile of J1 mESCs in the absence or presence of CHIR99021 (CHIR) or 6-bromoindirubin-3′-oxime (BIO) by small RNA deep-sequencing. The results demonstrate that CHIR and BIO decrease mature miRNAs of most miRNA species, 90.4% and 98.1% of the differentially expressed miRNAs in BIO and CHIR treated cells were downregulated respectively. CHIR and BIO treatment leads to a slight upregulation of the primary transcripts of the miR-302–367 cluster and miR-181 family of miRNAs, these miRNAs are activated by Wnt/β-catenin signalling. However, the precursor and mature form of the miR-302–367 cluster and miR-181 family of miRNAs are downregulated by CHIR, suggesting CHIR inhibits maturation of primary miRNA. Western blot analysis shows that BIO and CHIR treatment leads to a reduction of the RNase III enzyme Drosha in the nucleus. These data suggest that BIO and CHIR inhibit miRNA maturation by disturbing nuclear localisation of Drosha. Results also show that BIO and CHIR induce miR-211 expression in J1 mESCs. PMID:25727520

A Pilot Study of Circulating MicroRNA-125b as a Diagnostic and Prognostic Biomarker for Epithelial Ovarian Cancer.

PubMed

Zhu, Tao; Gao, Wen; Chen, Xi; Zhang, Ying; Wu, Meijuan; Zhang, Ping; Wang, Shihua

2017-01-01

Early diagnosis of epithelial ovarian cancer is critical for patient survival. The objective of this pilot study is to identify a circulating micro (mi)RNA as a potential biomarker for epithelial ovarian cancer. A total of 135 epithelial ovarian cancer patients and 54 benign ovarian tumor patients were recruited for this study. Using customized TaqMan low density miRNA arrays, we first screened expression levels of 48 miRNAs in sera from 18 epithelial ovarian cancer patients and 16 benign ovarian tumor patients. The most significantly and differentially expressed miRNA was then further examined in all serum samples using real-time polymerase chain reaction. Its expression was further analyzed in relationship with clinicopathological factors and patient survival. Array screening data showed that expression levels of serum miRNA-20a, miRNA-125b, miRNA-126, miRNA-355, and let-7c were significantly different between malignant and benign ovarian tumor patients. Subsequent real-time polymerase chain reaction results showed that serum miRNA-125b levels were significantly higher in epithelial ovarian cancer patients compared to benign controls. Moreover, serum miRNA-125b levels were significantly higher in ovarian cancer patients in early stages I and II, and in patients having no residual tumor following surgery, but were not associated with differentiation and histological types of ovarian cancer. Notably, the higher level of miR-125b was significantly positively correlated with progression-free survival (P = 0.035) and marginally, with overall survival (P = 0.069). miRNA-125b plays an important role in the pathogenesis and progression of epithelial ovarian cancer. Circulating miRNA-125b has the potential to become a novel biomarker for early diagnosis and prognosis prediction of epithelial ovarian cancer.

Exosomal and Non-Exosomal Transport of Extra-Cellular microRNAs in Follicular Fluid: Implications for Bovine Oocyte Developmental Competence

PubMed Central

Sohel, Md. Mahmodul Hasan; Hoelker, Michael; Noferesti, Sina Seifi; Salilew-Wondim, Dessie; Tholen, Ernst; Looft, Christian; Rings, Franca; Uddin, Muhammad Jasim; Spencer, Thomas E.; Schellander, Karl; Tesfaye, Dawit

2013-01-01

Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue (BCB) staining of the corresponding oocytes. Both Exoquick precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal fraction of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNA™ Universal RT miRNA PCR array system was used to profile miRNA expression. This analysis found that miRNAs were present in both exosomal and non-exosomal fraction of bovine follicular fluid. We found 25 miRNAs differentially expressed (16 up and 9 down) in exosomes and 30 miRNAs differentially expressed (21 up and 9 down) in non-exosomal fraction of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus oocyte complex. To further explore the potential roles of these follicular fluid derived extra-cellular miRNAs, the potential target genes were predicted, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. This study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment. PMID:24223816

Genome-wide discovery and differential regulation of conserved and novel microRNAs in chickpea via deep sequencing.

