Conserved miRNAs Are Candidate Post-Transcriptional Regulators of Developmental Arrest in Free-Living and Parasitic Nematodes

PubMed Central

Ahmed, Rina; Chang, Zisong; Younis, Abuelhassan Elshazly; Langnick, Claudia; Li, Na; Chen, Wei; Brattig, Norbert; Dieterich, Christoph

2013-01-01

Animal development is complex yet surprisingly robust. Animals may develop alternative phenotypes conditional on environmental changes. Under unfavorable conditions, Caenorhabditis elegans larvae enter the dauer stage, a developmentally arrested, long-lived, and stress-resistant state. Dauer larvae of free-living nematodes and infective larvae of parasitic nematodes share many traits including a conserved endocrine signaling module (DA/DAF-12), which is essential for the formation of dauer and infective larvae. We speculated that conserved post-transcriptional regulatory mechanism might also be involved in executing the dauer and infective larvae fate. We used an unbiased sequencing strategy to characterize the microRNA (miRNA) gene complement in C. elegans, Pristionchus pacificus, and Strongyloides ratti. Our study raised the number of described miRNA genes to 257 for C. elegans, tripled the known gene set for P. pacificus to 362 miRNAs, and is the first to describe miRNAs in a Strongyloides parasite. Moreover, we found a limited core set of 24 conserved miRNA families in all three species. Interestingly, our estimated expression fold changes between dauer versus nondauer stages and infective larvae versus free-living stages reveal that despite the speed of miRNA gene set evolution in nematodes, homologous gene families with conserved “dauer-infective” expression signatures are present. These findings suggest that common post-transcriptional regulatory mechanisms are at work and that the same miRNA families play important roles in developmental arrest and long-term survival in free-living and parasitic nematodes. PMID:23729632

RNA Deep Sequencing Reveals Differential MicroRNA Expression during Development of Sea Urchin and Sea Star

PubMed Central

Kadri, Sabah; Hinman, Veronica F.; Benos, Panayiotis V.

2011-01-01

microRNAs (miRNAs) are small (20–23 nt), non-coding single stranded RNA molecules that act as post-transcriptional regulators of mRNA gene expression. They have been implicated in regulation of developmental processes in diverse organisms. The echinoderms, Strongylocentrotus purpuratus (sea urchin) and Patiria miniata (sea star) are excellent model organisms for studying development with well-characterized transcriptional networks. However, to date, nothing is known about the role of miRNAs during development in these organisms, except that the genes that are involved in the miRNA biogenesis pathway are expressed during their developmental stages. In this paper, we used Illumina Genome Analyzer (Illumina, Inc.) to sequence small RNA libraries in mixed stage population of embryos from one to three days after fertilization of sea urchin and sea star (total of 22,670,000 reads). Analysis of these data revealed the miRNA populations in these two species. We found that 47 and 38 known miRNAs are expressed in sea urchin and sea star, respectively, during early development (32 in common). We also found 13 potentially novel miRNAs in the sea urchin embryonic library. miRNA expression is generally conserved between the two species during development, but 7 miRNAs are highly expressed in only one species. We expect that our two datasets will be a valuable resource for everyone working in the field of developmental biology and the regulatory networks that affect it. The computational pipeline to analyze Illumina reads is available at http://www.benoslab.pitt.edu/services.html. PMID:22216218

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

microRNAs Databases: Developmental Methodologies, Structural and Functional Annotations.

PubMed

Singh, Nagendra Kumar

2017-09-01

microRNA (miRNA) is an endogenous and evolutionary conserved non-coding RNA, involved in post-transcriptional process as gene repressor and mRNA cleavage through RNA-induced silencing complex (RISC) formation. In RISC, miRNA binds in complementary base pair with targeted mRNA along with Argonaut proteins complex, causes gene repression or endonucleolytic cleavage of mRNAs and results in many diseases and syndromes. After the discovery of miRNA lin-4 and let-7, subsequently large numbers of miRNAs were discovered by low-throughput and high-throughput experimental techniques along with computational process in various biological and metabolic processes. The miRNAs are important non-coding RNA for understanding the complex biological phenomena of organism because it controls the gene regulation. This paper reviews miRNA databases with structural and functional annotations developed by various researchers. These databases contain structural and functional information of animal, plant and virus miRNAs including miRNAs-associated diseases, stress resistance in plant, miRNAs take part in various biological processes, effect of miRNAs interaction on drugs and environment, effect of variance on miRNAs, miRNAs gene expression analysis, sequence of miRNAs, structure of miRNAs. This review focuses on the developmental methodology of miRNA databases such as computational tools and methods used for extraction of miRNAs annotation from different resources or through experiment. This study also discusses the efficiency of user interface design of every database along with current entry and annotations of miRNA (pathways, gene ontology, disease ontology, etc.). Here, an integrated schematic diagram of construction process for databases is also drawn along with tabular and graphical comparison of various types of entries in different databases. Aim of this paper is to present the importance of miRNAs-related resources at a single place.

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

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

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.

Genomewide analysis of Drosophila circular RNAs reveals their structural and sequence properties and age-dependent neural accumulation

PubMed Central

Westholm, Jakub O.; Miura, Pedro; Olson, Sara; Shenker, Sol; Joseph, Brian; Sanfilippo, Piero; Celniker, Susan E.; Graveley, Brenton R.; Lai, Eric C.

2014-01-01

Circularization was recently recognized to broadly expand transcriptome complexity. Here, we exploit massive Drosophila total RNA-sequencing data, >5 billion paired-end reads from >100 libraries covering diverse developmental stages, tissues and cultured cells, to rigorously annotate >2500 fruitfly circular RNAs. These mostly derive from back-splicing of protein-coding genes and lack poly(A) tails, and circularization of hundreds of genes is conserved across multiple Drosophila species. We elucidate structural and sequence properties of Drosophila circular RNAs, which exhibit commonalities and distinctions from mammalian circles. Notably, Drosophila circular RNAs harbor >1000 well-conserved canonical miRNA seed matches, especially within coding regions, and coding conserved miRNA sites reside preferentially within circularized exons. Finally, we analyze the developmental and tissue specificity of circular RNAs, and note their preferred derivation from neural genes and enhanced accumulation in neural tissues. Interestingly, circular isoforms increase dramatically relative to linear isoforms during CNS aging, and constitute a novel aging biomarker. PMID:25544350

Genome-wide Analysis of Drosophila Circular RNAs Reveals Their Structural and Sequence Properties and Age-Dependent Neural Accumulation

DOE PAGES

Westholm, Jakub  O.; Miura, Pedro; Olson, Sara; ...

2014-11-26

Circularization was recently recognized to broadly expand transcriptome complexity. Here, we exploit massive Drosophila total RNA-sequencing data, >5 billion paired-end reads from >100 libraries covering diverse developmental stages, tissues, and cultured cells, to rigorously annotate >2,500 fruit fly circular RNAs. These mostly derive from back-splicing of protein-coding genes and lack poly(A) tails, and the circularization of hundreds of genes is conserved across multiple Drosophila species. We elucidate structural and sequence properties of Drosophila circular RNAs, which exhibit commonalities and distinctions from mammalian circles. Notably, Drosophila circular RNAs harbor >1,000 well-conserved canonical miRNA seed matches, especially within coding regions, and codingmore » conserved miRNA sites reside preferentially within circularized exons. Finally, we analyze the developmental and tissue specificity of circular RNAs and note their preferred derivation from neural genes and enhanced accumulation in neural tissues. Interestingly, circular isoforms increase substantially relative to linear isoforms during CNS aging and constitute an aging biomarker.« less

Genome-wide Analysis of Drosophila Circular RNAs Reveals Their Structural and Sequence Properties and Age-Dependent Neural Accumulation

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

Westholm, Jakub  O.; Miura, Pedro; Olson, Sara

Circularization was recently recognized to broadly expand transcriptome complexity. Here, we exploit massive Drosophila total RNA-sequencing data, >5 billion paired-end reads from >100 libraries covering diverse developmental stages, tissues, and cultured cells, to rigorously annotate >2,500 fruit fly circular RNAs. These mostly derive from back-splicing of protein-coding genes and lack poly(A) tails, and the circularization of hundreds of genes is conserved across multiple Drosophila species. We elucidate structural and sequence properties of Drosophila circular RNAs, which exhibit commonalities and distinctions from mammalian circles. Notably, Drosophila circular RNAs harbor >1,000 well-conserved canonical miRNA seed matches, especially within coding regions, and codingmore » conserved miRNA sites reside preferentially within circularized exons. Finally, we analyze the developmental and tissue specificity of circular RNAs and note their preferred derivation from neural genes and enhanced accumulation in neural tissues. Interestingly, circular isoforms increase substantially relative to linear isoforms during CNS aging and constitute an aging biomarker.« less

Micro-RNAs and their roles in eye disorders.

PubMed

Raghunath, Azhwar; Perumal, Ekambaram

2015-01-01

Micro-RNAs (miRNAs) are members of the family of noncoding RNA molecules that regulate gene expression by translational repression and mRNA degradation. Initial identification of miRNAs revealed them only as developmental regulators; later, their radiated roles in various cellular processes have been established. They regulate several pathways, including developmental timing, hematopoiesis, organogenesis, apoptosis, cell differentiation and proliferation. Their roles in eye disorders are being explored by biologists around the world. Eye physiology requires the perfect orchestration of all the regulatory networks; any defect in any of the networks leads to eye disorders. The dysregulation of miRNA expression has been reported in many eye disorders, which paves the way for new therapeutics. This review summarizes the biogenesis of miRNAs and their role in eye disorders. miRNA studies also have implications for the understanding of various complex metabolic pathways leading to disorders of the eye. The ultimate understanding leads to potential opportunities in evaluating miRNAs as molecular biomarkers, prognostic tools, diagnostic tools and therapeutic agents for eye disorders. © 2015 S. Karger AG, Basel.

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.

Developmental transitions in C. elegans larval stages.

PubMed

Rougvie, Ann E; Moss, Eric G

2013-01-01

Molecular mechanisms control the timing, sequence, and synchrony of developmental events in multicellular organisms. In Caenorhabditis elegans, these mechanisms are revealed through the analysis of mutants with "heterochronic" defects: cell division or differentiation patterns that occur in the correct lineage, but simply at the wrong time. Subsets of cells in these mutants thus express temporal identities normally restricted to a different life stage. A seminal finding arising from studies of the heterochronic genes was the discovery of miRNAs; these tiny miRNAs are now a defining feature of the pathway. A series of sequentially expressed miRNAs guide larval transitions through stage-specific repression of key effector molecules. The wild-type lineage patterns are executed as discrete modules programmed between temporal borders imposed by the molting cycles. How these successive events are synchronized with the oscillatory molting cycle is just beginning to come to light. Progression through larval stages can be specifically, yet reversibly, halted in response to environmental cues, including nutrient availability. Here too, heterochronic genes and miRNAs play key roles. Remarkably, developmental arrest can, in some cases, either mask or reveal timing defects associated with mutations. In this chapter, we provide an overview of how the C. elegans heterochronic gene pathway guides developmental transitions during continuous and interrupted larval development. © 2013 Elsevier Inc. All rights reserved.

The hot pepper (Capsicum annuum) microRNA transcriptome reveals novel and conserved targets: a foundation for understanding MicroRNA functional roles in hot pepper.

PubMed

Hwang, Dong-Gyu; Park, June Hyun; Lim, Jae Yun; Kim, Donghyun; Choi, Yourim; Kim, Soyoung; Reeves, Gregory; Yeom, Seon-In; Lee, Jeong-Soo; Park, Minkyu; Kim, Seungill; Choi, Ik-Young; Choi, Doil; Shin, Chanseok

2013-01-01

MicroRNAs (miRNAs) are a class of non-coding RNAs approximately 21 nt in length which play important roles in regulating gene expression in plants. Although many miRNA studies have focused on a few model plants, miRNAs and their target genes remain largely unknown in hot pepper (Capsicum annuum), one of the most important crops cultivated worldwide. Here, we employed high-throughput sequencing technology to identify miRNAs in pepper extensively from 10 different libraries, including leaf, stem, root, flower, and six developmental stage fruits. Based on a bioinformatics pipeline, we successfully identified 29 and 35 families of conserved and novel miRNAs, respectively. Northern blot analysis was used to validate further the expression of representative miRNAs and to analyze their tissue-specific or developmental stage-specific expression patterns. Moreover, we computationally predicted miRNA targets, many of which were experimentally confirmed using 5' rapid amplification of cDNA ends analysis. One of the validated novel targets of miR-396 was a domain rearranged methyltransferase, the major de novo methylation enzyme, involved in RNA-directed DNA methylation in plants. This work provides the first reliable draft of the pepper miRNA transcriptome. It offers an expanded picture of pepper miRNAs in relation to other plants, providing a basis for understanding the functional roles of miRNAs in pepper.

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.

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.

The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development

PubMed Central

Vaucheret, Hervé; Vazquez, Franck; Crété, Patrice; Bartel, David P.

2004-01-01

MicroRNAs (miRNAs) are endogenous 21–24-nt RNAs that can down-regulate gene expression by pairing to the messages of protein-coding genes to specify mRNA cleavage or repression of productive translation. They act within the RNA-induced silencing complex (RISC), which in animals contains a member of the Argonaute family of proteins. In the present study, we show that Arabidopsis ago1 mutants have increased accumulation of mRNAs known to be targeted for cleavage by miRNAs. In hypomorphic ago1 alleles, this compromised miRNA function occurs without a substantial change in miRNA accumulation, whereas in null alleles it is accompanied by a drop in some of the miRNAs. Therefore, AGO1 acts within the Arabidopsis miRNA pathway, probably within the miRNA-programmed RISC, such that the absence of AGO1 destabilizes some of the miRNAs. We also show that targeting of AGO1 mRNA by miR168 is needed for proper plant development, illustrating the importance of feedback control by this miRNA. Transgenic plants expressing a mutant AGO1 mRNA with decreased complementarity to miR168 overaccumulate AGO1 mRNA and exhibit developmental defects partially overlapping with those of dcl1, hen1, and hyl1 mutants showing a decrease in miRNA accumulation. miRNA targets overaccumulate in miR168-resistant plants, suggesting that a large excess of AGO1 protein interferes with the function of RISC or sequesters miRNAs or other RISC components. Developmental defects induced by a miR168-resistant AGO1 mRNA can be rescued by a compensatory miRNA that is complementary to the mutant AGO1 mRNA, proving the regulatory relationship between miR168 and its target and opening the way for engineering artificial miRNAs in plants. PMID:15131082

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.

MicroRNA, mRNA, and protein expression link development and aging in human and macaque brain

PubMed Central

Somel, Mehmet; Guo, Song; Fu, Ning; Yan, Zheng; Hu, Hai Yang; Xu, Ying; Yuan, Yuan; Ning, Zhibin; Hu, Yuhui; Menzel, Corinna; Hu, Hao; Lachmann, Michael; Zeng, Rong; Chen, Wei; Khaitovich, Philipp

2010-01-01

Changes in gene expression levels determine differentiation of tissues involved in development and are associated with functional decline in aging. Although development is tightly regulated, the transition between development and aging, as well as regulation of post-developmental changes, are not well understood. Here, we measured messenger RNA (mRNA), microRNA (miRNA), and protein expression in the prefrontal cortex of humans and rhesus macaques over the species' life spans. We find that few gene expression changes are unique to aging. Instead, the vast majority of miRNA and gene expression changes that occur in aging represent reversals or extensions of developmental patterns. Surprisingly, many gene expression changes previously attributed to aging, such as down-regulation of neural genes, initiate in early childhood. Our results indicate that miRNA and transcription factors regulate not only developmental but also post-developmental expression changes, with a number of regulatory processes continuing throughout the entire life span. Differential evolutionary conservation of the corresponding genomic regions implies that these regulatory processes, although beneficial in development, might be detrimental in aging. These results suggest a direct link between developmental regulation and expression changes taking place in aging. PMID:20647238

Genome-Wide Small RNA Analysis of Soybean Reveals Auxin-Responsive microRNAs that are Differentially Expressed in Response to Salt Stress in Root Apex

PubMed Central

Sun, Zhengxi; Wang, Youning; Mou, Fupeng; Tian, Yinping; Chen, Liang; Zhang, Senlei; Jiang, Qiong; Li, Xia

2016-01-01

Root growth and the architecture of the root system in Arabidopsis are largely determined by root meristematic activity. Legume roots show strong developmental plasticity in response to both abiotic and biotic stimuli, including symbiotic rhizobia. However, a global analysis of gene regulation in the root meristem of soybean plants is lacking. In this study, we performed a global analysis of the small RNA transcriptome of root tips from soybean seedlings grown under normal and salt stress conditions. In total, 71 miRNA candidates, including known and novel variants of 59 miRNA families, were identified. We found 66 salt-responsive miRNAs in the soybean root meristem; among them, 22 are novel miRNAs. Interestingly, we found auxin-responsive cis-elements in the promoters of many salt-responsive miRNAs, implying that these miRNAs may be regulated by auxin and auxin signaling plays a key role in regulating the plasticity of the miRNAome and root development in soybean. A functional analysis of miR399, a salt-responsive miRNA in the root meristem, indicates the crucial role of this miRNA in modulating soybean root developmental plasticity. Our data provide novel insight into the miRNAome-mediated regulatory mechanism in soybean root growth under salt stress. PMID:26834773

MicroRNA regulation of immune events at conception.

PubMed

Robertson, Sarah A; Zhang, Bihong; Chan, Honyueng; Sharkey, David J; Barry, Simon C; Fullston, Tod; Schjenken, John E

2017-09-01

The reproductive tract environment at conception programs the developmental trajectory of the embryo, sets the course of pregnancy, and impacts offspring phenotype and health. Despite the fundamental importance of this stage of reproduction, the rate-limiting regulatory mechanisms operating locally to control fertility and fecundity are incompletely understood. Emerging studies highlight roles for microRNAs (miRNAs) in regulating reproductive and developmental processes and in modulating the quality and strength of the female immune response. Since endometrial receptivity and robust placentation require specific adaptation of the immune response, we hypothesize that miRNAs participate in establishing pregnancy through effects on key gene networks in immune cells. Our recent studies investigated miRNAs that are induced in the peri-conception environment, focusing on miRNAs that have immune-regulatory roles-particularly miR-223, miR-155, and miR-146a. Genetic mouse models deficient in individual miRNAs are proving informative in defining roles for these miRNAs in the generation and stabilization of regulatory T cells (Treg cells) that confer adaptive immune tolerance. Overlapping and redundant functions between miRNAs that target multiple genes, combined with multiple miRNAs targeting individual genes, indicate complex and sensitive regulatory networks. Although to date most data on miRNA regulation of reproductive events are from mice, conserved functions of miRNAs across species imply similar biological pathways operate in all mammals. Understanding the regulation and roles of miRNAs in the peri-conception immune response will advance our knowledge of how environmental determinants act at conception, and could have practical applications for animal breeding as well as human fertility. © 2017 Wiley Periodicals, Inc.

De novo characterization of microRNAs in oriental fruit moth Grapholita molesta and selection of reference genes for normalization of microRNA expression

PubMed Central

Zhang, Jing; Zhang, Qingwen; Liu, Xiaoxia; Li, Zhen

2017-01-01

MicroRNAs (miRNAs) are a group of endogenous non-coding small RNAs that have critical regulatory functions in almost all known biological processes at the post-transcriptional level in a variety of organisms. The oriental fruit moth Grapholita molesta is one of the most serious pests in orchards worldwide and threatens the production of Rosacea fruits. In this study, a de novo small RNA library constructed from mixed stages of G. molesta was sequenced through Illumina sequencing platform and a total of 536 mature miRNAs consisting of 291 conserved and 245 novel miRNAs were identified. Most of the conserved and novel miRNAs were detected with moderate abundance. The miRNAs in the same cluster normally showed correlated expressional profiles. A comparative analysis of the 79 conserved miRNA families within 31 arthropod species indicated that these miRNA families were more conserved among insects and within orders of closer phylogenetic relationships. The KEGG pathway analysis and network prediction of target genes indicated that the complex composed of miRNAs, clock genes and developmental regulation genes may play vital roles to regulate the developmental circadian rhythm of G. molesta. Furthermore, based on the sRNA library of G. molesta, suitable reference genes were selected and validated for study of miRNA transcriptional profile in G. molesta under two biotic and six abiotic experimental conditions. This study systematically documented the miRNA profile in G. molesta, which could lay a foundation for further understanding of the regulatory roles of miRNAs in the development and metabolism in this pest and might also suggest clues to the development of genetic-based techniques for agricultural pest control. PMID:28158242

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.

Turmeric (Curcuma longa): miRNAs and their regulating targets are involved in development and secondary metabolite pathways.