PubMed

Jain, Mukesh; Chevala, V V S Narayana; Garg, Rohini

2014-11-01

MicroRNAs (miRNAs) are essential components of complex gene regulatory networks that orchestrate plant development. Although several genomic resources have been developed for the legume crop chickpea, miRNAs have not been discovered until now. For genome-wide discovery of miRNAs in chickpea (Cicer arietinum), we sequenced the small RNA content from seven major tissues/organs employing Illumina technology. About 154 million reads were generated, which represented more than 20 million distinct small RNA sequences. We identified a total of 440 conserved miRNAs in chickpea based on sequence similarity with known miRNAs in other plants. In addition, 178 novel miRNAs were identified using a miRDeep pipeline with plant-specific scoring. Some of the conserved and novel miRNAs with significant sequence similarity were grouped into families. The chickpea miRNAs targeted a wide range of mRNAs involved in diverse cellular processes, including transcriptional regulation (transcription factors), protein modification and turnover, signal transduction, and metabolism. Our analysis revealed several miRNAs with differential spatial expression. Many of the chickpea miRNAs were expressed in a tissue-specific manner. The conserved and differential expression of members of the same miRNA family in different tissues was also observed. Some of the same family members were predicted to target different chickpea mRNAs, which suggested the specificity and complexity of miRNA-mediated developmental regulation. This study, for the first time, reveals a comprehensive set of conserved and novel miRNAs along with their expression patterns and putative targets in chickpea, and provides a framework for understanding regulation of developmental processes in legumes. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Identification of conserved microRNAs in peripheral blood from giant panda: expression of mammary gland-related microRNAs during late pregnancy and early lactation.

PubMed

Wang, C D; Long, K; Jin, L; Huang, S; Li, D H; Ma, X P; Wei, M; Gu, Y; Ma, J D; Zhang, H

2015-11-13

The giant panda (Ailuropoda melanoleuca) is one of the world's most endangered mammals, and it has evolved several unusual biological and behavioral traits. During puberty, pregnancy, lactation, and involution, the mammary gland undergoes profound morphological and functional changes. A large number of microRNAs (miRNAs) have been identified to be involved in mammary gland development and lactation. In this study, we identified 202 conserved mature miRNAs, corresponding to 147 pre-miRNAs, in giant panda peripheral blood using a small RNA-sequencing approach. In addition, 27 miRNA families and 29 miRNA clusters were identified. We analyzed the arm selection preference of pre-miRNAs and found that: 1) most giant panda pre-miRNAs generated one-strand miRNAs, and the 5p-arm only miRNAs have a higher expression level than 3p-arm only miRNAs; 2) there were more 5p-arm dominant miRNAs than 3p-arm dominant miRNAs; and 3) 5p-arm dominant miRNAs have a larger fold change within miRNA pairs than 3p-arm dominant miRNAs. Expression of 12 lactation-related miRNAs was detected across late pregnancy and early lactation stages by qPCR, and seven miRNAs were identified as clustered in one significant model. Most of these clustered miRNAs exhibited inhibitory roles in proliferation and differentiation of mammary epithelial cells. Functional analysis highlighted important roles of the seven as signed miRNAs in mammary development and metabolic changes, including blood vessel morphogenesis, macromolecule biosynthesis, cell cycle regulation, and protein transport.

Cryopreservation of boar sperm induces differential microRNAs expression.

PubMed

Zhang, Yan; Dai, Dinghui; Chang, Yu; Li, Yuan; Zhang, Ming; Zhou, Guangbin; Peng, Zhanghua; Zeng, Changjun

2017-06-01

Lower conception rates and litter sizes limit the wide use of artificial insemination with frozen-thawed boar sperm, due to a lack of understanding of the mechanisms that cause cryodamage and cryoinjury to sperm during cryopreservation. CryoMiRs, a family of freeze-related microRNAs (miRNAs), are associated with freeze tolerance, and regulate metabolism in mammalian hibernators and insects. Thus, we speculate that miRNAs maybe involved in the regulation of the freeze-thaw process and may affect boar sperm function. In this study, we studied the differential expression of 46 miRNAs that have roles in spermatogenesis, sperm maturation, and sperm quality in response to cryopreservation (with or without 3% glycerol). The results indicated that, in response to cryopreservation with 3% glycerol, 14 miRNAs were significantly up-regulated, but only two miRNAs (miR-22 and miR-450b-5p) were significantly down-regulated, relative to fresh sperm. Preservation with 3% glycerol caused up-regulation of 17 miRNAs, but only caused down-regulation of one miRNA (miR-24), relative to sperm cryopreserved without glycerol. Functional annotations of these differentially expressed miRNAs indicated that these miRNAs and their targets are mainly associated with metabolic and cellular processes. Therefore, our findings show that cryopreservation results in changes in miRNA expression, and suggest that the anti-freeze mechanisms of boar sperm need to be studied further. Copyright © 2017 Elsevier Inc. All rights reserved.

Profile of microRNA in Giant Panda Blood: A Resource for Immune-Related and Novel microRNAs

PubMed Central

Yang, Mingyu; Du, Lianming; Li, Wujiao; Shen, Fujun; Fan, Zhenxin; Jian, Zuoyi; Hou, Rong; Shen, Yongmei; Yue, Bisong; Zhang, Xiuyue

2015-01-01

The giant panda (Ailuropoda melanoleuca) is one of the world’s most beloved endangered mammals. Although the draft genome of this species had been assembled, little was known about the composition of its microRNAs (miRNAs) or their functional profiles. Recent studies demonstrated that changes in the expression of miRNAs are associated with immunity. In this study, miRNAs were extracted from the blood of four healthy giant pandas and sequenced by Illumina next generation sequencing technology. As determined by miRNA screening, a total of 276 conserved miRNAs and 51 novel putative miRNAs candidates were detected. After differential expression analysis, we noticed that the expressions of 7 miRNAs were significantly up-regulated in young giant pandas compared with that of adults. Moreover, 2 miRNAs were up-regulated in female giant pandas and 1 in the male individuals. Target gene prediction suggested that the miRNAs of giant panda might be relevant to the expressions of 4,602 downstream genes. Subseuqently, the predicted target genes were conducted to KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis and we found that these genes were mainly involved in host immunity, including the Ras signaling pathway, the PI3K-Akt signaling pathway, and the MAPK signaling pathway. In conclusion, our results provide the first miRNA profiles of giant panda blood, and the predicted functional analyses may open an avenue for further study of giant panda immunity. PMID:26599861

Splenic marginal zone lymphoma: comprehensive analysis of gene expression and miRNA profiling.

PubMed

Arribas, Alberto J; Gómez-Abad, Cristina; Sánchez-Beato, Margarita; Martinez, Nerea; Dilisio, Lorena; Casado, Felipe; Cruz, Miguel A; Algara, Patrocinio; Piris, Miguel A; Mollejo, Manuela

2013-07-01

Splenic marginal zone lymphoma is a small B-cell neoplasm whose molecular pathogenesis is still essentially unknown and whose differentiation from other small B-cell lymphomas is hampered by the lack of specific markers. We have analyzed the gene expression and miRNA profiles of 31 splenic marginal zone lymphoma cases. For comparison, 7 spleens with reactive lymphoid hyperplasia, 10 spleens infiltrated by chronic lymphocytic leukemia, 12 spleens with follicular lymphoma, 6 spleens infiltrated by mantle cell lymphoma and 15 lymph nodes infiltrated by nodal marginal zone lymphoma were included. The results were validated by qRT-PCR in an independent series including 77 paraffin-embedded splenic marginal zone lymphomas. The splenic marginal zone lymphoma miRNA signature had deregulated expression of 51 miRNAs. The most highly overexpressed miRNAs were miR-155, miR-21, miR-34a, miR-193b and miR-100, while the most repressed miRNAs were miR-377, miR-27b, miR-145, miR-376a and miR-424. MiRNAs located in 14q32-31 were underexpressed in splenic marginal zone lymphoma compared with reactive lymphoid tissues and other B-cell lymphomas. Finally, the gene expression data were integrated with the miRNA profile to identify functional relationships between genes and deregulated miRNAs. Our study reveals miRNAs that are deregulated in splenic marginal zone lymphoma and identifies new candidate diagnostic molecules for splenic marginal zone lymphoma.

Profile of microRNA in Giant Panda Blood: A Resource for Immune-Related and Novel microRNAs.