PubMed

Singh, Noopur; Sharma, Ashok

Turmeric has been used as a therapeutic herb over centuries in traditional medicinal systems due to the presence of several secondary metabolite compounds. microRNAs are known to regulate gene expression at the post-transcriptional level by transcriptional cleavage or translation repression. miRNAs have been demonstrated to play an active role in secondary metabolism regulation. The present work was focused on the identification of the miRNAs involved in the regulation of secondary metabolite and development process of turmeric. Eighteen miRNA families were identified for turmeric. Sixteen miRNA families were observed to regulate 238 target transcripts. LncRNAs targets of the putative miRNA candidates were also predicted. Our results indicated their role in binding, reproduction, stress, and other developmental processes. Gene annotation and pathway analysis illustrated the biological function of the targets regulated by the putative miRNAs. The miRNA-mediated gene regulatory network also revealed co-regulated targets that were regulated by two or more miRNA families. miR156 and miR5015 were observed to be involved in rhizome development. miR5021 showed regulation for terpenoid backbone biosynthesis and isoquinoline alkaloid biosynthesis pathways. The flavonoid biosynthesis pathway was observed to be regulated by miR2919. The analysis revealed the probable involvement of three miRNAs (miR1168.2, miR156b and miR1858) in curcumin biosynthesis. Other miRNAs were found to be involved in the growth and developmental process of turmeric. Phylogenetic analysis of selective miRNAs was also performed. Copyright © 2017 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.

The Inescapable Influence of Noncoding RNAs in Cancer

PubMed Central

Adams, Brian D.; Anastasiadou, Eleni; Esteller, Manel; He, Lin; Slack, Frank J.

2015-01-01

This report summarizes information presented at the 2015 Keystone Symposium on “MicroRNAs and Noncoding RNAs in Cancer”. Nearly two decades after the discovery of the first microRNA (miRNA), the role of noncoding RNAs in developmental processes and the mechanisms behind their dysregulation in cancer has been steadily elucidated. Excitingly, miRNAs have begun making their way into the clinic to combat disease such a hepatitis C, and various forms of cancer. Therefore, at this Keystone meeting novel findings were presented that enhance our view on how small and long noncoding RNAs control developmental timing and oncogenic processes. Recurring themes included, 1) how miRNAs can be differentially processed, degraded, and regulated by ribonucleoprotein (RNP) complexes, 2) how particular miRNA genetic networks that control developmental process, when disrupted, can result in cancer disease, 3) the technologies available to therapeutically deliver RNA to combat diseases such as cancer, and 4) the elucidation of the mechanism of actions for long noncoding RNAs, currently a poorly understood class of noncoding RNA. During the meeting there was an emphasis on presenting unpublished findings, and the breadth of topics covered reflected how inescapable the influence of noncoding RNAs are in development and cancer. PMID:26567137

Development-related expression patterns of protein-coding and miRNA genes involved in porcine muscle growth.

PubMed

Wang, F J; Jin, L; Guo, Y Q; Liu, R; He, M N; Li, M Z; Li, X W

2014-11-27

Muscle growth and development is associated with remarkable changes in protein-coding and microRNA (miRNA) gene expression. To determine the expression patterns of genes and miRNAs related to muscle growth and development, we measured the expression levels of 25 protein-coding and 16 miRNA genes in skeletal and cardiac muscles throughout 5 developmental stages by quantitative reverse transcription-polymerase chain reaction. The Short Time-Series Expression Miner (STEM) software clustering results showed that growth-related genes were downregulated at all developmental stages in both the psoas major and longissimus dorsi muscles, indicating their involvement in early developmental stages. Furthermore, genes related to muscle atrophy, such as forkhead box 1 and muscle ring finger, showed unregulated expression with increasing age, suggesting a decrease in protein synthesis during the later stages of skeletal muscle development. We found that development of the cardiac muscle was a complex process in which growth-related genes were highly expressed during embryonic development, but they did not show uniform postnatal expression patterns. Moreover, the expression level of miR-499, which enhances the expression of the β-myosin heavy chain, was significantly different in the psoas major and longissimus dorsi muscles, suggesting the involvement of miR-499 in the determination of skeletal muscle fiber types. We also performed correlation analyses of messenger RNA and miRNA expression. We found negative relationships between miR-486 and forkhead box 1, and miR-133a and serum response factor at all developmental stages, suggesting that forkhead box 1 and serum response factor are potential targets of miR-486 and miR-133a, respectively.

High-throughput characterization of Echinococcus spp. metacestode miRNomes.

PubMed

Cucher, Marcela; Macchiaroli, Natalia; Kamenetzky, Laura; Maldonado, Lucas; Brehm, Klaus; Rosenzvit, Mara Cecilia

2015-03-01

Echinococcosis is a worldwide zoonosis of great public health concern, considered a neglected disease by the World Health Organisation. The cestode parasites Echinococcus granulosus sensu lato (s. l.) and Echinococcus multilocularis are the main aetiological agents. In the intermediate host, these parasites display particular developmental traits that lead to different patterns of disease progression. In an attempt to understand the causes of these differences, we focused on the analysis of microRNAs (miRNAs), small non-coding regulatory RNAs with major roles in development of animals and plants. In this work, we analysed the small RNA expression pattern of the metacestode, the stage of sanitary relevance, and provide a detailed description of Echinococcus miRNAs. Using high-throughput small RNA sequencing, we believe that we have carried out the first experimental identification of miRNAs in E. multilocularis and have expanded the Echinococcus miRNA catalogue to 38 miRNA genes, including one miRNA only present in E. granulosus s. l. Our findings show that although both species share the top five highest expressed miRNAs, 13 are differentially expressed, which could be related to developmental differences. We also provide evidence that uridylation is the main miRNA processing mechanism in Echinococcus spp. These results provide detailed information on Echinococcus miRNAs, which is the first step in understanding their role in parasite biology and disease establishment and/or progression, and their future potential use as drug or diagnostic targets. Copyright © 2015 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Identification of novel drought-responsive microRNAs and trans-acting siRNAs from Sorghum bicolor (L.) Moench by high-throughput sequencing analysis

PubMed Central

Katiyar, Amit; Smita, Shuchi; Muthusamy, Senthilkumar K.; Chinnusamy, Viswanathan; Pandey, Dev M.; Bansal, Kailash C.

2015-01-01

Small non-coding RNAs (sRNAs) namely microRNAs (miRNAs) and trans-acting small interfering RNAs (tasi-RNAs) play a crucial role in post-transcriptional regulation of gene expression and thus the control plant development and stress responses. In order to identify drought-responsive miRNAs and tasi-RNAs in sorghum, we constructed small RNA libraries from a drought tolerant (M35-1) and susceptible (C43) sorghum genotypes grown under control and drought stress conditions, and sequenced by Illumina Genome Analyzer IIx. Ninety seven conserved and 526 novel miRNAs representing 472 unique miRNA families were identified from sorghum. Ninety-six unique miRNAs were found to be regulated by drought stress, of which 32 were up- and 49 were down-regulated (fold change ≥ 2 or ≤ −2) at least in one genotype, while the remaining 15 miRNAs showed contrasting drought-regulated expression pattern between genotypes. A maximum of 17 and 18 miRNAs was differentially regulated under drought stress condition in the sensitive and tolerant genotypes, respectively. These results suggest that genotype dependent stress responsive regulation of miRNAs may contribute, at least in part, to the differential drought tolerance of sorghum genotypes. We also identified two miR390-directed TAS3 gene homologs and the auxin response factors as tasi-RNA targets. We predicted more than 1300 unique target genes for the novel and conserved miRNAs. These target genes were predicted to be involved in different cellular, metabolic, response to stimulus, biological regulation, and developmental processes. Genome-wide identification of stress-responsive miRNAs, tasi-RNAs and their targets identified in this study will be useful in unraveling the molecular mechanisms underlying drought stress responses and genetic improvement of biomass production and stress tolerance in sorghum. PMID:26236318

Strategies to identify microRNA targets: New advances

USDA-ARS?s Scientific Manuscript database

MicroRNAs (miRNAs) are small regulatory RNA molecules functioning to modulate gene expression at the post-transcriptional level, and playing an important role in many developmental and physiological processes. Ten thousand miRNAs have been discovered in various organisms. Although considerable progr...

Coronary Heart Disease Alters Intercellular Communication by Modifying Microparticle-Mediated MicroRNA Transport

PubMed Central

Finn, Nnenna A.; Eapen, Danny; Manocha, Pankaj; Kassem, Hatem Al; Lassegue, Bernard; Ghasemzadeh, Nima; Quyyumi, Arshed; Searles, Charles D.

2013-01-01

Coronary heart disease (CHD) is characterized by abnormal intercellular communication and circulating microRNAs (miRNAs) are likely involved in this process. Here, we show that CHD was associated with changes in the transport of circulating miRNA, particularly decreased miRNA enrichment in microparticles (MPs). Additionally, MPs from CHD patients were less efficient at transferring miRNA to cultured HUVECs, which correlated with their diminished capacity to bind developmental endothelial locus-1 (Del-1). In summary, CHD was associated with distinct changes in circulating miRNA transport and these changes may contribute to the abnormal intercellular communication that underlies CHD initiation and progression. PMID:24042051

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.

Genome-wide identification of alternate bearing-associated microRNAs (miRNAs) in olive (Olea europaea L.)

PubMed Central

2013-01-01

Background Alternate bearing is a widespread phenomenon among crop plants, defined as the tendency of certain fruit trees to produce a high-yield crop one year ("on-year"), followed by a low-yield or even no crop the following year ("off-year"). Several factors may affect the balance between such developmental phase-transition processes. Among them are the microRNA (miRNA), being gene-expression regulators that have been found to be involved as key determinants in several physiological processes. Results Six olive (Olea europaea L. cv. Ayvalik variety) small RNA libraries were constructed from fruits (ripe and unripe) and leaves (”on year” and ”off year” leaves in July and in November, respectively) and sequenced by high-throughput Illumina sequencing. The RNA was retrotranscribed and sequenced using the high-throughput Illumina platform. Bioinformatics analyses of 93,526,915 reads identified 135 conserved miRNA, belonging to 22 miRNA families in the olive. In addition, 38 putative novel miRNAs were discovered in the datasets. Expression of olive tree miRNAs varied greatly among the six libraries, indicating the contribution of diverse miRNA in balancing between reproductive and vegetative phases. Predicted targets of miRNA were categorized into 108 process ontology groups with significance abundance. Among those, potential alternate bearing-associated processes were found, such as development, hormone-mediated signaling and organ morphogenesis. The KEGG analyses revealed that the miRNA-targeted genes are involved in seven main pathways, belonging to carbohydrate metabolism and hormone signal-transduction pathways. Conclusion A comprehensive study on olive miRNA related to alternate bearing was performed. Regulation of miRNA under different developmental phases and tissues indicated that control of nutrition and hormone, together with flowering processes had a noteworthy impact on the olive tree alternate bearing. Our results also provide significant data on the miRNA-fruit development interaction and advance perspectives in the miRNA profile of the olive tree. PMID:23320600

Comparative Analyses between Skeletal Muscle miRNAomes from Large White and Min Pigs Revealed MicroRNAs Associated with Postnatal Muscle Hypertrophy.

PubMed

Sheng, Xihui; Wang, Ligang; Ni, Hemin; Wang, Lixian; Qi, Xiaolong; Xing, Shuhan; Guo, Yong

2016-01-01

The molecular mechanism regulated by microRNAs (miRNAs) that underlies postnatal hypertrophy of skeletal muscle is complex and remains unclear. Here, the miRNAomes of longissimus dorsi muscle collected at five postnatal stages (60, 120, 150, 180, and 210 days after birth) from Large White (commercial breed) and Min pigs (indigenous breed of China) were analyzed by Illumina sequencing. We identified 734 miRNAs comprising 308 annotated miRNAs and 426 novel miRNAs, of which 307 could be considered pig-specific. Comparative analysis between two breeds suggested that 60 and 120 days after birth were important stages for skeletal muscle hypertrophy and intramuscular fat accumulation. A total of 263 miRNAs were significantly differentially expressed between two breeds at one or more developmental stages. In addition, the differentially expressed miRNAs between every two adjacent developmental stages in each breed were determined. Notably, ssc-miR-204 was significantly more highly expressed in Min pig skeletal muscle at all postnatal stages compared with its expression in Large White pig skeletal muscle. Based on gene ontology and KEGG pathway analyses of its predicted target genes, we concluded that ssc-miR-204 may exert an impact on postnatal hypertrophy of skeletal muscle by regulating myoblast proliferation. The results of this study will help in elucidating the mechanism underlying postnatal hypertrophy of skeletal muscle modulated by miRNAs, which could provide valuable information for improvement of pork quality and human myopathy.

Coronary heart disease alters intercellular communication by modifying microparticle-mediated microRNA transport.

PubMed

Finn, Nnenna A; Eapen, Danny; Manocha, Pankaj; Al Kassem, Hatem; Lassegue, Bernard; Ghasemzadeh, Nima; Quyyumi, Arshed; Searles, Charles D

2013-11-01

Coronary heart disease (CHD) is characterized by abnormal intercellular communication and circulating microRNAs (miRNAs) are likely involved in this process. Here, we show that CHD was associated with changes in the transport of circulating miRNA, particularly decreased miRNA enrichment in microparticles (MPs). Additionally, MPs from CHD patients were less efficient at transferring miRNA to cultured HUVECs, which correlated with their diminished capacity to bind developmental endothelial locus-1 (Del-1). In summary, CHD was associated with distinct changes in circulating miRNA transport and these changes may contribute to the abnormal intercellular communication that underlies CHD initiation and progression. Published by Elsevier B.V.

Overview of research on Bombyx mori microRNA

PubMed Central

Wang, Xin; Tang, Shun-ming; Shen, Xing-jia

2014-01-01

Abstract MicroRNAs (miRNAs) constitute some of the most significant regulatory factors involved at the post-transcriptional level after gene expression, contributing to the modulation of a large number of physiological processes such as development, metabolism, and disease occurrence. This review comprehensively and retrospectively explores the literature investigating silkworm, Bombyx mori L. (Lepidoptera: Bombicidae), miRNAs published to date, including discovery, identification, expression profiling analysis, target gene prediction, and the functional analysis of both miRNAs and their targets. It may provide experimental considerations and approaches for future study of miRNAs and benefit elucidation of the mechanisms of miRNAs involved in silkworm developmental processes and intracellular activities of other unknown non-coding RNAs. PMID:25368077

Functional screening for miRNAs targeting Smad4 identified miR-199a as a negative regulator of TGF-β signalling pathway

PubMed Central

Zhang, Yan; Fan, Kai-Ji; Sun, Qiang; Chen, Ai-Zhong; Shen, Wen-Long; Zhao, Zhi-Hu; Zheng, Xiao-Fei; Yang, Xiao

2012-01-01

The transforming growth factor-β (TGF-β) signalling pathway participates in various biological processes. Dysregulation of Smad4, a central cellular transducer of TGF-β signalling, is implicated in a wide range of human diseases and developmental disorders. However, the mechanisms underlying Smad4 dysregulation are not fully understood. Using a functional screening approach based on luciferase reporter assays, we identified 39 microRNAs (miRNAs) as potential regulators of Smad4 from an expression library of 388 human miRNAs. The screening was supported by bioinformatic analysis, as 24 of 39 identified miRNAs were also predicted to target Smad4. MiR-199a, one of the identified miRNAs, was inversely correlated with Smad4 expression in various human cancer cell lines and gastric cancer tissues, and repressed Smad4 expression and blocked canonical TGF-β transcriptional responses in cell lines. These effects were dependent on the presence of a conserved, but not perfect seed paired, miR-199a-binding site in the Smad4 3′-untranslated region (UTR). Overexpression of miR-199a significantly inhibited the ability of TGF-β to induce gastric cancer cell growth arrest and apoptosis in vitro, and promoted anchorage-independent growth in soft agar, suggesting that miR-199a plays an oncogenic role in human gastric tumourigenesis. In conclusion, our functional screening uncovers multiple miRNAs that regulate the cellular responsiveness to TGF-β signalling and reveals important roles of miR-199a in gastric cancer by directly targeting Smad4. PMID:22821565

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

Poudel, Saroj; Aryal, Niranjan; Lu, Chaofu

Camelina sativa is an annual oilseed crop that is under intensive development for renewable resources of biofuels and industrial oils. MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play key roles in diverse plant biological processes. Here, we conducted deep sequencing on small RNA libraries prepared from camelina leaves, flower buds and two stages of developing seeds corresponding to initial and peak storage products accumulation. Computational analyses identified 207 known miRNAs belonging to 63 families, as well as 5 novel miRNAs. These miRNAs, especially members of the miRNA families, varied greatly in different tissues and developmental stages. The predictedmore » miRNA target genes are involved in a broad range of physiological functions including lipid metabolism. This report is the first step toward elucidating roles of miRNAs in C. sativa and will provide additional tools to improve this oilseed crop for biofuels and biomaterials.« less

Virus-Based MicroRNA Silencing in Plants1[C][W][OPEN

PubMed Central

Sha, Aihua; Zhao, Jinping; Yin, Kangquan; Tang, Yang; Wang, Yan; Wei, Xiang; Hong, Yiguo; Liu, Yule

2014-01-01

MicroRNAs (miRNAs) play pivotal roles in various biological processes across kingdoms. Many plant miRNAs have been experimentally identified or predicted by bioinformatics mining of small RNA databases. However, the functions of these miRNAs remain largely unknown due to the lack of effective genetic tools. Here, we report a virus-based microRNA silencing (VbMS) system that can be used for functional analysis of plant miRNAs. VbMS is performed through tobacco rattle virus-based expression of miRNA target mimics to silence endogenous miRNAs. VbMS of either miR172 or miR165/166 caused developmental defects in Nicotiana benthamiana. VbMS of miR319 reduced the complexity of tomato (Solanum lycopersicum) compound leaves. These results demonstrate that tobacco rattle virus-based VbMS is a powerful tool to silence endogenous miRNAs and to dissect their functions in different plant species. PMID:24296072

MicroRNAs in the pineal gland: miR-483 regulates melatonin synthesis by targeting arylalkylamine N-acetyltransferase.

PubMed

Clokie, Samuel J H; Lau, Pierre; Kim, Hyun Hee; Coon, Steven L; Klein, David C

2012-07-20

MicroRNAs (miRNAs) play a broad range of roles in biological regulation. In this study, rat pineal miRNAs were profiled for the first time, and their importance was evaluated by focusing on the main function of the pineal gland, melatonin synthesis. Massively parallel sequencing and related methods revealed the miRNA population is dominated by a small group of miRNAs as follows: ~75% is accounted for by 15 miRNAs; miR-182 represents 28%. In addition to miR-182, miR-183 and miR-96 are also highly enriched in the pineal gland, a distinctive pattern also found in the retina. This effort also identified previously unrecognized miRNAs and other small noncoding RNAs. Pineal miRNAs do not exhibit a marked night/day difference in abundance with few exceptions (e.g. 2-fold night/day differences in the abundance of miR-96 and miR-182); this contrasts sharply with the dynamic 24-h pattern that characterizes the pineal transcriptome. During development, the abundance of most pineal gland-enriched miRNAs increases; however, there is a marked decrease in at least one, miR-483. miR-483 is a likely regulator of melatonin synthesis, based on the following. It inhibits melatonin synthesis by pinealocytes in culture; it acts via predicted binding sites in the 3"-UTR of arylalkylamine N-acetyltransferase (Aanat) mRNA, the penultimate enzyme in melatonin synthesis, and it exhibits a developmental profile opposite to that of Aanat transcripts. Additionally, a miR-483 targeted antagonist increased melatonin synthesis in neonatal pinealocytes. These observations support the hypothesis that miR-483 suppresses Aanat mRNA levels during development and that the developmental decrease in miR-483 abundance promotes melatonin synthesis.

MicroRNAs in the Pineal Gland

PubMed Central

Clokie, Samuel J. H.; Lau, Pierre; Kim, Hyun Hee; Coon, Steven L.; Klein, David C.

2012-01-01

MicroRNAs (miRNAs) play a broad range of roles in biological regulation. In this study, rat pineal miRNAs were profiled for the first time, and their importance was evaluated by focusing on the main function of the pineal gland, melatonin synthesis. Massively parallel sequencing and related methods revealed the miRNA population is dominated by a small group of miRNAs as follows: ∼75% is accounted for by 15 miRNAs; miR-182 represents 28%. In addition to miR-182, miR-183 and miR-96 are also highly enriched in the pineal gland, a distinctive pattern also found in the retina. This effort also identified previously unrecognized miRNAs and other small noncoding RNAs. Pineal miRNAs do not exhibit a marked night/day difference in abundance with few exceptions (e.g. 2-fold night/day differences in the abundance of miR-96 and miR-182); this contrasts sharply with the dynamic 24-h pattern that characterizes the pineal transcriptome. During development, the abundance of most pineal gland-enriched miRNAs increases; however, there is a marked decrease in at least one, miR-483. miR-483 is a likely regulator of melatonin synthesis, based on the following. It inhibits melatonin synthesis by pinealocytes in culture; it acts via predicted binding sites in the 3′-UTR of arylalkylamine N-acetyltransferase (Aanat) mRNA, the penultimate enzyme in melatonin synthesis, and it exhibits a developmental profile opposite to that of Aanat transcripts. Additionally, a miR-483 targeted antagonist increased melatonin synthesis in neonatal pinealocytes. These observations support the hypothesis that miR-483 suppresses Aanat mRNA levels during development and that the developmental decrease in miR-483 abundance promotes melatonin synthesis. PMID:22908386

Selective blockade of microRNA processing by Lin-28

PubMed Central

Viswanathan, Srinivas R.; Daley, George Q.; Gregory, Richard I.