PubMed

Yang, Mingyu; Du, Lianming; Li, Wujiao; Shen, Fujun; Fan, Zhenxin; Jian, Zuoyi; Hou, Rong; Shen, Yongmei; Yue, Bisong; Zhang, Xiuyue

2015-01-01

The giant panda (Ailuropoda melanoleuca) is one of the world's most beloved endangered mammals. Although the draft genome of this species had been assembled, little was known about the composition of its microRNAs (miRNAs) or their functional profiles. Recent studies demonstrated that changes in the expression of miRNAs are associated with immunity. In this study, miRNAs were extracted from the blood of four healthy giant pandas and sequenced by Illumina next generation sequencing technology. As determined by miRNA screening, a total of 276 conserved miRNAs and 51 novel putative miRNAs candidates were detected. After differential expression analysis, we noticed that the expressions of 7 miRNAs were significantly up-regulated in young giant pandas compared with that of adults. Moreover, 2 miRNAs were up-regulated in female giant pandas and 1 in the male individuals. Target gene prediction suggested that the miRNAs of giant panda might be relevant to the expressions of 4,602 downstream genes. Subseuqently, the predicted target genes were conducted to KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis and we found that these genes were mainly involved in host immunity, including the Ras signaling pathway, the PI3K-Akt signaling pathway, and the MAPK signaling pathway. In conclusion, our results provide the first miRNA profiles of giant panda blood, and the predicted functional analyses may open an avenue for further study of giant panda immunity.

Identification of neoblast- and regeneration-specific miRNAs in the planarian Schmidtea mediterranea

PubMed Central

Sasidharan, Vidyanand; Lu, Yi-Chien; Bansal, Dhiru; Dasari, Pranavi; Poduval, Deepak; Seshasayee, Aswin; Resch, Alissa M.; Graveley, Brenton R.; Palakodeti, Dasaradhi

2013-01-01

In recent years, the planarian Schmidtea mediterranea has emerged as a tractable model system to study stem cell biology and regeneration. MicroRNAs are small RNA species that control gene expression by modulating translational repression and mRNA stability and have been implicated in the regulation of various cellular processes. Though recent studies have identified several miRNAs in S. mediterranea, their expression in neoblast subpopulations and during regeneration has not been examined. Here, we identify several miRNAs whose expression is enriched in different neoblast subpopulations and in regenerating tissue at different time points in S. mediterranea. Some of these miRNAs were enriched within 3 h post-amputation and may, therefore, play a role in wound healing and/or neoblast migration. Our results also revealed miRNAs, such as sme-miR-2d-3p and the sme-miR-124 family, whose expression is enriched in the cephalic ganglia, are also expressed in the brain primordium during CNS regeneration. These results provide new insight into the potential biological functions of miRNAs in neoblasts and regeneration in planarians. PMID:23974438

miRNA expression profiling in formalin-fixed paraffin-embedded endometriosis and ovarian cancer samples

PubMed Central

Braicu, Ovidiu-Leonard; Budisan, Liviuta; Buiga, Rares; Jurj, Ancuta; Achimas-Cadariu, Patriciu; Pop, Laura Ancuta; Braicu, Cornelia; Irimie, Alexandru; Berindan-Neagoe, Ioana

2017-01-01

Endometriosis is an inflammatory pathology associated with a negative effect on life quality. Recently, this pathology was connected to ovarian cancer, in particular with endometrioid ovarian cancer. microRNAs (miRNAs) are a class of RNA transcripts ~19–22 nucleotides in length, the altered miRNA pattern being connected to pathological status. miRNAs are highly stable transcripts, and these can be assessed from formalin-fixed paraffin-embedded (FFPE) samples leading to the identification of miRNAs that could be developed as diagnostic and prognostic biomarkers, in particular those involved in malignant transformation. The aim of our study was to evaluate miRNA expression pattern in FFPE samples from endometriosis and ovarian cancer patients using PCR-array technology and also to compare the differential expression pattern in ovarian cancer versus endometriosis. For the PCR-array study, we have used nine macrodissected FFPE samples from endometriosis tissue, eight samples of ovarian cancers and five normal ovarian tissues. Quantitative real-time PCR (qRT-PCR) was used for data validation in a new patient cohort of 17 normal samples, 33 endometriosis samples and 28 ovarian cancer macrodissected FFPE samples. Considering 1.5-fold expression difference as a cut-off level and a P-value <0.05, we have identified four miRNAs being overexpressed in endometrial tissue, while in ovarian cancer 15 were differentially expressed (nine overexpressed and six downregulated). The expression level was confirmed by qRT-PCR for miR-93, miR-141, miR-155, miR-429, miR-200c, miR-205 and miR-492. Using the interpretative program Ingenuity Pathway Analysis revealed several deregulated pathways due to abnormal miRNA expression in endometriosis and ovarian cancer, which in turn is responsible for pathogenesis; this differential expression of miRNAs can be exploited as a therapeutic target. A higher number of altered miRNAs were detected in endometriosis versus ovarian cancer tissue, most of them being linked with epithelial-to-mesenchymal transition. PMID:28894379