2012-01-01

MicroRNAs (miRNAs) play critical roles in development, and dysregulation of miRNA expression has been observed in human malignancies. Recent evidence suggests that the processing of several primary miRNA transcripts (pri-miRNAs) is blocked post-transcriptionally in embryonic stem (ES) cells, embryonal carcinoma (EC) cells, and primary tumors. Here we show that Lin-28, a developmentally regulated RNA-binding protein, selectively blocks the processing of pri-let-7 miRNAs in embryonic cells. Using in vitro and in vivo studies, we demonstrate that Lin-28 is necessary and sufficient for blocking Microprocessor-mediated cleavage of pri-let-7 miRNAs. Our results identify Lin-28 as a negative regulator of miRNA biogenesis and suggest that Lin-28 may play a central role in blocking miRNA-mediated differentiation in stem cells and certain cancers. PMID:18292307

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

Bioinformatics resource manager v2.3: an integrated software environment for systems biology with microRNA and cross-species analysis tools

PubMed Central

2012-01-01

Background MicroRNAs (miRNAs) are noncoding RNAs that direct post-transcriptional regulation of protein coding genes. Recent studies have shown miRNAs are important for controlling many biological processes, including nervous system development, and are highly conserved across species. Given their importance, computational tools are necessary for analysis, interpretation and integration of high-throughput (HTP) miRNA data in an increasing number of model species. The Bioinformatics Resource Manager (BRM) v2.3 is a software environment for data management, mining, integration and functional annotation of HTP biological data. In this study, we report recent updates to BRM for miRNA data analysis and cross-species comparisons across datasets. Results BRM v2.3 has the capability to query predicted miRNA targets from multiple databases, retrieve potential regulatory miRNAs for known genes, integrate experimentally derived miRNA and mRNA datasets, perform ortholog mapping across species, and retrieve annotation and cross-reference identifiers for an expanded number of species. Here we use BRM to show that developmental exposure of zebrafish to 30 uM nicotine from 6–48 hours post fertilization (hpf) results in behavioral hyperactivity in larval zebrafish and alteration of putative miRNA gene targets in whole embryos at developmental stages that encompass early neurogenesis. We show typical workflows for using BRM to integrate experimental zebrafish miRNA and mRNA microarray datasets with example retrievals for zebrafish, including pathway annotation and mapping to human ortholog. Functional analysis of differentially regulated (p<0.05) gene targets in BRM indicates that nicotine exposure disrupts genes involved in neurogenesis, possibly through misregulation of nicotine-sensitive miRNAs. Conclusions BRM provides the ability to mine complex data for identification of candidate miRNAs or pathways that drive phenotypic outcome and, therefore, is a useful hypothesis generation tool for systems biology. The miRNA workflow in BRM allows for efficient processing of multiple miRNA and mRNA datasets in a single software environment with the added capability to interact with public data sources and visual analytic tools for HTP data analysis at a systems level. BRM is developed using Java™ and other open-source technologies for free distribution (http://www.sysbio.org/dataresources/brm.stm). PMID:23174015

IGF-1 deficiency in a critical period early in life influences the vascular aging phenotype in mice by altering miRNA-mediated post-transcriptional gene regulation: implications for the developmental origins of health and disease hypothesis.

PubMed

Tarantini, Stefano; Giles, Cory B; Wren, Jonathan D; Ashpole, Nicole M; Valcarcel-Ares, M Noa; Wei, Jeanne Y; Sonntag, William E; Ungvari, Zoltan; Csiszar, Anna

2016-08-01

Epidemiological findings support the concept of Developmental Origins of Health and Disease, suggesting that early-life hormonal influences during a sensitive period of development have a fundamental impact on vascular health later in life. The endocrine changes that occur during development are highly conserved across mammalian species and include dramatic increases in circulating IGF-1 levels during adolescence. The present study was designed to characterize the effect of developmental IGF-1 deficiency on the vascular aging phenotype. To achieve that goal, early-onset endocrine IGF-1 deficiency was induced in mice by knockdown of IGF-1 in the liver using Cre-lox technology (Igf1 f/f mice crossed with mice expressing albumin-driven Cre recombinase). This model exhibits low-circulating IGF-1 levels during the peripubertal phase of development, which is critical for the biology of aging. Due to the emergence of miRNAs as important regulators of the vascular aging phenotype, the effect of early-life IGF-1 deficiency on miRNA expression profile in the aorta was examined in animals at 27 months of age. We found that developmental IGF-1 deficiency elicits persisting late-life changes in miRNA expression in the vasculature, which significantly differed from those in mice with adult-onset IGF-1 deficiency (TBG-Cre-AAV8-mediated knockdown of IGF-1 at 5 month of age in Igf1 f/f mice). Using a novel computational approach, we identified miRNA target genes that are co-expressed with IGF-1 and associate with aging and vascular pathophysiology. We found that among the predicted targets, the expression of multiple extracellular matrix-related genes, including collagen-encoding genes, were downregulated in mice with developmental IGF-1 deficiency. Collectively, IGF-1 deficiency during a critical period during early in life results in persistent changes in post-transcriptional miRNA-mediated control of genes critical targets for vascular health, which likely contribute to the deleterious late-life cardiovascular effects known to occur with developmental IGF-1 deficiency.

Identification of mutant phenotypes associated with loss of individual microRNAs in sensitized genetic backgrounds in Caenorhabditis elegans

PubMed Central

Brenner, John L.; Jasiewicz, Kristen L.; Fahley, Alisha F.; Kemp, Benedict J.; Abbott, Allison L.

2010-01-01

Summary MicroRNAs (miRNAs) are small, non-coding RNAs that regulate the translation and/or the stability of their mRNA targets. Previous work showed that for most miRNA genes of C. elegans, single gene knockouts did not result in detectable mutant phenotypes [1]. This may be due, in part, to functional redundancy between miRNAs. However, in most cases, worms carrying deletions of all members of a miRNA family do not display strong mutant phenotypes [2]. They may function together with unrelated miRNAs or with non-miRNA genes in regulatory networks, possibly to ensure the robustness of developmental mechanisms. To test this, we examined worms lacking individual miRNAs in genetically sensitized backgrounds. These include genetic backgrounds with reduced processing and activity of all miRNAs or with reduced activity of a wide array of regulatory pathways [3]. Using these two approaches, mutant phenotypes were identified for 25 out of 31 miRNAs included in this analysis. Our findings describe biological roles for individual miRNAs and suggest that use of sensitized genetic backgrounds provides an efficient approach for miRNA functional analysis. PMID:20579881

Identification of MicroRNAs and transcript targets in Camelina sativa by deep sequencing and computational methods

DOE PAGES

Poudel, Saroj; Aryal, Niranjan; Lu, Chaofu; ...

2015-03-31

Camelina sativa is an annual oilseed crop that is under intensive development for renewable resources of biofuels and industrial oils. MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play key roles in diverse plant biological processes. Here, we conducted deep sequencing on small RNA libraries prepared from camelina leaves, flower buds and two stages of developing seeds corresponding to initial and peak storage products accumulation. Computational analyses identified 207 known miRNAs belonging to 63 families, as well as 5 novel miRNAs. These miRNAs, especially members of the miRNA families, varied greatly in different tissues and developmental stages. The predictedmore » miRNA target genes are involved in a broad range of physiological functions including lipid metabolism. This report is the first step toward elucidating roles of miRNAs in C. sativa and will provide additional tools to improve this oilseed crop for biofuels and biomaterials.« less

High-Throughput Sequencing of RNA Silencing-Associated Small RNAs in Olive (Olea europaea L.)

PubMed Central

Donaire, Livia; Pedrola, Laia; de la Rosa, Raúl; Llave, César

2011-01-01

Small RNAs (sRNAs) of 20 to 25 nucleotides (nt) in length maintain genome integrity and control gene expression in a multitude of developmental and physiological processes. Despite RNA silencing has been primarily studied in model plants, the advent of high-throughput sequencing technologies has enabled profiling of the sRNA component of more than 40 plant species. Here, we used deep sequencing and molecular methods to report the first inventory of sRNAs in olive (Olea europaea L.). sRNA libraries prepared from juvenile and adult shoots revealed that the 24-nt class dominates the sRNA transcriptome and atypically accumulates to levels never seen in other plant species, suggesting an active role of heterochromatin silencing in the maintenance and integrity of its large genome. A total of 18 known miRNA families were identified in the libraries. Also, 5 other sRNAs derived from potential hairpin-like precursors remain as plausible miRNA candidates. RNA blots confirmed miRNA expression and suggested tissue- and/or developmental-specific expression patterns. Target mRNAs of conserved miRNAs were computationally predicted among the olive cDNA collection and experimentally validated through endonucleolytic cleavage assays. Finally, we use expression data to uncover genetic components of the miR156, miR172 and miR390/TAS3-derived trans-acting small interfering RNA (tasiRNA) regulatory nodes, suggesting that these interactive networks controlling developmental transitions are fully operational in olive. PMID:22140484

Identification and Characteristics of microRNAs from Army Worm, Spodoptera frugiperda Cell Line Sf21

PubMed Central

Kakumani, Pavan Kumar; Chinnappan, Mahendran; Singh, Ashok K.; Malhotra, Pawan; Mukherjee, Sunil K.; Bhatnagar, Raj K.

2015-01-01

microRNAs play important regulatory role in all intrinsic cellular functions. Amongst lepidopteran insects, miRNAs from only Bombyx mori have been studied extensively with a little focus on Spodoptera sp. In the present study, we identified a total of 226 miRNAs from Spodoptera frugiperda cell line Sf21. Of the total, 116 miRNAs were well conserved within other insects, like B. mori, Drosophila melanogaster and Tribolium castenum while the remaining 110 miRNAs were identified as novel based on comparative analysis with the insect miRNA data set. Landscape distribution analysis based on Sf21 genome assembly revealed clustering of few novel miRNAs. A total of 5 miRNA clusters were identified and the largest one encodes 5 miRNA genes. In addition, 12 miRNAs were validated using northern blot analysis and putative functional role assignment for 6 Sf miRNAs was investigated by examining their relative abundance at different developmental stages of Spodoptera litura and body parts of 6th instar larvae. Further, we identified a total of 809 potential target genes with GO terms for selected miRNAs, involved in different metabolic and signalling pathways of the insect. The newly identified miRNAs greatly enrich the repertoire of insect miRNAs and analysis of expression profiles reveal their involvement at various steps of biochemical pathways of the army worm. PMID:25693181

Identification and characteristics of microRNAs from army worm, Spodoptera frugiperda cell line Sf21.

PubMed

Kakumani, Pavan Kumar; Chinnappan, Mahendran; Singh, Ashok K; Malhotra, Pawan; Mukherjee, Sunil K; Bhatnagar, Raj K

2015-01-01

microRNAs play important regulatory role in all intrinsic cellular functions. Amongst lepidopteran insects, miRNAs from only Bombyx mori have been studied extensively with a little focus on Spodoptera sp. In the present study, we identified a total of 226 miRNAs from Spodoptera frugiperda cell line Sf21. Of the total, 116 miRNAs were well conserved within other insects, like B. mori, Drosophila melanogaster and Tribolium castenum while the remaining 110 miRNAs were identified as novel based on comparative analysis with the insect miRNA data set. Landscape distribution analysis based on Sf21 genome assembly revealed clustering of few novel miRNAs. A total of 5 miRNA clusters were identified and the largest one encodes 5 miRNA genes. In addition, 12 miRNAs were validated using northern blot analysis and putative functional role assignment for 6 Sf miRNAs was investigated by examining their relative abundance at different developmental stages of Spodoptera litura and body parts of 6th instar larvae. Further, we identified a total of 809 potential target genes with GO terms for selected miRNAs, involved in different metabolic and signalling pathways of the insect. The newly identified miRNAs greatly enrich the repertoire of insect miRNAs and analysis of expression profiles reveal their involvement at various steps of biochemical pathways of the army worm.

Integrative analysis of genes and miRNA alterations in human embryonic stem cells-derived neural cells after exposure to silver nanoparticles

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

Oh, Jung-Hwa; Department of human and environmental toxicology, University of Science & Technology, Daejeon 34113; Son, Mi-Young

Given the rapid growth of engineered and customer products made of silver nanoparticles (Ag NPs), understanding their biological and toxicological effects on humans is critically important. The molecular developmental neurotoxic effects associated with exposure to Ag NPs were analyzed at the physiological and molecular levels, using an alternative cell model: human embryonic stem cell (hESC)-derived neural stem/progenitor cells (NPCs). In this study, the cytotoxic effects of Ag NPs (10–200 μg/ml) were examined in these hESC-derived NPCs, which have a capacity for neurogenesis in vitro, at 6 and 24 h. The results showed that Ag NPs evoked significant toxicity in hESC-derivedmore » NPCs at 24 h in a dose-dependent manner. In addition, Ag NPs induced cell cycle arrest and apoptosis following a significant increase in oxidative stress in these cells. To further clarify the molecular mechanisms of the toxicological effects of Ag NPs at the transcriptional and post-transcriptional levels, the global expression profiles of genes and miRNAs were analyzed in hESC-derived NPCs after Ag NP exposure. The results showed that Ag NPs induced oxidative stress and dysfunctional neurogenesis at the molecular level in hESC-derived NPCs. Based on this hESC-derived neural cell model, these findings have increased our understanding of the molecular events underlying developmental neurotoxicity induced by Ag NPs in humans. - Highlights: • This system served as a suitable model for developmental neurotoxicity testing. • Ag NPs induce the apoptosis in human neural cells by ROS generation. • Genes for development of neurons were dysregulated in response to Ag NPs. • Molecular events during early developmental neurotoxicity were proposed.« less

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.

Microarray-based analysis of cadmium-responsive microRNAs in rice (Oryza sativa)

PubMed Central

Ding, Yanfei; Chen, Zhen; Zhu, Cheng

2011-01-01

MicroRNAs (miRNAs) are a class of small non-coding RNAs that negatively regulate specific target mRNAs at the post-transcriptional level. Plant miRNAs have been implicated in developmental processes and adaptations to environmental stresses. Cadmium (Cd) is a non-essential heavy metal that is highly toxic to plants. To investigate the responsive functions of miRNAs under Cd stress, miRNA expression in Cd-stressed rice (Oryza sativa) was profiled using a microarray assay. A total of 19 Cd-responsive miRNAs were identified, of which six were further validated experimentally. Target genes were also predicted for these Cd-responsive miRNAs, which encoded transcription factors, and proteins associated with metabolic processes or stress responses. In addition, the mRNA levels of several targets were negatively correlated with the corresponding miRNAs under Cd stress. Promoter analysis showed that metal stress-responsive cis-elements tended to occur more frequently in the promoter regions of Cd-responsive miRNAs. These findings suggested that miRNAs played an important role in Cd tolerance in rice, and highlighted a novel molecular mechanism of heavy metal tolerance in plants. PMID:21362738

Functions of MicroRNAs in Cardiovascular Biology and Disease

PubMed Central

Hata, Akiko

2015-01-01

In 1993, lin-4 was discovered as a critical modulator of temporal development in Caenorhabditis elegans and, most notably, as the first in the class of small, single-stranded noncoding RNAs now defined as microRNAs (miRNAs). Another eight years elapsed before miRNA expression was detected in mammalian cells. Since then, explosive advancements in the field of miRNA biology have elucidated the basic mechanism of miRNA biogenesis, regulation, and gene-regulatory function. The discovery of this new class of small RNAs has augmented the complexity of gene-regulatory programs as well as the understanding of developmental and pathological processes in the cardiovascular system. Indeed, the contributions of miRNAs in cardiovascular development and function have been widely explored, revealing the extensive role of these small regulatory RNAs in cardiovascular physiology. PMID:23157557

Contributions of mRNA abundance, ribosome loading, and post- or peri-translational effects to temporal repression of C. elegans heterochronic miRNA targets

PubMed Central

Stadler, Michael; Artiles, Karen; Pak, Julia; Fire, Andrew

2012-01-01

miRNAs are post-transcriptional regulators of gene activity that reduce protein accumulation from target mRNAs. Elucidating precise molecular effects that animal miRNAs have on target transcripts has proven complex, with varied evidence indicating that miRNA regulation may produce different molecular outcomes in different species, systems, and/or physiological conditions. Here we use high-throughput ribosome profiling to analyze detailed translational parameters for five well-studied targets of miRNAs that regulate C. elegans developmental timing. For two targets of the miRNA lin-4 (lin-14 and lin-28), functional down-regulation was associated with decreases in both overall mRNA abundance and ribosome loading; however, these changes were of substantially smaller magnitude than corresponding changes observed in protein abundance. For three functional targets of the let-7 miRNA family for which down-regulation is critical in temporal progression of the animal (daf-12, hbl-1, and lin-41), we observed only modest changes in mRNA abundance and ribosome loading. lin-41 provides a striking example in that populations of ribosome-protected fragments from this gene remained essentially unchanged during the L3–L4 time interval when lin-41 activity is substantially down-regulated by let-7. Spectra of ribosomal positions were also examined for the five lin-4 and let-7 target mRNAs as a function of developmental time, with no indication of miRNA-induced ribosomal drop-off or significant pauses in translation. These data are consistent with models in which physiological regulation by this set of C. elegans miRNAs derives from combinatorial effects including suppressed recruitment/activation of translational machinery, compromised stability of target messages, and post- or peri-translational effects on lifetimes of polypeptide products. PMID:22855835

Human Milk Cells Contain Numerous miRNAs that May Change with Milk Removal and Regulate Multiple Physiological Processes

PubMed Central

Alsaweed, Mohammed; Lai, Ching Tat; Hartmann, Peter E.; Geddes, Donna T.; Kakulas, Foteini

2016-01-01

Human milk (HM) is a complex biofluid conferring nutritional, protective and developmental components for optimal infant growth. Amongst these are maternal cells, which change in response to feeding and were recently shown to be a rich source of miRNAs. We used next generation sequencing to characterize the cellular miRNA profile of HM collected before and after feeding. HM cells conserved higher miRNA content than the lipid and skim HM fractions or other body fluids, in accordance with previous studies. In total, 1467 known mature and 1996 novel miRNAs were identified, with 89 high-confidence novel miRNAs. HM cell content was higher post-feeding (p < 0.05), and was positively associated with total miRNA content (p = 0.014) and species number (p < 0.001). This coincided with upregulation of 29 known and 2 novel miRNAs, and downregulation of 4 known and 1 novel miRNAs post-feeding, but no statistically significant change in expression was found for the remaining miRNAs. These findings suggest that feeding may influence the miRNA content of HM cells. The most highly and differentially expressed miRNAs were key regulators of milk components, with potential diagnostic value in lactation performance. They are also involved in the control of body fluid balance, thirst, appetite, immune response, and development, implicating their functional significance for the infant. PMID:27322254

Integrative Analysis of Porcine microRNAome during Skeletal Muscle Development

PubMed Central

Qin, Lijun; Chen, Yaosheng; Liu, Xiaohong; Ye, Sanxing; Yu, Kaifan; Huang, Zheng; Yu, Jingwei; Zhou, Xingyu; Chen, Hu; Mo, Delin

2013-01-01

Pig is an important agricultural animal for meat production and provides a valuable model for many human diseases. Functional studies have demonstrated that microRNAs (miRNAs) play critical roles in almost all aspects of skeletal muscle development and disease pathogenesis. To investigate the miRNAs involved in regulating different periods of skeletal muscle development, we herein performed a comprehensive research for porcine microRNAome (miRNAome) during 10 skeletal muscle developmental stages including 35, 49, 63, 77, 91 dpc (days post coitum) and 2, 28, 90, 120, 180 dpn (days postnatal) using Solexa sequencing technology. Our results extend the repertoire of pig miRNAome to 247 known miRNAs processed from 210 pre-miRNAs and 297 candidate novel miRNAs through comparison with known miRNAs in the miRBase. Expression analysis of the 15 most abundant miRNAs in every library indicated that functional miRNAome may be smaller and tend to be highly expressed. A series of muscle-related miRNAs summarized in our study present different patterns between myofibers formation phase and muscle maturation phase, providing valuable reference for investigation of functional miRNAs during skeletal muscle development. Analysis of temporal profiles of miRNA expression identifies 18 novel candidate myogenic miRNAs in pig, which might provide new insight into regulation mechanism mediated by miRNAs underlying muscle development. PMID:24039761

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.

Analysis of the microRNA transcriptome of Daphnia pulex during aging.

PubMed

Hu, Jiabao; Lin, Chongyuan; Liu, Mengdi; Tong, Qiaoqiong; Xu, Shanliang; Wang, Danli; Zhao, Yunlong

2018-07-20

Daphnia pulex is an important food organism that exhibits a particular mode of reproduction known as cyclical parthenogenesis (asexual) and sexual reproduction. Regulation of the aging process by microRNAs (miRNAs) is a research hotspot in miRNA studies. To investigate a possible role of miRNAs in regulating aging and senescence, we used Illumina HiSeq to sequence two miRNA libraries from 1-day-old (1d) and 25-day-old (25d) D. pulex specimens. In total, we obtained 11,218,097 clean reads and 28,569 unique miRNAs from 1d specimens and 11,819,106 clean reads and 44,709 unique miRNAs from 25d specimens. Bioinformatic analyses was used to identify 1335 differentially expressed miRNAs from known miRNAs, including 127 miRNAs that exhibited statistically significant differences (P < 0.01); 92 miRNAs were upregulated and 35 were downregulated. Quantitative real-time (qRT)-PCR experiments were performed for nine miRNAs from five samples (1d, 5d, 10d, 15d, 20d and 25d) during the aging process, and the sequencing and qRT-PCR data were found to be consistent. Ninety-four miRNAs were predicted to correspond to 2014 target genes in known miRNAs with 4032 target gene sites. Sixteen pathways changed significantly (P < 0.05) at different developmental stages, revealing many important principles of the miRNA regulatory aging network of D. pulex. Overall, the difference in miRNA expression profile during aging of D. pulex forms a basis for further studies aimed at understanding the role of miRNAs in regulating aging, reproductive transformation, senescence, and longevity. Copyright © 2018 Elsevier B.V. All rights reserved.

Computational and transcriptional evidence for microRNAs in the honey bee genome

PubMed Central

Weaver, Daniel B; Anzola, Juan M; Evans, Jay D; Reid, Jeffrey G; Reese, Justin T; Childs, Kevin L; Zdobnov, Evgeny M; Samanta, Manoj P; Miller, Jonathan; Elsik, Christine G

2007-01-01

Background Non-coding microRNAs (miRNAs) are key regulators of gene expression in eukaryotes. Insect miRNAs help regulate the levels of proteins involved with development, metabolism, and other life history traits. The recently sequenced honey bee genome provides an opportunity to detect novel miRNAs in both this species and others, and to begin to infer the roles of miRNAs in honey bee development. Results Three independent computational surveys of the assembled honey bee genome identified a total of 65 non-redundant candidate miRNAs, several of which appear to have previously unrecognized orthologs in the Drosophila genome. A subset of these candidate miRNAs were screened for expression by quantitative RT-PCR and/or genome tiling arrays and most predicted miRNAs were confirmed as being expressed in at least one honey bee tissue. Interestingly, the transcript abundance for several known and novel miRNAs displayed caste or age-related differences in honey bees. Genes in proximity to miRNAs in the bee genome are disproportionately associated with the Gene Ontology terms 'physiological process', 'nucleus' and 'response to stress'. Conclusion Computational approaches successfully identified miRNAs in the honey bee and indicated previously unrecognized miRNAs in the well-studied Drosophila melanogaster genome despite the 280 million year distance between these insects. Differentially transcribed miRNAs are likely to be involved in regulating honey bee development, and arguably in the extreme developmental switch between sterile worker bees and highly fertile queens. PMID:17543122

Identification of evolutionarily conserved Momordica charantia microRNAs using computational approach and its utility in phylogeny analysis.

PubMed

Thirugnanasambantham, Krishnaraj; Saravanan, Subramanian; Karikalan, Kulandaivelu; Bharanidharan, Rajaraman; Lalitha, Perumal; Ilango, S; HairulIslam, Villianur Ibrahim

2015-10-01

Momordica charantia (bitter gourd, bitter melon) is a monoecious Cucurbitaceae with anti-oxidant, anti-microbial, anti-viral and anti-diabetic potential. Molecular studies on this economically valuable plant are very essential to understand its phylogeny and evolution. MicroRNAs (miRNAs) are conserved, small, non-coding RNA with ability to regulate gene expression by bind the 3' UTR region of target mRNA and are evolved at different rates in different plant species. In this study we have utilized homology based computational approach and identified 27 mature miRNAs for the first time from this bio-medically important plant. The phylogenetic tree developed from binary data derived from the data on presence/absence of the identified miRNAs were noticed to be uncertain and biased. Most of the identified miRNAs were highly conserved among the plant species and sequence based phylogeny analysis of miRNAs resolved the above difficulties in phylogeny approach using miRNA. Predicted gene targets of the identified miRNAs revealed their importance in regulation of plant developmental process. Reported miRNAs held sequence conservation in mature miRNAs and the detailed phylogeny analysis of pre-miRNA sequences revealed genus specific segregation of clusters. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

Genome-wide identification of microRNAs in pomegranate (Punica granatum L.) by high-throughput sequencing.

PubMed

Saminathan, Thangasamy; Bodunrin, Abiodun; Singh, Nripendra V; Devarajan, Ramajayam; Nimmakayala, Padma; Jeff, Moersfelder; Aradhya, Mallikarjuna; Reddy, Umesh K

2016-05-26

MicroRNAs (miRNAs), a class of small non-coding endogenous RNAs that regulate gene expression post-transcriptionally, play multiple key roles in plant growth and development and in biotic and abiotic stress response. Knowledge and roles of miRNAs in pomegranate fruit development have not been explored. Pomegranate, which accumulates a large amount of anthocyanins in skin and arils, is valuable to human health, mainly because of its antioxidant properties. In this study, we developed a small RNA library from pooled RNA samples from young seedlings to mature fruits and identified both conserved and pomegranate-specific miRNA from 29,948,480 high-quality reads. For the pool of 15- to 30-nt small RNAs, ~50 % were 24 nt. The miR157 family was the most abundant, followed by miR156, miR166, and miR168, with variants within each family. The base bias at the first position from the 5' end had a strong preference for U for most 18- to 26-nt sRNAs but a preference for A for 18-nt sRNAs. In addition, for all 24-nt sRNAs, the nucleotide U was preferred (97 %) in the first position. Stem-loop RT-qPCR was used to validate the expression of the predominant miRNAs and novel miRNAs in leaves, male and female flowers, and multiple fruit developmental stages; miR156, miR156a, miR159a, miR159b, and miR319b were upregulated during the later stages of fruit development. Higher expression of miR156 in later fruit developmental may positively regulate anthocyanin biosynthesis by reducing SPL transcription factor. Novel miRNAs showed variation in expression among different tissues. These novel miRNAs targeted different transcription factors and hormone related regulators. Gene ontology and KEGG pathway analyses revealed predominant metabolic processes and catalytic activities, important for fruit development. In addition, KEGG pathway analyses revealed the involvement of miRNAs in ascorbate and linolenic acid, starch and sucrose metabolism; RNA transport; plant hormone signaling pathways; and circadian clock. Our first and preliminary report of miRNAs will provide information on the synthesis of biochemical compounds of pomegranate for future research. The functions of the targets of the novel miRNAs need further investigation.

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.

Multiple microRNAs regulate human FOXP2 gene expression by targeting sequences in its 3' untranslated region.

PubMed

Fu, Lijuan; Shi, Zhimin; Luo, Guanzheng; Tu, Weihong; Wang, XiuJie; Fang, Zhide; Li, XiaoChing

2014-10-01

Mutations in the human FOXP2 gene cause speech and language impairments. The FOXP2 protein is a transcription factor that regulates the expression of many downstream genes, which may have important roles in nervous system development and function. An adequate amount of functional FOXP2 protein is thought to be critical for the proper development of the neural circuitry underlying speech and language. However, how FOXP2 gene expression is regulated is not clearly understood. The FOXP2 mRNA has an approximately 4-kb-long 3' untranslated region (3' UTR), twice as long as its protein coding region, indicating that FOXP2 can be regulated by microRNAs (miRNAs). We identified multiple miRNAs that regulate the expression of the human FOXP2 gene using sequence analysis and in vitro cell systems. Focusing on let-7a, miR-9, and miR-129-5p, three brain-enriched miRNAs, we show that these miRNAs regulate human FOXP2 expression in a dosage-dependent manner and target specific sequences in the FOXP2 3' UTR. We further show that these three miRNAs are expressed in the cerebellum of the human fetal brain, where FOXP2 is known to be expressed. Our results reveal novel regulatory functions of the human FOXP2 3' UTR sequence and regulatory interactions between multiple miRNAs and the human FOXP2 gene. The expression of let-7a, miR-9, and miR-129-5p in the human fetal cerebellum is consistent with their roles in regulating FOXP2 expression during early cerebellum development. These results suggest that various genetic and environmental factors may contribute to speech and language development and related neural developmental disorders via the miRNA-FOXP2 regulatory network.

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.

Conditional deletion of Dicer in vascular smooth muscle cells leads to the developmental delay and embryonic mortality

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

Pan, Yaoqian; Center for Cancer Research, University of Tennessee Health Science Center, Memphis, TN 38163; Balazs, Louisa

2011-05-13

Highlights: {yields} Deletion of Dicer in vascular smooth muscle cells(VSMCs) leads to embryonic mortality. {yields} Loss of Dicer in VSMCs leads to developmental delay. {yields} Loss of Dicer in VSMCs leads to hemorrhage in various organs including brain, skin and liver. {yields} Loss of Dicer in VSMCs leads to vascular wall remodeling. {yields} Loss of Dicer in VSMCs dysregulates the expression of miRNA and VSMC marker genes. -- Abstract: Dicer is a RNAase III enzyme that cleaves double stranded RNA and generates small interfering RNA (siRNA) and microRNA (miRNA). The goal of this study is to examine the role ofmore » Dicer and miRNAs in vascular smooth muscle cells (VSMCs). We deleted Dicer in VSMCs of mice, which caused a developmental delay that manifested as early as embryonic day E12.5, leading to embryonic death between E14.5 and E15.5 due to extensive hemorrhage in the liver, brain, and skin. Dicer KO embryos showed dilated blood vessels and a disarray of vascular architecture between E14.5 and E15.5. VSMC proliferation was significantly inhibited in Dicer KOs. The expression of VSMC marker genes were significantly downregulated in Dicer cKO embryos. The vascular structure of the yolk sac and embryo in Dicer KOs was lost to an extent that no blood vessels could be identified after E15.5. Expression of most miRNAs examined was compromised in VSMCs of Dicer KO. Our results indicate that Dicer is required for vascular development and regulates vascular remodeling by modulating VSMC proliferation and differentiation.« less

Recent advances in signal amplification strategy based on oligonucleotide and nanomaterials for microRNA detection-a review.

PubMed

Chen, Ying-Xu; Huang, Ke-Jing; Niu, Ke-Xin

2018-01-15

MicroRNAs (MiRNAs) play multiple crucial regulating roles in cell which can regulate one third of protein-coding genes. MiRNAs participate in the developmental and physiological processes of human body, while their aberrant adjustment will be more likely to trigger diseases such as cancers, kidney disease, central nervous system diseases, cardiovascular diseases, diabetes, viral infections and so on. What's worse, for the detection of miRNAs, their small size, high sequence similarity, low abundance and difficult extraction from cells impose great challenges in the analysis. Hence, it's necessary to fabricate accurate and sensitive biosensing platform for miRNAs detection. Up to now, researchers have developed many signal-amplification strategies for miRNAs detection, including hybridization chain reaction, nuclease amplification, rolling circle amplification, catalyzed hairpin assembly amplification and nanomaterials based amplification. These methods are typical, feasible and frequently used. In this review, we retrospect recent advances in signal amplification strategies for detecting miRNAs and point out the pros and cons of them. Furthermore, further prospects and promising developments of the signal-amplification strategies for detecting miRNAs are proposed. Copyright © 2017 Elsevier B.V. All rights reserved.

A Systematic Genetic Screen to Dissect the MicroRNA Pathway in Drosophila.

PubMed

Pressman, Sigal; Reinke, Catherine A; Wang, Xiaohong; Carthew, Richard W

2012-04-01

A central goal of microRNA biology is to elucidate the genetic program of miRNA function and regulation. However, relatively few of the effectors that execute miRNA repression have been identified. Because such genes may function in many developmental processes, mutations in them are expected to be pleiotropic and thus are discarded in most standard genetic screens. Here, we describe a systematic screen designed to identify all Drosophila genes in ∼40% of the genome that function in the miRNA pathway. To identify potentially pleiotropic genes, the screen analyzed clones of homozygous mutant cells in heterozygous animals. We identified 45 mutations representing 24 genes, and we molecularly characterized 9 genes. These include 4 previously known genes that encode core components of the miRNA pathway, including Drosha, Pasha, Dicer-1, and Ago1. The rest are new genes that function through chromatin remodeling, signaling, and mRNA decapping. The results suggest genetic screens that use clonal analysis can elucidate the miRNA program and that ∼100 genes are required to execute the miRNA program.

Adaptive evolution and functional innovation of Populus-specific recently evolved microRNAs.

PubMed

Xie, Jianbo; Yang, Xiaohui; Song, Yuepeng; Du, Qingzhang; Li, Ying; Chen, Jinhui; Zhang, Deqiang

2017-01-01

Lineage-specific microRNAs (miRNAs) undergo rapid turnover during evolution; however, their origin and functional importance have remained controversial. Here, we examine the origin, evolution, and potential roles in local adaptation of Populus-specific miRNAs, which originated after the recent salicoid-specific, whole-genome duplication. RNA sequencing was used to generate extensive, comparable miRNA and gene expression data for six tissues. A natural population of Populus trichocarpa and closely related species were used to study the divergence rates, evolution, and adaptive variation of miRNAs. MiRNAs that originated in 5' untranslated regions had higher expression levels and their expression showed high correlation with their host genes. Compared with conserved miRNAs, a significantly higher proportion of Populus-specific miRNAs appear to target genes that were duplicated in salicoids. Examination of single nucleotide polymorphisms in Populus-specific miRNA precursors showed high amounts of population differentiation. We also characterized the newly emerged MIR6445 family, which could trigger the production of phased small interfering RNAs from NAC mRNAs, which encode a transcription factor with primary roles in a variety of plant developmental processes. Together, these observations provide evolutionary insights into the birth and potential roles of Populus-specific miRNAs in genome maintenance, local adaptation, and functional innovation. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

NPK macronutrients and microRNA homeostasis.

PubMed

Kulcheski, Franceli R; Côrrea, Régis; Gomes, Igor A; de Lima, Júlio C; Margis, Rogerio

2015-01-01

Macronutrients are essential elements for plant growth and development. In natural, non-cultivated systems, the availability of macronutrients is not a limiting factor of growth, due to fast recycling mechanisms. However, their availability might be an issue in modern agricultural practices, since soil has been frequently over exploited. From a crop management perspective, the nitrogen (N), phosphorus (P), and potassium (K) are three important limiting factors and therefore frequently added as fertilizers. NPK are among the nutrients that have been reported to alter post-embryonic root developmental processes and consequently, impairs crop yield. To cope with nutrients scarcity, plants have evolved several mechanisms involved in metabolic, physiological, and developmental adaptations. In this scenario, microRNAs (miRNAs) have emerged as additional key regulators of nutrients uptake and assimilation. Some studies have demonstrated the intrinsic relation between miRNAs and their targets, and how they can modulate plants to deal with the NPK availability. In this review, we focus on miRNAs and their regulation of targets involved in NPK metabolism. In general, NPK starvation is related with miRNAs that are involved in root-architectural changes and uptake activity modulation. We further show that several miRNAs were discovered to be involved in plant-microbe symbiosis during N and P uptake, and in this way we present a global view of some studies that were conducted in the last years. The integration of current knowledge about miRNA-NPK signaling may help future studies to focus in good candidates genes for the development of important tools for plant nutritional breeding.

The 3-to-5 Exoribonuclease Knabber Shapes the 32 Ends of MicroRNAs Bound to Drosophila Argonaute1

PubMed Central

Han, Bo W.; Hung, Jui-Hung; Weng, Zhiping; Zamore, Phillip D.; Ameres, Stefan L.

2011-01-01

Summary Background MicroRNAs (miRNAs) are ~22 nt small RNAs that control development, physiology and pathology in animals and plants. Production of miRNAs involves the sequential processing of primary hairpin -containing RNA polymerase II transcripts by the RNase III enzymes Drosha in the nucleus and Dicer in the cytoplasm. miRNA duplexes then assemble into Argonaute proteins to form the RNA-induced silencing complex (RISC). In mature RISC, a single-stranded miRNA directs the Argonaute protein to bind partially complementary sequences, typically in the 32 untranslated regions of messenger RNAs, repressing their expression. Results Here, we show that after loading into Ago1 more than a quarter of all Drosophila miRNAs undergo 32 end trimming by the 32-to-5′ exoribonuclease Knabber (CG9247). Depletion of Knabber by RNAi reveals that miRNAs are frequently produced by Dicer-1 as intermediates that are longer than ~22 nucleotides. Trimming of miRNA 32 ends occurs after removal of the miRNA* strand from pre-RISC and may be the final step in RISC assembly, ultim ately enhancing target mRNA repression. In vivo, depletion of Knabber by RNAi causes developmental defects. Conclusions We provide a molecular explanation for the previously reported heterogeneity of miRNA 32 ends and propose a model in which Knabber converts miRNAs into isoforms that are compatible with the preferred length of Ago1-bound small RNAs. PMID:22055293

Identification and comparative profiling of miRNAs in an early flowering mutant of trifoliate orange and its wild type by genome-wide deep sequencing.

PubMed

Sun, Lei-Ming; Ai, Xiao-Yan; Li, Wen-Yang; Guo, Wen-Wu; Deng, Xiu-Xin; Hu, Chun-Gen; Zhang, Jin-Zhi

2012-01-01

MicroRNAs (miRNAs) are a new class of small, endogenous RNAs that play a regulatory role in various biological and metabolic processes by negatively affecting gene expression at the post-transcriptional level. While the number of known Arabidopsis and rice miRNAs is continuously increasing, information regarding miRNAs from woody plants such as citrus remains limited. Solexa sequencing was performed at different developmental stages on both an early flowering mutant of trifoliate orange (precocious trifoliate orange, Poncirus trifoliata L. Raf.) and its wild-type in this study, resulting in the obtainment of 141 known miRNAs belonging to 99 families and 75 novel miRNAs in four libraries. A total of 317 potential target genes were predicted based on the 51 novel miRNAs families, GO and KEGG annotation revealed that high ranked miRNA-target genes are those implicated in diverse cellular processes in plants, including development, transcription, protein degradation and cross adaptation. To characterize those miRNAs expressed at the juvenile and adult development stages of the mutant and its wild-type, further analysis on the expression profiles of several miRNAs through real-time PCR was performed. The results revealed that most miRNAs were down-regulated at adult stage compared with juvenile stage for both the mutant and its wild-type. These results indicate that both conserved and novel miRNAs may play important roles in citrus growth and development, stress responses and other physiological processes.

Biocomputational identification and validation of novel microRNAs predicted from bubaline whole genome shotgun sequences.

PubMed

Manku, H K; Dhanoa, J K; Kaur, S; Arora, J S; Mukhopadhyay, C S

2017-10-01

MicroRNAs (miRNAs) are small (19-25 base long), non-coding RNAs that regulate post-transcriptional gene expression by cleaving targeted mRNAs in several eukaryotes. The miRNAs play vital roles in multiple biological and metabolic processes, including developmental timing, signal transduction, cell maintenance and differentiation, diseases and cancers. Experimental identification of microRNAs is expensive and lab-intensive. Alternatively, computational approaches for predicting putative miRNAs from genomic or exomic sequences rely on features of miRNAs viz. secondary structures, sequence conservation, minimum free energy index (MFEI) etc. To date, not a single miRNA has been identified in bubaline (Bubalus bubalis), which is an economically important livestock. The present study aims at predicting the putative miRNAs of buffalo using comparative computational approach from buffalo whole genome shotgun sequencing data (INSDC: AWWX00000000.1). The sequences were blasted against the known mammalian miRNA. The obtained miRNAs were then passed through a series of filtration criteria to obtain the set of predicted (putative and novel) bubaline miRNA. Eight miRNAs were selected based on lowest E-value and validated by real time PCR (SYBR green chemistry) using RNU6 as endogenous control. The results from different trails of real time PCR shows that out of selected 8 miRNAs, only 2 (hsa-miR-1277-5p; bta-miR-2285b) are not expressed in bubaline PBMCs. The potential target genes based on their sequence complementarities were then predicted using miRanda. This work is the first report on prediction of bubaline miRNA from whole genome sequencing data followed by experimental validation. The finding could pave the way to future studies in economically important traits in buffalo. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

An Exportin-1–dependent microRNA biogenesis pathway during human cell quiescence

PubMed Central

Martinez, Ivan; Hayes, Karen E.; Barr, Jamie A.; Harold, Abby D.; Xie, Mingyi; Bukhari, Syed I. A.; Vasudevan, Shobha; Steitz, Joan A.; DiMaio, Daniel

2017-01-01

The reversible state of proliferative arrest known as “cellular quiescence” plays an important role in tissue homeostasis and stem cell biology. By analyzing the expression of miRNAs and miRNA-processing factors during quiescence in primary human fibroblasts, we identified a group of miRNAs that are induced during quiescence despite markedly reduced expression of Exportin-5, a protein required for canonical miRNA biogenesis. The biogenesis of these quiescence-induced miRNAs is independent of Exportin-5 and depends instead on Exportin-1. Moreover, these quiescence-induced primary miRNAs (pri-miRNAs) are modified with a 2,2,7-trimethylguanosine (TMG)-cap, which is known to bind Exportin-1, and knockdown of Exportin-1 or trimethylguanosine synthase 1, responsible for (TMG)-capping, inhibits their biogenesis. Surprisingly, in quiescent cells Exportin-1–dependent pri-miR-34a is present in the cytoplasm together with a small isoform of Drosha, implying the existence of a different miRNA processing pathway in these cells. Our findings suggest that during quiescence the canonical miRNA biogenesis pathway is down-regulated and specific miRNAs are generated by an alternative pathway to regulate genes involved in cellular growth arrest. PMID:28584122

Design and interpretation of microRNA-reporter gene activity.