High-Throughput Sequencing Reveals Circulating miRNAs as Potential Biomarkers for Measuring Puberty Onset in Chicken (Gallus gallus).

PubMed

Han, Wei; Zhu, Yunfen; Su, Yijun; Li, Guohui; Qu, Liang; Zhang, Huiyong; Wang, Kehua; Zou, Jianmin; Liu, Honglin

2016-01-01

There are still no highly sensitive and unique biomarkers for measurement of puberty onset. Circulating miRNAs have been shown to be promising biomarkers for diagnosis of various diseases. To identify circulating miRNAs that could be served as biomarkers for measuring chicken (Gallus gallus) puberty onset, the Solexa deep sequencing was performed to analyze the miRNA expression profiles in serum and plasma of hens from two different pubertal stages, before puberty onset (BO) and after puberty onset (AO). 197 conserved and 19 novel miRNAs (reads > 10) were identified as serum/plasma-expressed miRNAs in the chicken. The common miRNA amounts and their expression changes from BO to AO between serum and plasma were very similar, indicating the different treatments to generate serum and plasma had quite small influence on the miRNAs. 130 conserved serum-miRNAs were showed to be differentially expressed (reads > 10, P < 0.05) from BO to AO, with 68 up-regulated and 62 down-regulated. 4829 putative genes were predicted as the targets of the 40 most differentially expressed miRNAs (|log2(fold-change)|>1.0, P < 0.01). Functional analysis revealed several pathways that were associated with puberty onset. Further quantitative real-time PCR (RT-qPCR) test found that a seven-miRNA panel, including miR-29c, miR-375, miR-215, miR-217, miR-19b, miR-133a and let-7a, had great potentials to serve as novel biomarkers for measuring puberty onset in chicken. Due to highly conserved nature of miRNAs, the findings could provide cues for measurement of puberty onset in other animals as well as humans.

High-Throughput Sequencing Reveals Circulating miRNAs as Potential Biomarkers for Measuring Puberty Onset in Chicken (Gallus gallus)

PubMed Central

Su, Yijun; Li, Guohui; Qu, Liang; Zhang, Huiyong; Wang, Kehua; Zou, Jianmin; Liu, Honglin

2016-01-01

There are still no highly sensitive and unique biomarkers for measurement of puberty onset. Circulating miRNAs have been shown to be promising biomarkers for diagnosis of various diseases. To identify circulating miRNAs that could be served as biomarkers for measuring chicken (Gallus gallus) puberty onset, the Solexa deep sequencing was performed to analyze the miRNA expression profiles in serum and plasma of hens from two different pubertal stages, before puberty onset (BO) and after puberty onset (AO). 197 conserved and 19 novel miRNAs (reads > 10) were identified as serum/plasma-expressed miRNAs in the chicken. The common miRNA amounts and their expression changes from BO to AO between serum and plasma were very similar, indicating the different treatments to generate serum and plasma had quite small influence on the miRNAs. 130 conserved serum-miRNAs were showed to be differentially expressed (reads > 10, P < 0.05) from BO to AO, with 68 up-regulated and 62 down-regulated. 4829 putative genes were predicted as the targets of the 40 most differentially expressed miRNAs (|log2(fold-change)|>1.0, P < 0.01). Functional analysis revealed several pathways that were associated with puberty onset. Further quantitative real-time PCR (RT-qPCR) test found that a seven-miRNA panel, including miR-29c, miR-375, miR-215, miR-217, miR-19b, miR-133a and let-7a, had great potentials to serve as novel biomarkers for measuring puberty onset in chicken. Due to highly conserved nature of miRNAs, the findings could provide cues for measurement of puberty onset in other animals as well as humans. PMID:27149515