PubMed

Carroll, Adam P; Tooney, Paul A; Cairns, Murray J

2013-06-15

MicroRNAs (miRNAs) are small noncoding RNA molecules that act as sequence specificity guides to direct post-transcriptional gene silencing. In doing so, miRNAs regulate many critical developmental processes, including cellular proliferation, differentiation, migration, and apoptosis, as well as more specialized biological functions such as dendritic spine development and synaptogenesis. Interactions between miRNAs and their miRNA recognition elements occur via partial complementarity, rendering tremendous redundancy in targeting such that miRNAs are predicted to regulate 60% of the genome, with each miRNA estimated to regulate more than 200 genes. Because these predictions are prone to false positives and false negatives, there is an ever present need to provide material support to these assertions to firmly establish the biological function of specific miRNAs in both normal and pathophysiological contexts. Using schizophrenia-associated miR-181b as an example, we present detailed guidelines and novel insights for the rapid establishment of a streamlined miRNA-reporter gene assay and explore various design concepts for miRNA-reporter gene applications, including bidirectional miRNA modulation. In exemplifying this approach, we report seven novel miR-181b target sites for five schizophrenia candidate genes (DISC1, BDNF, ENKUR, GRIA1, and GRIK1) and dissect a number of vital concepts regarding future developments for miRNA-reporter gene assays and the interpretation of their results. Copyright © 2013 Elsevier Inc. All rights reserved.

Argonaute identity defines the length of mature mammalian microRNAs.

PubMed

Juvvuna, Prasanna Kumar; Khandelia, Piyush; Lee, Li Ming; Makeyev, Eugene V

2012-08-01

MicroRNAs (miRNAs) are 19- to 25-nt-long non-coding RNAs that regulate gene expression by base-pairing with target mRNAs and reducing their stability or translational efficiency. Mammalian miRNAs function in association with four closely related Argonaute proteins, AGO1-4. All four proteins contain the PAZ and the MID domains interacting with the miRNA 3' and 5' termini, respectively, as well as the PIWI domain comprising an mRNA 'slicing' activity in the case of AGO2 but not AGO1, AGO3 and AGO4. However, the slicing mode of the miRNA-programmed AGO2 is rarely realized in vivo and the four Argonautes are thought to play largely overlapping roles in the mammalian miRNA pathway. Here, we show that the average length of many miRNAs is diminished during nervous system development as a result of progressive shortening of the miRNA 3' ends. We link this modification with an increase in the fractional abundance of Ago2 in the adult brain and identify a specific structural motif within the PAZ domain that enables efficient trimming of miRNAs associated with this but not the other three Argonautes. Taken together, our data suggest that mammalian Argonautes may define the length and possibly biological activity of mature mammalian miRNAs in a developmentally controlled manner.

Argonaute identity defines the length of mature mammalian microRNAs

PubMed Central

Juvvuna, Prasanna Kumar; Khandelia, Piyush; Lee, Li Ming; Makeyev, Eugene V.

2012-01-01

MicroRNAs (miRNAs) are 19- to 25-nt-long non-coding RNAs that regulate gene expression by base-pairing with target mRNAs and reducing their stability or translational efficiency. Mammalian miRNAs function in association with four closely related Argonaute proteins, AGO1–4. All four proteins contain the PAZ and the MID domains interacting with the miRNA 3′ and 5′ termini, respectively, as well as the PIWI domain comprising an mRNA ‘slicing’ activity in the case of AGO2 but not AGO1, AGO3 and AGO4. However, the slicing mode of the miRNA-programmed AGO2 is rarely realized in vivo and the four Argonautes are thought to play largely overlapping roles in the mammalian miRNA pathway. Here, we show that the average length of many miRNAs is diminished during nervous system development as a result of progressive shortening of the miRNA 3′ ends. We link this modification with an increase in the fractional abundance of Ago2 in the adult brain and identify a specific structural motif within the PAZ domain that enables efficient trimming of miRNAs associated with this but not the other three Argonautes. Taken together, our data suggest that mammalian Argonautes may define the length and possibly biological activity of mature mammalian miRNAs in a developmentally controlled manner. PMID:22505576

Myostatin regulates miR-431 expression via the Ras-Mek-Erk signaling pathway.

PubMed

Wu, Rimao; Li, Hu; Li, Tingting; Zhang, Yong; Zhu, Dahai

2015-05-29

MicroRNAs (miRNAs) play critical regulatory roles in controlling myogenic development both in vitro and in vivo; however, the molecular mechanisms underlying transcriptional regulation of miRNA genes in skeletal muscle cells are largely unknown. Here, using a microarray hybridization approach, we identified myostatin-regulated miRNA genes in skeletal muscle tissues by systematically searching miRNAs that are differentially expressed between wild-type and myostatin-null mice during development. We found that 116 miRNA genes were differentially expressed in muscles between these mice across different developmental stages. We further characterized myostatin-regulated miR-431 was upregulated in skeletal muscle tissues of myostatin-null mice. In functional studies, we found that overexpression of miR-431 in C2C12 myoblast cells attenuated myostatin-induced suppression of myogenic differentiation. Mechanistic studies further demonstrated that myostatin acted through the Ras-Mek-Erk signaling pathway to transcriptionally regulate miR-431 expression C2C12 cells. Our findings provide new insight into the mechanisms underlying transcriptional regulation of miRNA genes by myostatin during skeletal muscle development. Copyright © 2015 Elsevier Inc. All rights reserved.

Nanotechnology based approaches for detection and delivery of microRNA in healthcare and crop protection.

PubMed

Chaudhary, Vrantika; Jangra, Sumit; Yadav, Neelam R

2018-04-13

Nanobiotechnology has the potential to revolutionize diverse sectors including medicine, agriculture, food, textile and pharmaceuticals. Disease diagnostics, therapeutics and crop protection strategies are fast emerging using nanomaterials preferably nanobiomaterials. It has potential for development of novel nanobiomolecules which offer several advantages over conventional treatment methods. RNA nanoparticles with many unique features are promising candidates in disease treatment. The miRNAs are involved in many biochemical and developmental pathways and their regulation in plants and animals. These appear to be a powerful tool for controlling various pathological diseases in human, plants and animals, however there are challenges associated with miRNA based nanotechnology. Several advancements made in the field of miRNA therapeutics make it an attractive approach, but a lot more has to be explored in nanotechnology assisted miRNA therapy. The miRNA based technologies can be employed for detection and combating crop diseases as well. Despite these potential advantages, nanobiotechnology applications in the agricultural sector are still in its infancy and have not yet made its mark in comparison with healthcare sector. The review provides a platform to discuss nature, role and use of miRNAs in nanobiotechnology applications.

A biogenesis step upstream of Microprocessor controls miR-17~92 expression

PubMed Central

Du, Peng; Wang, Longfei; Sliz, Piotr; Gregory, Richard I.

2015-01-01

SUMMARY The precise control of miR-17~92 microRNA (miRNA) is essential for normal development and overexpression of certain miRNAs from this cluster is oncogenic. Here we find the relative expression of the six miRNAs processed from the primary (pri-miR-17~92) transcript is dynamically regulated during embryonic stem cell (ESC) differentiation. Pri-miR-17~92 is processed to a biogenesis intermediate, termed ‘progenitor-miRNA’ (pro-miRNA). Pro-miRNA is an efficient substrate for Microprocessor and is required to selectively license production of pre-miR-17, -18a, -19a, 20a, and -19b from this cluster. Two complementary cis-regulatory repression domains within pri-miR-17~92 are required for the blockade of miRNA processing through the formation of an autoinhibitory RNA conformation. The endonuclease CPSF3 (CPSF73), and the Spliceosome-associated ISY1 are responsible for pro-miRNA biogenesis and expression of all miRNAs within the cluster except miR-92. Thus, developmentally regulated pro-miRNA processing is key step controlling miRNA expression and explains the posttranscriptional control of miR-17~92 expression in development. PMID:26255770

History and current status of wheat miRNAs using next-generation sequencing and their roles in development and stress.

PubMed

Budak, Hikmet; Khan, Zaeema; Kantar, Melda

2015-05-01

As small molecules that aid in posttranscriptional silencing, microRNA (miRNA) discovery and characterization have vastly benefited from the recent development and widespread application of next-generation sequencing (NGS) technologies. Several miRNAs were identified through sequencing of constructed small RNA libraries, whereas others were predicted by in silico methods using the recently accumulating sequence data. NGS was a major breakthrough in efforts to sequence and dissect the genomes of plants, including bread wheat and its progenitors, which have large, repetitive and complex genomes. Availability of survey sequences of wheat whole genome and its individual chromosomes enabled researchers to predict and assess wheat miRNAs both in the subgenomic and whole genome levels. Moreover, small RNA construction and sequencing-based studies identified several putative development- and stress-related wheat miRNAs, revealing their differential expression patterns in specific developmental stages and/or in response to stress conditions. With the vast amount of wheat miRNAs identified in recent years, we are approaching to an overall knowledge on the wheat miRNA repertoire. In the following years, more comprehensive research in relation to miRNA conservation or divergence across wheat and its close relatives or progenitors should be performed. Results may serve valuable in understanding both the significant roles of species-specific miRNAs and also provide us information in relation to the dynamics between miRNAs and evolution in wheat. Furthermore, putative development- or stress-related miRNAs identified should be subjected to further functional analysis, which may be valuable in efforts to develop wheat with better resistance and/or yield. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Identification and comparative analyses of myocardial miRNAs involved in the fetal response to maternal obesity.

PubMed

Maloyan, Alina; Muralimanoharan, Sribalasubashini; Huffman, Steven; Cox, Laura A; Nathanielsz, Peter W; Myatt, Leslie; Nijland, Mark J

2013-10-01

Human and animal studies show that suboptimal intrauterine environments lead to fetal programming, predisposing offspring to disease in later life. Maternal obesity has been shown to program offspring for cardiovascular disease (CVD), diabetes, and obesity. MicroRNAs (miRNAs) are small, noncoding RNA molecules that act as key regulators of numerous cellular processes. Compelling evidence links miRNAs to the control of cardiac development and etiology of cardiac pathology; however, little is known about their role in the fetal cardiac response to maternal obesity. Our aim was to sequence and profile the cardiac miRNAs that are dysregulated in the hearts of baboon fetuses born to high fat/high fructose-diet (HFD) fed mothers for comparison with fetal hearts from mothers eating a regular diet. Eighty miRNAs were differentially expressed. Of those, 55 miRNAs were upregulated and 25 downregulated with HFD. Twenty-two miRNAs were mapped to human; 14 of these miRNAs were previously reported to be dysregulated in experimental or human CVD. We used an Ingenuity Pathway Analysis to integrate miRNA profiling and bioinformatics predictions to determine miRNA-regulated processes and genes potentially involved in fetal programming. We found a correlation between miRNA expression and putative gene targets involved in developmental disorders and CVD. Cellular death, growth, and proliferation were the most affected cellular functions in response to maternal obesity. Thus, the current study reveals significant alterations in cardiac miRNA expression in the fetus of obese baboons. The epigenetic modifications caused by adverse prenatal environment may represent one of the mechanisms underlying fetal programming of CVD.

The mir-279/996 cluster represses receptor tyrosine kinase signaling to determine cell fates in the Drosophila eye.

PubMed

Duan, Hong; de Navas, Luis F; Hu, Fuqu; Sun, Kailiang; Mavromatakis, Yannis E; Viets, Kayla; Zhou, Cyrus; Kavaler, Joshua; Johnston, Robert J; Tomlinson, Andrew; Lai, Eric C

2018-04-09

Photoreceptors in the crystalline Drosophila eye are recruited by receptor tyrosine kinase (RTK)/Ras signaling mediated by Epidermal growth factor receptor (EGFR) and the Sevenless (Sev) receptor. Analyses of an allelic deletion series of the mir-279/996 locus, along with a panel of modified genomic rescue transgenes, show that Drosophila eye patterning depends on both miRNAs. Transcriptional reporter and activity sensor transgenes reveal expression and function of miR-279/996 in non-neural cells of the developing eye. Moreover, mir-279/996 mutants exhibit substantial numbers of ectopic photoreceptors, particularly of R7, and cone cell loss. These miRNAs restrict RTK signaling in the eye, since mir-279/996 nulls are dominantly suppressed by positive components of the EGFR pathway and enhanced by heterozygosity for an EGFR repressor. miR-279/996 limit photoreceptor recruitment by targeting multiple positive RTK/Ras signaling components that promote photoreceptor/R7 specification. Strikingly, deletion of mir-279/996 sufficiently derepresses RTK/Ras signaling so as to rescue a population of R7 cells in R7-specific RTK null mutants boss and sev , which otherwise completely lack this cell fate. Altogether, we reveal a rare setting of developmental cell specification that involves substantial miRNA control. © 2018. Published by The Company of Biologists Ltd.

Let-7b regulates the expression of the growth hormone receptor gene in deletion-type dwarf chickens.

PubMed

Lin, Shumao; Li, Hongmei; Mu, Heping; Luo, Wen; Li, Ying; Jia, Xinzheng; Wang, Sibing; Jia, Xiaolu; Nie, Qinghua; Li, Yugu; Zhang, Xiquan

2012-07-10

A deletion mutation in the growth hormone receptor (GHR) gene results in the inhibition of skeletal muscle growth and fat deposition in dwarf chickens. We used microarray techniques to determine microRNA (miRNA) and mRNA expression profiles of GHR in the skeletal muscles of 14-day-old embryos as well as 7-week-old deletion-type dwarf and normal-type chickens. Our aim was to elucidate the miRNA regulation of GHR expression with respect to growth inhibition and fat deposition. At the same developmental stages, different expression profiles in skeletal muscles of dwarf and normal chickens occurred for four miRNAs (miR-1623, miR-181b, let-7b, and miR-128). At different developmental stages, there was a significant difference in the expression profiles of a greater number of miRNAs. Eleven miRNAs were up-regulated and 18 down-regulated in the 7-week-old dwarf chickens when compared with profiles in 14-day-old embryos. In 7-week-old normal chickens, seven miRNAs were up-regulated and nine down-regulated compared with those in 14-day-old embryos. In skeletal muscles, 22 genes were up-regulated and 33 down-regulated in 14-day-old embryos compared with 7-week-old dwarf chickens. Sixty-five mRNAs were up-regulated and 108 down-regulated in 14-day-old embryos as compared with 7-week-old normal chickens. Thirty-four differentially expressed miRNAs were grouped into 18 categories based on overlapping seed and target sequences. Only let-7b was found to be complementary to its target in the 3' untranslated region of GHR, and was able to inhibit its expression. Kyoto Encyclopedia of Genes and Genomes pathway analysis and quantitative polymerase chain reactions indicated there were three main signaling pathways regulating skeletal muscle growth and fat deposition of chickens. These were influenced by let-7b-regulated GHR. Suppression of the cytokine signaling 3 (SOCS3) gene was found to be involved in the signaling pathway of adipocytokines. There is a critical miRNA, let-7b, involved in the regulation of GHR. SOCS3 plays a critical role in regulating skeletal muscle growth and fat deposition via let-7b-mediated GHR expression.

Let-7b regulates the expression of the growth hormone receptor gene in deletion-type dwarf chickens

PubMed Central

2012-01-01

Background A deletion mutation in the growth hormone receptor (GHR) gene results in the inhibition of skeletal muscle growth and fat deposition in dwarf chickens. We used microarray techniques to determine microRNA (miRNA) and mRNA expression profiles of GHR in the skeletal muscles of 14-day-old embryos as well as 7-week-old deletion-type dwarf and normal-type chickens. Our aim was to elucidate the miRNA regulation of GHR expression with respect to growth inhibition and fat deposition. Results At the same developmental stages, different expression profiles in skeletal muscles of dwarf and normal chickens occurred for four miRNAs (miR-1623, miR-181b, let-7b, and miR-128). At different developmental stages, there was a significant difference in the expression profiles of a greater number of miRNAs. Eleven miRNAs were up-regulated and 18 down-regulated in the 7-week-old dwarf chickens when compared with profiles in 14-day-old embryos. In 7-week-old normal chickens, seven miRNAs were up-regulated and nine down-regulated compared with those in 14-day-old embryos. In skeletal muscles, 22 genes were up-regulated and 33 down-regulated in 14-day-old embryos compared with 7-week-old dwarf chickens. Sixty-five mRNAs were up-regulated and 108 down-regulated in 14-day-old embryos as compared with 7-week-old normal chickens. Thirty-four differentially expressed miRNAs were grouped into 18 categories based on overlapping seed and target sequences. Only let-7b was found to be complementary to its target in the 3′ untranslated region of GHR, and was able to inhibit its expression. Kyoto Encyclopedia of Genes and Genomes pathway analysis and quantitative polymerase chain reactions indicated there were three main signaling pathways regulating skeletal muscle growth and fat deposition of chickens. These were influenced by let-7b-regulated GHR. Suppression of the cytokine signaling 3 (SOCS3) gene was found to be involved in the signaling pathway of adipocytokines. Conclusions There is a critical miRNA, let-7b, involved in the regulation of GHR. SOCS3 plays a critical role in regulating skeletal muscle growth and fat deposition via let-7b-mediated GHR expression. PMID:22781587

Conditional deletion of Dicer in vascular smooth muscle cells leads to the developmental delay and embryonic mortality

PubMed Central

Pan, Yaoqian; Balazs, Louisa; Tigyi, Gabor; Yue, Junming

2013-01-01

Dicer is a RNAase III enzyme that cleaves double stranded RNA and generates small interfering RNA (siRNA) and microRNA (miRNA). The goal of this study is to examine the role of Dicer and miRNAs in vascular smooth muscle cells (VSMCs). We deleted Dicer in VSMCs of mice, which caused a developmental delay that manifested as early as embryonic day E12.5, leading to embryonic death between E14.5 and E15.5 due to extensive hemorrhage in the liver, brain, and skin. Dicer KO embryos showed dilated blood vessels and a disarray of vascular architecture between E14.5 and E15.5. VSMC proliferation was significantly inhibited in Dicer KOs. The expression of VSMC marker genes were significantly downregulated in Dicer cKO embryos. The vascular structure of the yolk sac and embryo in Dicer KOs was lost to an extent that no blood vessels could be identified after E15.5. Expression of most miRNAs examined was compromised in VSMCs of Dicer KO. Our results indicate that Dicer is required for vascular development and regulates vascular remodeling by modulating VSMC proliferation and differentiation. PMID:21371421

Integrative analysis of genes and miRNA alterations in human embryonic stem cells-derived neural cells after exposure to silver nanoparticles.

PubMed

Oh, Jung-Hwa; Son, Mi-Young; Choi, Mi-Sun; Kim, Soojin; Choi, A-Young; Lee, Hyang-Ae; Kim, Ki-Suk; Kim, Janghwan; Song, Chang Woo; Yoon, Seokjoo

2016-05-15

Given the rapid growth of engineered and customer products made of silver nanoparticles (Ag NPs), understanding their biological and toxicological effects on humans is critically important. The molecular developmental neurotoxic effects associated with exposure to Ag NPs were analyzed at the physiological and molecular levels, using an alternative cell model: human embryonic stem cell (hESC)-derived neural stem/progenitor cells (NPCs). In this study, the cytotoxic effects of Ag NPs (10-200μg/ml) were examined in these hESC-derived NPCs, which have a capacity for neurogenesis in vitro, at 6 and 24h. The results showed that Ag NPs evoked significant toxicity in hESC-derived NPCs at 24h in a dose-dependent manner. In addition, Ag NPs induced cell cycle arrest and apoptosis following a significant increase in oxidative stress in these cells. To further clarify the molecular mechanisms of the toxicological effects of Ag NPs at the transcriptional and post-transcriptional levels, the global expression profiles of genes and miRNAs were analyzed in hESC-derived NPCs after Ag NP exposure. The results showed that Ag NPs induced oxidative stress and dysfunctional neurogenesis at the molecular level in hESC-derived NPCs. Based on this hESC-derived neural cell model, these findings have increased our understanding of the molecular events underlying developmental neurotoxicity induced by Ag NPs in humans. Copyright © 2015 Elsevier Inc. All rights reserved.

In silico identification and characterization of conserved miRNAs and their target genes in sweet potato (Ipomoea batatas L.) Expressed Sequence Tags (ESTs)

PubMed Central

Dehury, Budheswar; Panda, Debashis; Sahu, Jagajjit; Sahu, Mousumi; Sarma, Kishore; Barooah, Madhumita; Sen, Priyabrata; Modi, Mahendra Kumar

2013-01-01

The endogenous small non-coding micro RNAs (miRNAs), which are typically ~21–24 nt nucleotides, play a crucial role in regulating the intrinsic normal growth of cells and development of the plants as well as in maintaining the integrity of genomes. These small non-coding RNAs function as the universal specificity factors in post-transcriptional gene silencing. Discovering miRNAs, identifying their targets, and further inferring miRNA functions is a routine process to understand normal biological processes of miRNAs and their roles in the development of plants. Comparative genomics based approach using expressed sequence tags (EST) and genome survey sequences (GSS) offer a cost-effective platform for identification and characterization of miRNAs and their target genes in plants. Despite the fact that sweet potato (Ipomoea batatas L.) is an important staple food source for poor small farmers throughout the world, the role of miRNA in various developmental processes remains largely unknown. In this paper, we report the computational identification of miRNAs and their target genes in sweet potato from their ESTs. Using comparative genomics-based approach, 8 potential miRNA candidates belonging to miR168, miR2911, and miR156 families were identified from 23 406 ESTs in sweet potato. A total of 42 target genes were predicted and their probable functions were illustrated. Most of the newly identified miRNAs target transcription factors as well as genes involved in plant growth and development, signal transduction, metabolism, defense, and stress response. The identification of miRNAs and their targets is expected to accelerate the pace of miRNA discovery, leading to an improved understanding of the role of miRNA in development and physiology of sweet potato, as well as stress response. PMID:24067297

A resource of vectors and ES cells for targeted deletion of microRNAs in mice

PubMed Central

Prosser, Haydn M.; Koike-Yusa, Hiroko; Cooper, James D.; Law, Frances C.; Bradley, Allan

2011-01-01

The 21-23 nucleotide single-stranded RNAs classified as microRNAs (miRNA) perform fundamental roles in a wide range of cellular and developmental processes. miRNAs regulate protein expression through sequence-specific base pairing with target messenger RNAs (mRNA) reducing both their stability and the process of protein translation1, 2. At least 30% of protein coding genes appear to be conserved targets for miRNAs1. In contrast to the protein coding genes3, 4, no public resource of miRNA mouse mutant alleles exists. We have generated a library of highly germ-line transmissible C57BL/6N mouse mutant embryonic stem (ES) cells with targeted deletions for the majority of miRNA genes currently annotated within the miRBase registry5. These alleles have been designed to be highly adaptable research tools that can be efficiently altered to create reporter, conditional and other allelic variants. This ES cell resource can be searched electronically and is available from ES cell repositories for distribution to the scientific community6. PMID:21822254

MicroRNA biogenesis pathway from the salmon louse (Caligus rogercresseyi): emerging role in delousing drug response.

PubMed

Valenzuela-Miranda, Diego; Nuñez-Acuña, Gustavo; Valenzuela-Muñoz, Valentina; Asgari, Sassan; Gallardo-Escárate, Cristian

2015-01-25

Despite the increasing evidence of the importance of microRNAs (miRNAs) in the regulation of multiple biological processes, the molecular bases supporting this regulation are still barely understood in crustaceans. Therefore, the molecular characterization and transcriptome modulation of the miRNA biogenesis pathway were evaluated in the salmon louse Caligus rogercresseyi, an ectoparasite that constitutes one of the biggest concerns for salmonid aquaculture industry. Hence, RNA-Seq analysis was conducted from six different developmental stages, and also after bioassays with delousing drugs Deltamethrin and Azamethiphos using adult individuals. In silico analysis evidenced 24 putative genes involved in the miRNA pathway such as biogenesis, transport, maturation and miRNA-target interaction. Moreover, 243 putative single nucleotide polymorphisms (SNPs) were identified, 15 of which showed non-synonym mutations. RNA-Seq analysis revealed that CCR4-Not complex subunit 3 (CNOT3) was upregulated at earlier developmental stages (nauplius I-II and copepodid), and also after the exposure to Azamethiphos, but not to Deltamethrin. In contrast, the subunit 7 (CNOT7) showed an inverse expression pattern. Different Argonaute transcripts were associated to chalimus and adult stages, revealing specific expression patterns in response to antiparasitic drugs. Our results suggest novel insights into the regulatory network of the post-transcriptional gene regulation in C. rogercresseyi mediated by miRNAs, evidencing a putative role during the ontogeny and drug response. Copyright © 2014 Elsevier B.V. All rights reserved.

Next-generation small RNA sequencing for microRNAs profiling in the honey bee Apis mellifera.

PubMed

Chen, X; Yu, X; Cai, Y; Zheng, H; Yu, D; Liu, G; Zhou, Q; Hu, S; Hu, F

2010-12-01

MicroRNAs (miRNAs) are key regulators in various physiological and pathological processes via post-transcriptional regulation of gene expression. The honey bee (Apis mellifera) is a key model for highly social species, and its complex social behaviour can be interpreted theoretically as changes in gene regulation, in which miRNAs are thought to be involved. We used the SOLiD sequencing system to identify the repertoire of miRNAs in the honey bee by sequencing a mixed small RNA library from different developmental stages. We obtained a total of 36,796,459 raw sequences; of which 5,491,100 short sequences were fragments of mRNA and other noncoding RNAs (ncRNA), and 1,759,346 reads mapped to the known miRNAs. We predicted 267 novel honey bee miRNAs representing 380,182 short reads, including eight miRNAs of other insects in 14,107,583 genome-mapped sequences. We verified 50 of them using stem-loop reverse-transcription PCR (RT-PCR), in which 35 yielded PCR products. Cross-species analyses showed 81 novel miRNAs with homologues in other insects, suggesting that they were authentic miRNAs and have similar functions. The results of this study provide a basis for studies of the miRNA-modulating networks in development and some intriguing phenomena such as caste differentiation in A. mellifera. © 2010 The Authors. Insect Molecular Biology © 2010 The Royal Entomological Society.

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.

Conservation and divergence of microRNAs in Populus

PubMed Central

Barakat, Abdelali; Wall, Phillip K; DiLoreto, Scott; dePamphilis, Claude W; Carlson, John E

2007-01-01

Background MicroRNAs (miRNAs) are small RNAs (sRNA) ~21 nucleotides in length that negatively control gene expression by cleaving or inhibiting the translation of target gene transcripts. miRNAs have been extensively analyzed in Arabidopsis and rice and partially investigated in other non-model plant species. To date, 109 and 62 miRNA families have been identified in Arabidopsis and rice respectively. However, only 33 miRNAs have been identified from the genome of the model tree species (Populus trichocarpa), of which 11 are Populus specific. The low number of miRNA families previously identified in Populus, compared with the number of families identified in Arabidopsis and rice, suggests that many miRNAs still remain to be discovered in Populus. In this study, we analyzed expressed small RNAs from leaves and vegetative buds of Populus using high throughput pyrosequencing. Results Analysis of almost eighty thousand small RNA reads allowed us to identify 123 new sequences belonging to previously identified miRNA families as well as 48 new miRNA families that could be Populus-specific. Comparison of the organization of miRNA families in Populus, Arabidopsis and rice showed that miRNA family sizes were generally expanded in Populus. The putative targets of non-conserved miRNA include both previously identified targets as well as several new putative target genes involved in development, resistance to stress, and other cellular processes. Moreover, almost half of the genes predicted to be targeted by non-conserved miRNAs appear to be Populus-specific. Comparative analyses showed that genes targeted by conserved and non-conserved miRNAs are biased mainly towards development, electron transport and signal transduction processes. Similar results were found for non-conserved miRNAs from Arabidopsis. Conclusion Our results suggest that while there is a conserved set of miRNAs among plant species, a large fraction of miRNAs vary among species. The non-conserved miRNAs may regulate cellular, physiological or developmental processes specific to the taxa that produce them, as appears likely to be the case for those miRNAs that have only been observed in Populus. Non-conserved and conserved miRNAs seem to target genes with similar biological functions indicating that similar selection pressures are acting on both types of miRNAs. The expansion in the number of most conserved miRNAs in Populus relative to Arabidopsis, may be linked to the recent genome duplication in Populus, the slow evolution of the Populus genome, or to differences in the selection pressure on duplicated miRNAs in these species. PMID:18166134

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.

miRNAs involved in the development and differentiation of fertile and sterile flowers in Viburnum macrocephalum f. keteleeri.

PubMed

Li, Weixing; He, Zhichong; Zhang, Li; Lu, Zhaogeng; Xu, Jing; Cui, Jiawen; Wang, Li; Jin, Biao

2017-10-13

Sterile and fertile flowers are important evolutionary developmental phenotypes in angiosperm flowers. The development of floral organs, critical in angiosperm reproduction, is regulated by microRNAs (miRNAs). However, the mechanisms underpinning the miRNA regulation of the differentiation and development of sterile and fertile flowers remain unclear. Here, based on investigations of the morphological differences between fertile and sterile flowers, we used high-throughput sequencing to characterize the miRNAs in the differentiated floral organs of Viburnum macrocephalum f. keteleeri. We identified 49 known miRNAs and 67 novel miRNAs by small RNA (sRNA) sequencing and bioinformatics analysis, and 17 of these known and novel miRNA precursors were validated by polymerase chain reaction (PCR) and Sanger sequencing. Furthermore, by comparing the sequencing results of two sRNA libraries, we found that 30 known and 39 novel miRNA sequences were differentially expressed, and 35 were upregulated and 34 downregulated in sterile compared with fertile flowers. Combined with their predicted targets, the potential roles of miRNAs in V. macrocephalum f. keteleeri flowers include involvement in floral organogenesis, cell proliferation, hormonal pathways, and stress responses. miRNA precursors and targets were further validated by quantitative real-time PCR (qRT-PCR). Specifically, miR156a-5p, miR156g, and miR156j expression levels were significantly higher in fertile flowers than in sterile flowers, while SPL genes displayed the opposite expression pattern. Considering that the targets of miR156 are predicted to be SPL genes, we propose that miR156 may be involved in the regulation of stamen development in V. macrocephalum f. keteleeri. We identified miRNAs differentially expressed between fertile and sterile flowers in V. macrocephalum f. keteleeri and provided new insights into the important regulatory roles of miRNAs in the differentiation and development of fertile and sterile flowers.

Engineering of a conditional allele reveals multiple roles of XRN2 in Caenorhabditis elegans development and substrate specificity in microRNA turnover.

PubMed

Miki, Takashi S; Rüegger, Stefan; Gaidatzis, Dimos; Stadler, Michael B; Großhans, Helge

2014-04-01

Although XRN2 proteins are highly conserved eukaryotic 5'→3' exonucleases, little is known about their function in animals. Here, we characterize Caenorhabditis elegans XRN2, which we find to be a broadly and constitutively expressed nuclear protein. An xrn-2 null mutation or loss of XRN2 catalytic activity causes a molting defect and early larval arrest. However, by generating a conditionally mutant xrn-2ts strain de novo through an approach that may be also applicable to other genes of interest, we reveal further functions in fertility, during embryogenesis and during additional larval stages. Consistent with the known role of XRN2 in controlling microRNA (miRNA) levels, we can demonstrate that loss of XRN2 activity stabilizes some rapidly decaying miRNAs. Surprisingly, however, other miRNAs continue to decay rapidly in xrn-2ts animals. Thus, XRN2 has unanticipated miRNA specificity in vivo, and its diverse developmental functions may relate to distinct substrates. Finally, our global analysis of miRNA stability during larval stage 1 reveals that miRNA passenger strands (miR*s) are substantially less stable than guide strands (miRs), supporting the notion that the former are mostly byproducts of biogenesis rather than a less abundant functional species.

Milk’s Role as an Epigenetic Regulator in Health and Disease

PubMed Central

Melnik, Bodo C.; Schmitz, Gerd

2017-01-01

It is the intention of this review to characterize milk’s role as an epigenetic regulator in health and disease. Based on translational research, we identify milk as a major epigenetic modulator of gene expression of the milk recipient. Milk is presented as an epigenetic “doping system” of mammalian development. Milk exosome-derived micro-ribonucleic acids (miRNAs) that target DNA methyltransferases are implicated to play the key role in the upregulation of developmental genes such as FTO, INS, and IGF1. In contrast to miRNA-deficient infant formula, breastfeeding via physiological miRNA transfer provides the appropriate signals for adequate epigenetic programming of the newborn infant. Whereas breastfeeding is restricted to the lactation period, continued consumption of cow’s milk results in persistent epigenetic upregulation of genes critically involved in the development of diseases of civilization such as diabesity, neurodegeneration, and cancer. We hypothesize that the same miRNAs that epigenetically increase lactation, upregulate gene expression of the milk recipient via milk-derived miRNAs. It is of critical concern that persistent consumption of pasteurized cow’s milk contaminates the human food chain with bovine miRNAs, that are identical to their human analogs. Commercial interest to enhance dairy lactation performance may further increase the epigenetic miRNA burden for the milk consumer. PMID:28933365

Hippo signaling regulates Microprocessor and links cell density-dependent miRNA biogenesis to cancer

PubMed Central

Mori, Masaki; Triboulet, Robinson; Mohseni, Morvarid; Schlegelmilch, Karin; Shrestha, Kriti; Camargo, Fernando D.; Gregory, Richard I.

2014-01-01

SUMMARY Global downregulation of microRNAs (miRNAs) is commonly observed in human cancers and can have a causative role in tumorigenesis. The mechanisms responsible for this phenomenon remain poorly understood. Here we show that YAP, the downstream target of the tumor-suppressive Hippo signaling pathway regulates miRNA biogenesis in a cell density-dependent manner. At low cell density, nuclear YAP binds and sequesters p72 (DDX17), a regulatory component of the miRNA processing machinery. At high cell density, Hippo-mediated cytoplasmic retention of YAP facilitates p72 association with Microprocessor and binding to a specific sequence motif in pri-miRNAs. Inactivation of the Hippo pathway or expression of constitutively active YAP causes widespread miRNA suppression in cells and tumors and a corresponding post-transcriptional induction of MYC expression. Thus, the Hippo pathway links contact-inhibition regulation to miRNA biogenesis and may be responsible for the widespread miRNA repression observed in cancer. PMID:24581491

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.

Extracellular small RNAs: what, where, why?

PubMed Central

Hoy, Anna M.; Buck, Amy H.

2012-01-01

miRNAs (microRNAs) are a class of small RNA that regulate gene expression by binding to mRNAs and modulating the precise amount of proteins that get expressed in a cell at a given time. This form of gene regulation plays an important role in developmental systems and is critical for the proper function of numerous biological pathways. Although miRNAs exert their functions inside the cell, these and other classes of RNA are found in body fluids in a cell-free form that is resistant to degradation by RNases. A broad range of cell types have also been shown to secrete miRNAs in association with components of the RISC (RNA-induced silencing complex) and/or encapsulation within vesicles, which can be taken up by other cells. In the present paper, we provide an overview of the properties of extracellular miRNAs in relation to their capacity as biomarkers, stability against degradation and mediators of cell–cell communication. PMID:22817753

Implication of microRNAs in the development and potential treatment of radiation-induced heart disease.

PubMed

Kura, Branislav; Babal, Pavel; Slezak, Jan

2017-10-01

Radiotherapy is the most commonly used methodology to treat oncological disease, one of the most widespread causes of death worldwide. Oncological patients cured by radiotherapy applied to the mediastinal area have been shown to suffer from cardiovascular disease. The increase in the prevalence of radiation-induced heart disease has emphasized the need to seek new therapeutic targets to mitigate the negative impact of radiation on the heart. In this regard, microRNAs (miRNAs) have received considerable interest. miRNAs regulate post-transcriptional gene expression by their ability to target various mRNA sequences because of their imperfect pairing with mRNAs. It has been recognized that miRNAs modulate a diverse spectrum of cardiac functions with developmental, pathophysiological, and clinical implications. This makes them promising potential targets for diagnosis and treatment. This review summarizes the recent findings about the possible involvement of miRNAs in radiation-induced heart disease and their potential use as diagnostic or treatment targets in this respect.

Bisphenol A exposure leads to specific microRNA alterations in placental cells.

PubMed

Avissar-Whiting, Michele; Veiga, Keila R; Uhl, Kristen M; Maccani, Matthew A; Gagne, Luc A; Moen, Erika L; Marsit, Carmen J

2010-07-01

Exposure to bisphenol A (BPA) has been observed to alter developmental pathways and cell processes, at least in part, through epigenetic mechanisms. This study sought to investigate the effect of BPA on microRNAs (miRNAs) in human placental cells. miRNA microarray was performed following BPA treatment in three immortalized cytotrophoblast cell lines and the results validated using quantitative real-time PCR. For functional analysis, overexpression constructs were stably transfected into cells that were then assayed for changes in proliferation and response to toxicants. Microarray analysis revealed several miRNAs to be significantly altered in response to BPA treatment in two cell lines. Real-time PCR results confirmed that miR-146a was particularly strongly induced and its overexpression in cells led to slower proliferation as well as higher sensitivity to the DNA damaging agent, bleomycin. Overall, these results suggest that BPA can alter miRNA expression in placental cells, a potentially novel mode of BPA toxicity.

Bisphenol A Exposure Leads to Specific MicroRNA Alterations in Placental Cells

PubMed Central

Avissar-Whiting, Michele; Veiga, Keila; Uhl, Kristen; Maccani, Matthew; Gagne, Luc; Moen, Erika; Marsit, Carmen J.

2010-01-01

Exposure to bisphenol-A (BPA) has been observed to alter developmental pathways and cell processes, at least in part, through epigenetic mechanisms. This study sought to investigate the effect of BPA on microRNAs (miRNAs) in human placental cells. miRNA microarray was performed following BPA treatment in three immortalized cytotrophoblast cell lines and the results validated using quantitative real-time PCR. For functional analysis, overexpression constructs were stably transfected into cells that were then assayed for changes in proliferation and response to toxicants. Microarray analysis revealed several miRNAs to be significantly altered in response to BPA treatment in two cell lines. Real-time PCR results confirmed that miR-146a was particularly strongly induced and its overexpression in cells led to slower proliferation as well as higher sensitivity to the DNA damaging agent, bleomycin. Overall, these results suggest that BPA can alter miRNA expression in placental cells, a potentially novel mode of BPA toxicity. PMID:20417706

Identification and Characterization of the miRNA Transcriptome of Ovis aries

PubMed Central

Wei, Caihong; Sheng, Xihui; Ren, Hangxing; Xu, Lingyang; Lu, Jian; Liu, Jiasen; Zhang, Li; Du, Lixin

2013-01-01

The discovery and identification of Ovis aries (sheep) miRNAs will further promote the study of miRNA functions and gene regulatory mechanisms. To explore the microRNAome (miRNAome) of sheep in depth, samples were collected that included eight developmental stages: the longissimus dorsi muscles of Texel fetuses at 70, 85, 100, 120, and 135 days, and the longissimus dorsi muscles of Ujumqin fetuses at 70, 85, 100, 120, and 135 d, and lambs at 0 (birth), 35, and 70 d. These samples covered all of the representative periods of Ovis aries growth and development throughout gestation (about 150 d) and 70 d after birth. Texel and Ujumqin libraries were separately subjected to Solexa deep sequencing; 35,700,772 raw reads were obtained overall. We used ACGT101-miR v4.2 to analyze the sequence data. Following meticulous comparisons with mammalian mature miRNAs, precursor hairpins (pre-miRNAs), and the latest sheep genome, we substantially extended the Ovis aries miRNAome. The list of pre-miRNAs was extended to 2,319, expressing 2,914 mature miRNAs. Among those, 1,879 were genome mapped to unique miRNAs, representing 2,436 genome locations, and 1,754 pre-miRNAs were mapped to chromosomes. Furthermore, the Ovis aries miRNAome was processed using an elaborate bioinformatic analysis that examined multiple end sequence variation in miRNAs, precursors, chromosomal localizations, species-specific expressions, and conservative properties. Taken together, this study provides the most comprehensive and accurate exploration of the sheep miRNAome, and draws conclusions about numerous characteristics of Ovis aries miRNAs, including miRNAs and isomiRs. PMID:23516575

Identification of an miRNA candidate reflects the possible significance of transcribed microsatellites in the hairpin precursors of black pepper.

PubMed

Joy, Nisha; Soniya, Eppurathu Vasudevan

2012-06-01

Plant miRNAs (18-24nt) are generated by the RNase III-type Dicer endonuclease from the endogenous hairpin precursors ('pre-miRNAs') with significant regulatory functions. The transcribed regions display a higher frequency of microsatellites, when compared to other regions of the genomic DNA. Simple sequence repeats (SSRs) resulting from replication slippage occurring in transcripts affect the expression of genes. The available experimental evidence for the incidence of SSRs in the miRNA precursors is limited. Considering the potential significance of SSRs in the miRNA genes, we carried out a preliminary analysis to verify the presence of SSRs in the pri-miRNAs of black pepper (Piper nigrum L.). We isolated a (CT) dinucleotide SSR bearing transcript using SMART strategy. The transcript was predicted to be a 'pri-miRNA candidate' with Dicer sites based on miRNA prediction tools and MFOLD structural predictions. The presence of this 'miRNA candidate' was confirmed by real-time TaqMan assays. The upstream sequence of the 'miRNA candidate' by genome walking when subjected to PlantCARE showed the presence of certain promoter elements, and the deduced amino acid showed significant similarity with NAP1 gene, which affects the transcription of many genes. Moreover the hairpin-like precursor overlapped the neighbouring NAP1 gene. In silico analysis revealed distinct putative functions for the 'miRNA candidate', of which majority were related to growth. Hence, we assume that this 'miRNA candidate' may get activated during transcription of NAP gene, thereby regulating the expression of many genes involved in developmental processes.