Global miRNA expression and correlation with mRNA levels in primary human bone cells

PubMed Central

Laxman, Navya; Rubin, Carl-Johan; Mallmin, Hans; Nilsson, Olle; Pastinen, Tomi; Grundberg, Elin; Kindmark, Andreas

2015-01-01

MicroRNAs (miRNAs) are important post-transcriptional regulators that have recently introduced an additional level of intricacy to our understanding of gene regulation. The aim of this study was to investigate miRNA–mRNA interactions that may be relevant for bone metabolism by assessing correlations and interindividual variability in miRNA levels as well as global correlations between miRNA and mRNA levels in a large cohort of primary human osteoblasts (HOBs) obtained during orthopedic surgery in otherwise healthy individuals. We identified differential expression (DE) of 24 miRNAs, and found 9 miRNAs exhibiting DE between males and females. We identified hsa-miR-29b, hsa-miR-30c2, and hsa-miR-125b and their target genes as important modulators of bone metabolism. Further, we used an integrated analysis of global miRNA–mRNA correlations, mRNA-expression profiling, DE, bioinformatics analysis, and functional studies to identify novel target genes for miRNAs with the potential to regulate osteoblast differentiation and extracellular matrix production. Functional studies by overexpression and knockdown of miRNAs showed that, the differentially expressed miRNAs hsa-miR-29b, hsa-miR-30c2, and hsa-miR-125b target genes highly relevant to bone metabolism, e.g., collagen, type I, α1 (COL1A1), osteonectin (SPARC), Runt-related transcription factor 2 (RUNX2), osteocalcin (BGLAP), and frizzled-related protein (FRZB). These miRNAs orchestrate the activities of key regulators of osteoblast differentiation and extracellular matrix proteins by their convergent action on target genes and pathways to control the skeletal gene expression. PMID:26078267

Methylation of microRNA genes regulates gene expression in bisexual flower development in andromonoecious poplar.

PubMed

Song, Yuepeng; Tian, Min; Ci, Dong; Zhang, Deqiang

2015-04-01

Previous studies showed sex-specific DNA methylation and expression of candidate genes in bisexual flowers of andromonoecious poplar, but the regulatory relationship between methylation and microRNAs (miRNAs) remains unclear. To investigate whether the methylation of miRNA genes regulates gene expression in bisexual flower development, the methylome, microRNA, and transcriptome were examined in female and male flowers of andromonoecious poplar. 27 636 methylated coding genes and 113 methylated miRNA genes were identified. In the coding genes, 64.5% of the methylated reads mapped to the gene body region; by contrast, 60.7% of methylated reads in miRNA genes mainly mapped in the 5' and 3' flanking regions. CHH methylation showed the highest methylation levels and CHG showed the lowest methylation levels. Correlation analysis showed a significant, negative, strand-specific correlation of methylation and miRNA gene expression (r=0.79, P <0.05). The methylated miRNA genes included eight long miRNAs (lmiRNAs) of 24 nucleotides and 11 miRNAs related to flower development. miRNA172b might play an important role in the regulation of bisexual flower development-related gene expression in andromonoecious poplar, via modification of methylation. Gynomonoecious, female, and male poplars were used to validate the methylation patterns of the miRNA172b gene, implying that hyper-methylation in andromonoecious and gynomonoecious poplar might function as an important regulator in bisexual flower development. Our data provide a useful resource for the study of flower development in poplar and improve our understanding of the effect of epigenetic regulation on genes other than protein-coding genes. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Methylation of microRNA genes regulates gene expression in bisexual flower development in andromonoecious poplar