Identification of microRNA Genes in Three Opisthorchiids

PubMed Central

Ovchinnikov, Vladimir Y.; Afonnikov, Dmitry A.; Vasiliev, Gennady V.; Kashina, Elena V.; Sripa, Banchob; Mordvinov, Viacheslav A.; Katokhin, Alexey V.

2015-01-01

Background Opisthorchis felineus, O. viverrini, and Clonorchis sinensis (family Opisthorchiidae) are parasitic flatworms that pose a serious threat to humans in some countries and cause opisthorchiasis/clonorchiasis. Chronic disease may lead to a risk of carcinogenesis in the biliary ducts. MicroRNAs (miRNAs) are small noncoding RNAs that control gene expression at post-transcriptional level and are implicated in the regulation of various cellular processes during the parasite- host interplay. However, to date, the miRNAs of opisthorchiid flukes, in particular those essential for maintaining their complex biology and parasitic mode of existence, have not been satisfactorily described. Methodology/Principal Findings Using a SOLiD deep sequencing-bioinformatic approach, we identified 43 novel and 18 conserved miRNAs for O. felineus (miracidia, metacercariae and adult worms), 20 novel and 16 conserved miRNAs for O. viverrini (adult worms), and 33 novel and 18 conserved miRNAs for C. sinensis (adult worms). The analysis of the data revealed differences in the expression level of conserved miRNAs among the three species and among three the developmental stages of O. felineus. Analysis of miRNA genes revealed two gene clusters, one cluster-like region and one intronic miRNA in the genome. The presence and structure of the two gene clusters were validated using a PCR-based approach in the three flukes. Conclusions This study represents a comprehensive description of miRNAs in three members of the family Opistorchiidae, significantly expands our knowledge of miRNAs in multicellular parasites and provides a basis for understanding the structural and functional evolution of miRNAs in these metazoan parasites. Results of this study also provides novel resources for deeper understanding the complex parasite biology, for further research on the pathogenesis and molecular events of disease induced by the liver flukes. The present data may also facilitate the development of novel approaches for the prevention and treatment of opisthorchiasis/clonorchiasis. PMID:25898350

MicroRNA networks in mouse lung organogenesis.

PubMed

Dong, Jie; Jiang, Guoqian; Asmann, Yan W; Tomaszek, Sandra; Jen, Jin; Kislinger, Thomas; Wigle, Dennis A

2010-05-26

MicroRNAs (miRNAs) are known to be important regulators of both organ development and tumorigenesis. MiRNA networks and their regulation of messenger RNA (mRNA) translation and protein expression in specific biological processes are poorly understood. We explored the dynamic regulation of miRNAs in mouse lung organogenesis. Comprehensive miRNA and mRNA profiling was performed encompassing all recognized stages of lung development beginning at embryonic day 12 and continuing to adulthood. We analyzed the expression patterns of dynamically regulated miRNAs and mRNAs using a number of statistical and computational approaches, and in an integrated manner with protein levels from an existing mass-spectrometry derived protein database for lung development. In total, 117 statistically significant miRNAs were dynamically regulated during mouse lung organogenesis and clustered into distinct temporal expression patterns. 11,220 mRNA probes were also shown to be dynamically regulated and clustered into distinct temporal expression patterns, with 3 major patterns accounting for 75% of all probes. 3,067 direct miRNA-mRNA correlation pairs were identified involving 37 miRNAs. Two defined correlation patterns were observed upon integration with protein data: 1) increased levels of specific miRNAs directly correlating with downregulation of predicted mRNA targets; and 2) increased levels of specific miRNAs directly correlating with downregulation of translated target proteins without detectable changes in mRNA levels. Of 1345 proteins analyzed, 55% appeared to be regulated in this manner with a direct correlation between miRNA and protein level, but without detectable change in mRNA levels. Systematic analysis of microRNA, mRNA, and protein levels over the time course of lung organogenesis demonstrates dynamic regulation and reveals 2 distinct patterns of miRNA-mRNA interaction. The translation of target proteins affected by miRNAs independent of changes in mRNA level appears to be a prominent mechanism of developmental regulation in lung organogenesis.

Identification of MicroRNAs and their Targets Associated with Embryo Abortion during Chrysanthemum Cross Breeding via High-Throughput Sequencing.

PubMed

Zhang, Fengjiao; Dong, Wen; Huang, Lulu; Song, Aiping; Wang, Haibin; Fang, Weimin; Chen, Fadi; Teng, Nianjun

2015-01-01

MicroRNAs (miRNAs) are important regulators in plant development. They post-transcriptionally regulate gene expression during various biological and metabolic processes by binding to the 3'-untranslated region of target mRNAs to facilitate mRNA degradation or inhibit translation. Chrysanthemum (Chrysanthemum morifolium) is one of the most important ornamental flowers with increasing demand each year. However, embryo abortion is the main reason for chrysanthemum cross breeding failure. To date, there have been no experiments examining the expression of miRNAs associated with chrysanthemum embryo development. Therefore, we sequenced three small RNA libraries to identify miRNAs and their functions. Our results will provide molecular insights into chrysanthemum embryo abortion. Three small RNA libraries were built from normal chrysanthemum ovules at 12 days after pollination (DAP), and normal and abnormal chrysanthemum ovules at 18 DAP. We validated 228 miRNAs with significant changes in expression frequency during embryonic development. Comparative profiling revealed that 69 miRNAs exhibited significant differential expression between normal and abnormal embryos at 18 DAP. In addition, a total of 1037 miRNA target genes were predicted, and their annotations were defined by transcriptome data. Target genes associated with metabolic pathways were most highly represented according to the annotation. Moreover, 52 predicted target genes were identified to be associated with embryonic development, including 31 transcription factors and 21 additional genes. Gene ontology (GO) annotation also revealed that high-ranking miRNA target genes related to cellular processes and metabolic processes were involved in transcription regulation and the embryo developmental process. The present study generated three miRNA libraries and gained information on miRNAs and their targets in the chrysanthemum embryo. These results enrich the growing database of new miRNAs and lay the foundation for the further understanding of miRNA biological function in the regulation of chrysanthemum embryo abortion.

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

Discovery and Analysis of MicroRNAs in Leymus chinensis under Saline-Alkali and Drought Stress Using High-Throughput Sequencing

PubMed Central

Wang, Qi; Wang, Nan; Wang, Fawei; Liu, Weican; Li, Xiaowei; Chen, Huan; Yao, Na; Guan, Lili; Chen, Kai; Cui, Xiyan; Yang, Meiying; Li, Haiyan

2014-01-01

Leymus chinensis (Trin.) Tzvel. is a perennial rhizome grass of the Poaceae (also called Gramineae) family, which adapts well to drought, saline and alkaline conditions. However, little is known about the stress tolerance of L. chinensis at the molecular level. microRNAs (miRNAs) are known to play critical roles in nutrient homeostasis, developmental processes, pathogen responses, and abiotic stress in plants. In this study, we used Solexa sequencing technology to generate high-quality small RNA data from three L. chinensis groups: a control group, a saline-alkaline stress group (100 mM NaCl and 200 mM NaHCO3), and a drought stress group (20% polyethylene glycol 2000). From these data we identified 132 known miRNAs and 16 novel miRNAs candidates. For these miRNAs we also identified target genes that encode a broad range of proteins that may be correlated with abiotic stress regulation. This is the first study to demonstrate differentially expressed miRNAs in L. chinensis under saline-alkali and drought stress. These findings may help explain the saline-alkaline and drought stress responses in L. chinensis. PMID:25369004

Discovery and analysis of microRNAs in Leymus chinensis under saline-alkali and drought stress using high-throughput sequencing.

PubMed

Zhai, Junfeng; Dong, Yuanyuan; Sun, Yepeng; Wang, Qi; Wang, Nan; Wang, Fawei; Liu, Weican; Li, Xiaowei; Chen, Huan; Yao, Na; Guan, Lili; Chen, Kai; Cui, Xiyan; Yang, Meiying; Li, Haiyan

2014-01-01

Leymus chinensis (Trin.) Tzvel. is a perennial rhizome grass of the Poaceae (also called Gramineae) family, which adapts well to drought, saline and alkaline conditions. However, little is known about the stress tolerance of L. chinensis at the molecular level. microRNAs (miRNAs) are known to play critical roles in nutrient homeostasis, developmental processes, pathogen responses, and abiotic stress in plants. In this study, we used Solexa sequencing technology to generate high-quality small RNA data from three L. chinensis groups: a control group, a saline-alkaline stress group (100 mM NaCl and 200 mM NaHCO3), and a drought stress group (20% polyethylene glycol 2000). From these data we identified 132 known miRNAs and 16 novel miRNAs candidates. For these miRNAs we also identified target genes that encode a broad range of proteins that may be correlated with abiotic stress regulation. This is the first study to demonstrate differentially expressed miRNAs in L. chinensis under saline-alkali and drought stress. These findings may help explain the saline-alkaline and drought stress responses in L. chinensis.

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.

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.

Changes in microRNA abundance may regulate diapause in the flesh fly, Sarcophaga bullata.

PubMed

Reynolds, Julie A; Peyton, Justin T; Denlinger, David L

2017-05-01

Diapause, an alternative developmental pathway characterized by changes in developmental timing and metabolism, is coordinated by molecular mechanisms that are not completely understood. MicroRNA (miRNA) mediated gene silencing is emerging as a key component of animal development and may have a significant role in initiating, maintaining, and terminating insect diapause. In the present study, we test this possibility by using high-throughput sequencing and qRT-PCR to discover diapause-related shifts in miRNA abundance in the flesh fly, Sarcophaga bullata. We identified ten evolutionarily conserved miRNAs that were differentially expressed in diapausing pupae compared to their nondiapausing counterparts. miR-289-5p and miR-1-3p were overexpressed in diapausing pupae and may be responsible for silencing expression of candidate genes during diapause. miR-9c-5p, miR-13b-3p, miR-31a-5p, miR-92b-3p, miR-275-3p, miR-276a-3p, miR-277-3p, and miR-305-5p were underexpressed in diapausing pupae and may contribute to increased expression of heat shock proteins and other factors necessary for the enhanced environmental stress-response that is a feature of diapause. In S. bullata, a maternal effect blocks the programming of diapause in progeny of females that have experienced pupal diapause, and in this study we report that several miRNAs, including miR-263a-5p, miR-100-5p, miR-125-5p, and let-7-5p were significantly overexpressed in such nondiapausing flies and may prevent entry into diapause. Together these miRNAs appear to be integral to the molecular processes that mediate entry into diapause. Copyright © 2017. Published by Elsevier Ltd.

The let-7 microRNA interfaces extensively with the translation machinery to regulate cell differentiation

PubMed Central

Ding, Xavier C.; Slack, Frank J.; Großhans, Helge

2010-01-01

MicroRNAs (miRNAs) are noncoding RNAs that regulate numerous target genes through a posttranscriptional mechanism and thus control major developmental pathways. The phylogenetically conserved let-7 miRNA regulates cell proliferation and differentiation, thus functioning as a key regulator of developmental timing in C. elegans and a tumor suppressor gene in humans. Using a reverse genetic screen, we have identified genetic interaction partners of C. elegans let-7, including known and novel potential target genes. Initial identification of several translation initiation factors as suppressors of a let-7 mutation led us to systematically examine genetic interaction between let-7 and the translational machinery, which we found to be widespread. In the presence of wild-type let-7, depletion of the translation initiation factor eIF3 resulted in precocious cell differentiation, suggesting that developmental timing is translationally regulated, possibly by let-7. As overexpression of eIF3 in humans promotes translation of mRNAs that are also targets of let-7-mediated repression, we suggest that eIF3 may directly or indirectly oppose let-7 activity. This might provide an explanation for the opposite functions of let-7 and eIF3 in regulating tumorigenesis. PMID:18818519

Characterisation of mouse epididymosomes reveals a complex profile of microRNAs and a potential mechanism for modification of the sperm epigenome

PubMed Central

Reilly, Jackson N.; McLaughlin, Eileen A.; Stanger, Simone J.; Anderson, Amanda L.; Hutcheon, Kate; Church, Kiralee; Mihalas, Bettina P.; Tyagi, Sonika; Holt, Janet E.; Eamens, Andrew L.; Nixon, Brett

2016-01-01

Recent evidence has shown that the sperm epigenome is vulnerable to dynamic modifications arising from a variety of paternal environment exposures and that this legacy can serve as an important determinant of intergenerational inheritance. It has been postulated that such exchange is communicated to maturing spermatozoa via the transfer of small non-protein-coding RNAs (sRNAs) in a mechanism mediated by epididymosomes; small membrane bound vesicles released by the soma of the male reproductive tract (epididymis). Here we confirm that mouse epididymosomes encapsulate an impressive cargo of >350 microRNAs (miRNAs), a developmentally important sRNA class, the majority (~60%) of which are also represented by the miRNA signature of spermatozoa. This includes >50 miRNAs that were found exclusively in epididymal sperm and epididymosomes, but not in the surrounding soma. We also documented substantial changes in the epididymosome miRNA cargo, including significant fold changes in almost half of the miRNAs along the length of the epididymis. Finally, we provide the first direct evidence for the transfer of several prominent miRNA species between mouse epididymosomes and spermatozoa to afford novel insight into a mechanism of intercellular communication by which the sRNA payload of sperm can be selectively modified during their post-testicular maturation. PMID:27549865

Human embryonic stem cells express a unique set of microRNAs.

PubMed

Suh, Mi-Ra; Lee, Yoontae; Kim, Jung Yeon; Kim, Soo-Kyoung; Moon, Sung-Hwan; Lee, Ji Yeon; Cha, Kwang-Yul; Chung, Hyung Min; Yoon, Hyun Soo; Moon, Shin Yong; Kim, V Narry; Kim, Kye-Seong

2004-06-15

Human embryonic stem (hES) cells are pluripotent cell lines established from the explanted inner cell mass of human blastocysts. Despite their importance for human embryology and regenerative medicine, studies on hES cells, unlike those on mouse ES (mES) cells, have been hampered by difficulties in culture and by scant knowledge concerning the regulatory mechanism. Recent evidence from plants and animals indicates small RNAs of approximately 22 nucleotides (nt), collectively named microRNAs, play important roles in developmental regulation. Here we describe 36 miRNAs (from 32 stem-loops) identified by cDNA cloning in hES cells. Importantly, most of the newly cloned miRNAs are specifically expressed in hES cells and downregulated during development into embryoid bodies (EBs), while miRNAs previously reported from other human cell types are poorly expressed in hES cells. We further show that some of the ES-specific miRNA genes are highly related to each other, organized as clusters, and transcribed as polycistronic primary transcripts. These miRNA gene families have murine homologues that have similar genomic organizations and expression patterns, suggesting that they may operate key regulatory networks conserved in mammalian pluripotent stem cells. The newly identified hES-specific miRNAs may also serve as molecular markers for the early embryonic stage and for undifferentiated hES cells.

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.

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

MicroRNAs enriched in hematopoietic stem cells differentially regulate long-term hematopoietic output.

PubMed

O'Connell, Ryan M; Chaudhuri, Aadel A; Rao, Dinesh S; Gibson, William S J; Balazs, Alejandro B; Baltimore, David

2010-08-10

The production of blood cells depends on a rare hematopoietic stem-cell (HSC) population, but the molecular mechanisms underlying HSC biology remain incompletely understood. Here, we identify a subset of microRNAs (miRNAs) that is enriched in HSCs compared with other bone-marrow cells. An in vivo gain-of-function screen found that three of these miRNAs conferred a competitive advantage to engrafting hematopoietic cells, whereas other HSC miRNAs attenuated production of blood cells. Overexpression of the most advantageous miRNA, miR-125b, caused a dose-dependent myeloproliferative disorder that progressed to a lethal myeloid leukemia in mice and also enhanced hematopoietic engraftment in human immune system mice. Our study identifies an evolutionarily conserved subset of miRNAs that is expressed in HSCs and functions to modulate hematopoietic output.

Identification of MicroRNA Targets of Capsicum spp. Using MiRTrans—a Trans-Omics Approach

PubMed Central

Zhang, Lu; Qin, Cheng; Mei, Junpu; Chen, Xiaocui; Wu, Zhiming; Luo, Xirong; Cheng, Jiaowen; Tang, Xiangqun; Hu, Kailin; Li, Shuai C.

2017-01-01

The microRNA (miRNA) can regulate the transcripts that are involved in eukaryotic cell proliferation, differentiation, and metabolism. Especially for plants, our understanding of miRNA targets, is still limited. Early attempts of prediction on sequence alignments have been plagued by enormous false positives. It is helpful to improve target prediction specificity by incorporating the other data sources such as the dependency between miRNA and transcript expression or even cleaved transcripts by miRNA regulations, which are referred to as trans-omics data. In this paper, we developed MiRTrans (Prediction of MiRNA targets by Trans-omics data) to explore miRNA targets by incorporating miRNA sequencing, transcriptome sequencing, and degradome sequencing. MiRTrans consisted of three major steps. First, the target transcripts of miRNAs were predicted by scrutinizing their sequence characteristics and collected as an initial potential targets pool. Second, false positive targets were eliminated if the expression of miRNA and its targets were weakly correlated by lasso regression. Third, degradome sequencing was utilized to capture the miRNA targets by examining the cleaved transcripts that regulated by miRNAs. Finally, the predicted targets from the second and third step were combined by Fisher's combination test. MiRTrans was applied to identify the miRNA targets for Capsicum spp. (i.e., pepper). It can generate more functional miRNA targets than sequence-based predictions by evaluating functional enrichment. MiRTrans identified 58 miRNA-transcript pairs with high confidence from 18 miRNA families conserved in eudicots. Most of these targets were transcription factors; this lent support to the role of miRNA as key regulator in pepper. To our best knowledge, this work is the first attempt to investigate the miRNA targets of pepper, as well as their regulatory networks. Surprisingly, only a small proportion of miRNA-transcript pairs were shared between degradome sequencing and expression dependency predictions, suggesting that miRNA targets predicted by a single technology alone may be prone to report false negatives. PMID:28443105

Common developmental genome deprogramming in schizophrenia - Role of Integrative Nuclear FGFR1 Signaling (INFS).

PubMed

Narla, S T; Lee, Y-W; Benson, C A; Sarder, P; Brennand, K J; Stachowiak, E K; Stachowiak, M K

2017-07-01

The watershed-hypothesis of schizophrenia asserts that over 200 different mutations dysregulate distinct pathways that converge on an unspecified common mechanism(s) that controls disease ontogeny. Consistent with this hypothesis, our RNA-sequencing of neuron committed cells (NCCs) differentiated from established iPSCs of 4 schizophrenia patients and 4 control subjects uncovered a dysregulated transcriptome of 1349 mRNAs common to all patients. Data reveals a global dysregulation of developmental genome, deconstruction of coordinated mRNA networks, and the formation of aberrant, new coordinated mRNA networks indicating a concerted action of the responsible factor(s). Sequencing of miRNA transcriptomes demonstrated an overexpression of 16 miRNAs and deconstruction of interactive miRNA-mRNA networks in schizophrenia NCCs. ChiPseq revealed that the nuclear (n) form of FGFR1, a pan-ontogenic regulator, is overexpressed in schizophrenia NCCs and overtargets dysregulated mRNA and miRNA genes. The nFGFR1 targeted 54% of all human gene promoters and 84.4% of schizophrenia dysregulated genes. The upregulated genes reside within major developmental pathways that control neurogenesis and neuron formation, whereas downregulated genes are involved in oligodendrogenesis. Our results indicate (i) an early (preneuronal) genomic etiology of schizophrenia, (ii) dysregulated genes and new coordinated gene networks are common to unrelated cases of schizophrenia, (iii) gene dysregulations are accompanied by increased nFGFR1-genome interactions, and (iv) modeling of increased nFGFR1 by an overexpression of a nFGFR1 lead to up or downregulation of selected genes as observed in schizophrenia NCCs. Together our results designate nFGFR1 signaling as a potential common dysregulated mechanism in investigated patients and potential therapeutic target in schizophrenia. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Hypoxia regulates microRNA expression in the human carotid body

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

Mkrtchian, Souren, E-mail: souren.mkrtchian@ki.se; Lee, Kian Leong, E-mail: csilkl@nus.edu.sg; Kåhlin, Jessica

The carotid body (CB) is the key sensing organ for physiological oxygen levels in the body. Under conditions of low oxygen (hypoxia), the CB plays crucial roles in signaling to the cardiorespiratory center in the medulla oblongata for the restoration of oxygen homeostasis. How hypoxia regulates gene expression in the human CB remains poorly understood. While limited information on transcriptional regulation in animal CBs is available, the identity and impact of important post-transcriptional regulators such as non-coding RNAs, and in particular miRNAs are not known. Here we show using ex vivo experiments that indeed a number of miRNAs are differentiallymore » regulated in surgically removed human CB slices when acute hypoxic conditions were applied. Analysis of the hypoxia-regulated miRNAs shows that they target biological pathways with upregulation of functions related to cell proliferation and immune response and downregulation of cell differentiation and cell death functions. Comparative analysis of the human CB miRNAome with the global miRNA expression patterns of a large number of different human tissues showed that the CB miRNAome had a unique profile which reflects its highly specialized functional status. Nevertheless, the human CB miRNAome is most closely related to the miRNA expression pattern of brain tissues indicating that they may have the most similar developmental origins. - Highlights: • Hypoxia triggers differential expression of many miRNAs in the human carotid body. • This can lead to the upregulation of proliferation and immune response functions. • CB expression profile in the carotid body resembles the miRNA expression pattern in the brain. • miRNAs are involved in the regulation of carotid body functions including oxygen sensing.« less

The microRNA signature of mouse spermatozoa is substantially modified during epididymal maturation.