PubMed Central

Song, Yuepeng; Tian, Min; Ci, Dong; Zhang, Deqiang

2015-01-01

Previous studies showed sex-specific DNA methylation and expression of candidate genes in bisexual flowers of andromonoecious poplar, but the regulatory relationship between methylation and microRNAs (miRNAs) remains unclear. To investigate whether the methylation of miRNA genes regulates gene expression in bisexual flower development, the methylome, microRNA, and transcriptome were examined in female and male flowers of andromonoecious poplar. 27 636 methylated coding genes and 113 methylated miRNA genes were identified. In the coding genes, 64.5% of the methylated reads mapped to the gene body region; by contrast, 60.7% of methylated reads in miRNA genes mainly mapped in the 5′ and 3′ flanking regions. CHH methylation showed the highest methylation levels and CHG showed the lowest methylation levels. Correlation analysis showed a significant, negative, strand-specific correlation of methylation and miRNA gene expression (r=0.79, P <0.05). The methylated miRNA genes included eight long miRNAs (lmiRNAs) of 24 nucleotides and 11 miRNAs related to flower development. miRNA172b might play an important role in the regulation of bisexual flower development-related gene expression in andromonoecious poplar, via modification of methylation. Gynomonoecious, female, and male poplars were used to validate the methylation patterns of the miRNA172b gene, implying that hyper-methylation in andromonoecious and gynomonoecious poplar might function as an important regulator in bisexual flower development. Our data provide a useful resource for the study of flower development in poplar and improve our understanding of the effect of epigenetic regulation on genes other than protein-coding genes. PMID:25617468

Identification of microRNAs in PCV2 subclinically infected pigs by high throughput sequencing.

PubMed

Núñez-Hernández, Fernando; Pérez, Lester J; Muñoz, Marta; Vera, Gonzalo; Tomás, Anna; Egea, Raquel; Córdoba, Sarai; Segalés, Joaquim; Sánchez, Armand; Núñez, José I

2015-03-03

Porcine circovirus type 2 (PCV2) is the essential etiological infectious agent of PCV2-systemic disease and has been associated with other swine diseases, all of them collectively known as porcine circovirus diseases. MicroRNAs (miRNAs) are a new class of small non-coding RNAs that regulate gene expression post-transcriptionally. miRNAs play an increasing role in many biological processes. The study of miRNA-mediated host-pathogen interactions has emerged in the last decade due to the important role that miRNAs play in antiviral defense. The objective of this study was to identify the miRNA expression pattern in PCV2 subclinically infected and non-infected pigs. For this purpose an experimental PCV2 infection was carried out and small-RNA libraries were constructed from tonsil and mediastinal lymph node (MLN) of infected and non-infected pigs. High throughput sequencing determined differences in miRNA expression in MLN between infected and non-infected while, in tonsil, a very conserved pattern was observed. In MLN, miRNA 126-3p, miRNA 126-5p, let-7d-3p, mir-129a and mir-let-7b-3p were up-regulated whereas mir-193a-5p, mir-574-5p and mir-34a down-regulated. Prediction of functional analysis showed that these miRNAs can be involved in pathways related to immune system and in processes related to the pathogenesis of PCV2, although functional assays are needed to support these predictions. This is the first study on miRNA gene expression in pigs infected with PCV2 using a high throughput sequencing approach in which several host miRNAs were differentially expressed in response to PCV2 infection.

miR2Pathway: A novel analytical method to discover MicroRNA-mediated dysregulated pathways involved in hepatocellular carcinoma.

PubMed

Li, Chaoxing; Dinu, Valentin

2018-05-01

MicroRNAs (miRNAs) are small, non-coding RNAs involved in the regulation of gene expression at a post-transcriptional level. Recent studies have shown miRNAs as key regulators of a variety of biological processes, such as proliferation, differentiation, apoptosis, metabolism, etc. Aberrantly expressed miRNAs influence individual gene expression level, but rewired miRNA-mRNA connections can influence the activity of biological pathways. Here, we define rewired miRNA-mRNA connections as the differential (rewiring) effects on the activity of biological pathways between hepatocellular carcinoma (HCC) and normal phenotypes. Our work presented here uses a PageRank-based approach to measure the degree of miRNA-mediated dysregulation of biological pathways between HCC and normal samples based on rewired miRNA-mRNA connections. In our study, we regard the degree of miRNA-mediated dysregulation of biological pathways as disease risk of biological pathways. Therefore, we propose a new method, miR2Pathway, to measure and rank the degree of miRNA-mediated dysregulation of biological pathways by measuring the total differential influence of miRNAs on the activity of pathways between HCC and normal states. miR2Pathway proposed here systematically shows the first evidence for a mechanism of biological pathways being dysregulated by rewired miRNA-mRNA connections, and provides new insight into exploring mechanisms behind HCC. Thus, miR2Pathway is a novel method to identify and rank miRNA-dysregulated pathways in HCC. Copyright © 2018 Elsevier Inc. All rights reserved.