PubMed

Nixon, Brett; Stanger, Simone J; Mihalas, Bettina P; Reilly, Jackson N; Anderson, Amanda L; Tyagi, Sonika; Holt, Janet E; McLaughlin, Eileen A

2015-10-01

In recent years considerable effort has been devoted to understanding the epigenetic control of sperm development, leading to an increased appreciation of the importance of RNA interference pathways, and in particular miRNAs, as key regulators of spermatogenesis and epididymal maturation. It has also been shown that sperm are endowed with an impressive array of miRNA that have been implicated in various aspects of fertilization and embryo development. However, to date there have been no reports on whether the sperm miRNA signature is static or whether it is influenced by their prolonged maturation within the male reproductive tract. To investigate this phenomenon, we employed next-generation sequencing to systematically profile the miRNA signature of maturing mouse spermatozoa. In so doing we have provided the first evidence for the posttesticular modification of the sperm miRNA profile under normal physiological conditions. Such modifications include the apparent loss and acquisition of an impressive cohort of some 113 and 115 miRNAs, respectively, between the proximal and distal epididymal segments. Interestingly, the majority of these changes occur late in maturation and include the uptake of novel miRNA species in addition to a significant increase in many miRNAs natively expressed in immature sperm. Because sperm are not capable of de novo transcription, these findings identify the epididymis as an important site in establishing the sperm epigenome with the potential to influence the peri-conceptual environment of the female reproductive tract, contribute to the inheritance of acquired characteristics, and/or alter the developmental trajectory of the resulting offspring. © 2015 by the Society for the Study of Reproduction, Inc.

Small RNA profiling and degradome analysis reveal regulation of microRNA in peanut embryogenesis and early pod development.

PubMed

Gao, Chao; Wang, Pengfei; Zhao, Shuzhen; Zhao, Chuanzhi; Xia, Han; Hou, Lei; Ju, Zheng; Zhang, Ye; Li, Changsheng; Wang, Xingjun

2017-03-02

As a typical geocarpic plant, peanut embryogenesis and pod development are complex processes involving many gene regulatory pathways and controlled by appropriate hormone level. MicroRNAs (miRNAs) are small non-coding RNAs that play indispensable roles in post-transcriptional gene regulation. Recently, identification and characterization of peanut miRNAs has been described. However, whether miRNAs participate in the regulation of peanut embryogenesis and pod development has yet to be explored. In this study, small RNA and degradome libraries from peanut early pod of different developmental stages were constructed and sequenced. A total of 70 known and 24 novel miRNA families were discovered. Among them, 16 miRNA families were legume-specific and 12 families were peanut-specific. 30 known and 10 novel miRNA families were differentially expressed during pod development. In addition, 115 target genes were identified for 47 miRNA families by degradome sequencing. Several new targets that might be specific to peanut were found and further validated by RNA ligase-mediated rapid amplification of 5' cDNA ends (RLM 5'-RACE). Furthermore, we performed profiling analysis of intact and total transcripts of several target genes, demonstrating that SPL (miR156/157), NAC (miR164), PPRP (miR167 and miR1088), AP2 (miR172) and GRF (miR396) are actively modulated during early pod development, respectively. Large numbers of miRNAs and their related target genes were identified through deep sequencing. These findings provided new information on miRNA-mediated regulatory pathways in peanut pod, which will contribute to the comprehensive understanding of the molecular mechanisms that governing peanut embryo and early pod development.

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

PubMed Central

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. PMID:28450878

Identification and Characterization of FGF2-Dependent mRNA: microRNA Networks During Lens Fiber Cell Differentiation

PubMed Central

Wolf, Louise; Gao, Chun S.; Gueta, Karen; Xie, Qing; Chevallier, Tiphaine; Podduturi, Nikhil R.; Sun, Jian; Conte, Ivan; Zelenka, Peggy S.; Ashery-Padan, Ruth; Zavadil, Jiri; Cvekl, Ales

2013-01-01

MicroRNAs (miRNAs) and fibroblast growth factor (FGF) signaling regulate a wide range of cellular functions, including cell specification, proliferation, migration, differentiation, and survival. In lens, both these systems control lens fiber cell differentiation; however, a possible link between these processes remains to be examined. Herein, the functional requirement for miRNAs in differentiating lens fiber cells was demonstrated via conditional inactivation of Dicer1 in mouse (Mus musculus) lens. To dissect the miRNA-dependent pathways during lens differentiation, we used a rat (Rattus norvegicus) lens epithelial explant system, induced by FGF2 to differentiate, followed by mRNA and miRNA expression profiling. Transcriptome and miRNome analysis identified extensive FGF2-regulated cellular responses that were both independent and dependent on miRNAs. We identified 131 FGF2-regulated miRNAs. Seventy-six of these miRNAs had at least two in silico predicted and inversely regulated target mRNAs. Genes modulated by the greatest number of FGF-regulated miRNAs include DNA-binding transcription factors Nfib, Nfat5/OREBP, c-Maf, Ets1, and N-Myc. Activated FGF signaling influenced bone morphogenetic factor/transforming growth factor-β, Notch, and Wnt signaling cascades implicated earlier in lens differentiation. Specific miRNA:mRNA interaction networks were predicted for c-Maf, N-Myc, and Nfib (DNA-binding transcription factors); Cnot6, Cpsf6, Dicer1, and Tnrc6b (RNA to miRNA processing); and Ash1l, Med1/PBP, and Kdm5b/Jarid1b/Plu1 (chromatin remodeling). Three miRNAs, including miR-143, miR-155, and miR-301a, down-regulated expression of c-Maf in the 3′-UTR luciferase reporter assays. These present studies demonstrate for the first time global impact of activated FGF signaling in lens cell culture system and predicted novel gene regulatory networks connected by multiple miRNAs that regulate lens differentiation. PMID:24142921

Site-Dependent Differences in DNA Methylation and Their Impact on Plant Establishment and Phosphorus Nutrition in Populus trichocarpa.

PubMed

Schönberger, Brigitte; Chen, Xiaochao; Mager, Svenja; Ludewig, Uwe

2016-01-01

The propagation via clonal stem cuttings is a frequent practice in tree plantations. Despite their clonal origin, the trees establish differently according to weather, temperature and nutrient availability, as well as the presence of various stresses. Here, clonal Populus trichocarpa (cv. Muhle Larson) cuttings from different sites were transferred into a common, fully nutrient supplied environment. Despite identical underlying genetics, stem cuttings derived from sites with lower phosphorus availability established worse, independent of phosphorus (P) level after transplantation. Differential growth of material from the sites was reflected in differences in the whole genome DNA methylome. Methylation differences were sequence context-dependent, but differentially methylated regions (DMRs) were apparently unrelated to P nutrition genes. Despite the undisputed negative general correlation of DNA promoter methylation with gene repression, only few of the top-ranked DMRs resulted in differential gene expression in roots or shoots. However, differential methylation was associated with site-dependent, different total amounts of microRNAs (miRNAs), with few miRNAs sequences directly targeted by differential methylation. Interestingly, in roots and shoots, the miRNA amount was dependent on the previous habitat and changed in roots in a habitat-dependent way under phosphate starvation conditions. Differentially methylated miRNAs, together with their target genes, showed P-dependent expression profiles, indicating miRNA expression differences as a P-related epigenetic modification in poplar. Together with differences in DNA methylation, such epigenetic mechanisms may explain habitat or seasonal memory in perennials and site-dependent growth performances.

Regional and subtype-dependent miRNA signatures in sporadic Creutzfeldt-Jakob disease are accompanied by alterations in miRNA silencing machinery and biogenesis

PubMed Central

Kanata, Eirini; Dafou, Dimitra; Díaz-Lucena, Daniela; Vivancos, Ana; Shomroni, Orr; Zafar, Saima; Schmitz, Matthias; Fernández-Borges, Natalia; Andréoletti, Olivier; Díez, Juana; Fischer, Andre; Sklaviadis, Theodoros; Ferrer, Isidre; Zerr, Inga

2018-01-01

Increasing evidence indicates that microRNAs (miRNAs) are contributing factors to neurodegeneration. Alterations in miRNA signatures have been reported in several neurodegenerative dementias, but data in prion diseases are restricted to ex vivo and animal models. The present study identified significant miRNA expression pattern alterations in the frontal cortex and cerebellum of sporadic Creutzfeldt-Jakob disease (sCJD) patients. These changes display a highly regional and disease subtype-dependent regulation that correlates with brain pathology. We demonstrate that selected miRNAs are enriched in sCJD isolated Argonaute(Ago)-binding complexes in disease, indicating their incorporation into RNA-induced silencing complexes, and further suggesting their contribution to disease-associated gene expression changes. Alterations in the miRNA-mRNA regulatory machinery and perturbed levels of miRNA biogenesis key components in sCJD brain samples reported here further implicate miRNAs in sCJD gene expression (de)regulation. We also show that a subset of sCJD-altered miRNAs are commonly changed in Alzheimer’s disease, dementia with Lewy bodies and fatal familial insomnia, suggesting potential common mechanisms underlying these neurodegenerative processes. Additionally, we report no correlation between brain and cerebrospinal fluid (CSF) miRNA-profiles in sCJD, indicating that CSF-miRNA profiles do not faithfully mirror miRNA alterations detected in brain tissue of human prion diseases. Finally, utilizing a sCJD MM1 mouse model, we analyzed the miRNA deregulation patterns observed in sCJD in a temporal manner. While fourteen sCJD-related miRNAs were validated at clinical stages, only two of those were changed at early symptomatic phase, suggesting that the miRNAs altered in sCJD may contribute to later pathogenic processes. Altogether, the present work identifies alterations in the miRNA network, biogenesis and miRNA-mRNA silencing machinery in sCJD, whereby contributions to disease mechanisms deserve further investigation. PMID:29357384

Regional and subtype-dependent miRNA signatures in sporadic Creutzfeldt-Jakob disease are accompanied by alterations in miRNA silencing machinery and biogenesis.

PubMed

Llorens, Franc; Thüne, Katrin; Martí, Eulàlia; Kanata, Eirini; Dafou, Dimitra; Díaz-Lucena, Daniela; Vivancos, Ana; Shomroni, Orr; Zafar, Saima; Schmitz, Matthias; Michel, Uwe; Fernández-Borges, Natalia; Andréoletti, Olivier; Del Río, José Antonio; Díez, Juana; Fischer, Andre; Bonn, Stefan; Sklaviadis, Theodoros; Torres, Juan Maria; Ferrer, Isidre; Zerr, Inga

2018-01-01

Increasing evidence indicates that microRNAs (miRNAs) are contributing factors to neurodegeneration. Alterations in miRNA signatures have been reported in several neurodegenerative dementias, but data in prion diseases are restricted to ex vivo and animal models. The present study identified significant miRNA expression pattern alterations in the frontal cortex and cerebellum of sporadic Creutzfeldt-Jakob disease (sCJD) patients. These changes display a highly regional and disease subtype-dependent regulation that correlates with brain pathology. We demonstrate that selected miRNAs are enriched in sCJD isolated Argonaute(Ago)-binding complexes in disease, indicating their incorporation into RNA-induced silencing complexes, and further suggesting their contribution to disease-associated gene expression changes. Alterations in the miRNA-mRNA regulatory machinery and perturbed levels of miRNA biogenesis key components in sCJD brain samples reported here further implicate miRNAs in sCJD gene expression (de)regulation. We also show that a subset of sCJD-altered miRNAs are commonly changed in Alzheimer's disease, dementia with Lewy bodies and fatal familial insomnia, suggesting potential common mechanisms underlying these neurodegenerative processes. Additionally, we report no correlation between brain and cerebrospinal fluid (CSF) miRNA-profiles in sCJD, indicating that CSF-miRNA profiles do not faithfully mirror miRNA alterations detected in brain tissue of human prion diseases. Finally, utilizing a sCJD MM1 mouse model, we analyzed the miRNA deregulation patterns observed in sCJD in a temporal manner. While fourteen sCJD-related miRNAs were validated at clinical stages, only two of those were changed at early symptomatic phase, suggesting that the miRNAs altered in sCJD may contribute to later pathogenic processes. Altogether, the present work identifies alterations in the miRNA network, biogenesis and miRNA-mRNA silencing machinery in sCJD, whereby contributions to disease mechanisms deserve further investigation.

Defining Transcriptional Regulatory Mechanisms for Primary let-7 miRNAs

PubMed Central

Gaeta, Xavier; Le, Luat; Lin, Ying; Xie, Yuan; Lowry, William E.

2017-01-01

The let-7 family of miRNAs have been shown to control developmental timing in organisms from C. elegans to humans; their function in several essential cell processes throughout development is also well conserved. Numerous studies have defined several steps of post-transcriptional regulation of let-7 production; from pri-miRNA through pre-miRNA, to the mature miRNA that targets endogenous mRNAs for degradation or translational inhibition. Less-well defined are modes of transcriptional regulation of the pri-miRNAs for let-7. let-7 pri-miRNAs are expressed in polycistronic fashion, in long transcripts newly annotated based on chromatin-associated RNA-sequencing. Upon differentiation, we found that some let-7 pri-miRNAs are regulated at the transcriptional level, while others appear to be constitutively transcribed. Using the Epigenetic Roadmap database, we further annotated regulatory elements of each polycistron identified putative promoters and enhancers. Probing these regulatory elements for transcription factor binding sites identified factors that regulate transcription of let-7 in both promoter and enhancer regions, and identified novel regulatory mechanisms for this important class of miRNAs. PMID:28052101

microRNA and Autism.

PubMed

Anitha, Ayyappan; Thanseem, Ismail

2015-01-01

Autism is a complex neurodevelopmental disorder characterized by deficiencies in social interaction and communication, and by repetitive and stereotyped behaviors. According to a recent report, the prevalence of this pervasive developmental disorder has risen to 1 in 88. This will have enormous public health implications in the future, and has necessitated the need to discover predictive biomarkers that could index for autism before the onset of symptoms. microRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression at the posttranscriptional level. They have recently emerged as prominent epigenetic regulators of various cellular processes including neurodevelopment. They are abundantly present in the brain, and their dysfunction has been implicated in an array of neuropathological conditions including autism. miRNAs, previously known to be expressed only in cells and tissues, have also been detected in extracellular body fluids such as serum, plasma, saliva, and urine. Altered expression of cellular and circulating miRNAs have been observed in autistic individuals compared to healthy controls. miRNAs are now being considered as potential targets for the development of novel therapeutic strategies for autism.

Tissue slide-based microRNA characterization of tumors: how detailed could diagnosis become for cancer medicine?

PubMed Central

Sempere, Lorenzo F

2014-01-01

miRNAs are short, non-coding, regulatory RNAs that exert cell type-dependent, context-dependent, transcriptome-wide gene expression control under physiological and pathological conditions. Tissue slide-based assays provide qualitative (tumor compartment) and semi-quantitative (expression levels) information about altered miRNA expression at single-cell resolution in clinical tumor specimens. Reviewed here are key technological advances in the last 5 years that have led to implementation of fully automated, robust and reproducible tissue slide-based assays for in situ miRNA detection on US FDA-approved instruments; recent tissue slide-based discovery studies that suggest potential clinical applications of specific miRNAs in cancer medicine are highlighted; and the challenges in bringing tissue slide-based miRNA assays into the clinic are discussed, including clinical validation, biomarker performance, biomarker space and integration with other biomarkers. PMID:25090088

Tuning of major signaling networks (TGF-β, Wnt, Notch and Hedgehog) by miRNAs in human stem cells commitment to different lineages: Possible clinical application.

PubMed

Aval, Sedigheh Fekri; Lotfi, Hajie; Sheervalilou, Roghayeh; Zarghami, Nosratollah

2017-07-01

Two distinguishing characteristics of stem cells, their continuous division in the undifferentiated state and growth into any cell types, are orchestrated by a number of cell signaling pathways. These pathways act as a niche factor in controlling variety of stem cells. The core stem cell signaling pathways include Wingless-type (Wnt), Hedgehog (HH), and Notch. Additionally, they critically regulate the self-renewal and survival of cancer stem cells. Conversely, stem cells' main properties, lineage commitment and stemness, are tightly controlled by epigenetic mechanisms such as DNA methylation, histone modifications and non-coding RNA-mediated regulatory events. MicroRNAs (miRNAs) are cellular switches that modulate stem cells outcomes in response to diverse extracellular signals. Numerous scientific evidences implicating miRNAs in major signal transduction pathways highlight new crosstalks of cellular processes. Aberrant signaling pathways and miRNAs levels result in developmental defects and diverse human pathologies. This review discusses the crosstalk between the components of main signaling networks and the miRNA machinery, which plays a role in the context of stem cells development and provides a set of examples to illustrate the extensive relevance of potential novel therapeutic targets. Copyright © 2017. Published by Elsevier Masson SAS.

MicroRNAs and atherosclerosis: new actors for an old movie.

PubMed

Santovito, D; Mezzetti, A; Cipollone, F

2012-11-01

To date, cardiovascular diseases (CVDs) are the leading causes of morbidity and mortality worldwide. MicroRNAs (miRNAs) are endogenous, short, non-coding RNA sequences able to regulate gene expression principally at the post-transcriptional level. Initially, they were thought to be involved only in developmental timing of worms. Their involvement in human biology was recently discovered and many studies have been performed to demonstrate the role of miRNA in human cancer. Since the first observation in 2005 of their implication in cardiac biology, many studies have demonstrated their role in the genetic modulation of cardiovascular development and in cardiovascular diseases such as cardial remodeling and heart failure, cardiac arrhythmias, cardiac ischaemia, cardiac fibrosis, atherosclerosis and stroke. Thus, the aim of this review is to describe the role of miRNA in atherosclerosis development and evolution and to individuate their role as potential therapeutic target. Copyright © 2012 Elsevier B.V. All rights reserved.

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

Listeria monocytogenes Induces a Virulence-Dependent microRNA Signature That Regulates the Immune Response in Galleria mellonella

PubMed Central

Mannala, Gopala K.; Izar, Benjamin; Rupp, Oliver; Schultze, Tilman; Goesmann, Alexander; Chakraborty, Trinad; Hain, Torsten

2017-01-01

microRNAs (miRNAs) coordinate several physiological and pathological processes by regulating the fate of mRNAs. Studies conducted in vitro indicate a role of microRNAs in the control of host-microbe interactions. However, there is limited understanding of miRNA functions in in vivo models of bacterial infections. In this study, we systematically explored changes in miRNA expression levels of Galleria mellonella larvae (greater-wax moth), a model system that recapitulates the vertebrate innate immunity, following infection with L. monocytogenes. Using an insect-specific miRNA microarray with more than 2000 probes, we found differential expression of 90 miRNAs (39 upregulated and 51 downregulated) in response to infection with L. monocytogenes. We validated the expression of a subset of miRNAs which have mammalian homologs of known or predicted function. In contrast, non-pathogenic L. innocua failed to induce these miRNAs, indicating a virulence-dependent miRNA deregulation. To predict miRNA targets using established algorithms, we generated a publically available G. mellonella transcriptome database. We identified miRNA targets involved in innate immunity, signal transduction and autophagy, including spätzle, MAP kinase, and optineurin, respectively, which exhibited a virulence-specific differential expression. Finally, in silico estimation of minimum free energy of miRNA-mRNA duplexes of validated microRNAs and target transcripts revealed a regulatory network of the host immune response to L. monocytogenes. In conclusion, this study provides evidence for a role of miRNAs in the regulation of the innate immune response following bacterial infection in a simple, rapid and scalable in vivo model that may predict host-microbe interactions in higher vertebrates. PMID:29312175

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.

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.

Discovery of novel and differentially expressed microRNAs between fetal and adult backfat in cattle.

PubMed

Sun, Jiajie; Zhou, Yang; Cai, Hanfang; Lan, Xianyong; Lei, Chuzhao; Zhao, Xin; Zhang, Chunlei; Chen, Hong

2014-01-01

The posttranscriptional gene regulation mediated by microRNAs (miRNAs) plays an important role in various species. Recently, a large number of miRNAs and their expression patterns have been identified. However, to date, limited miRNAs have been reported to modulate adipogenesis and lipid deposition in beef cattle. Total RNAs from Chinese Qinchuan bovine backfat at fetal and adult stages were used to construct small RNA libraries for Illumina next-generation sequencing. A total of 13,915,411 clean reads were obtained from a fetal library and 14,244,946 clean reads from an adult library. In total, 475 known and 36 novel miRNA candidates from backfat were identified. The nucleotide bias, base editing, and family of the known miRNAs were also analyzed. Based on stem-loop qPCR, 15 specific miRNAs were detected, and the results showed that bta-miRNAn25 and miRNAn26 were highly expressed in backfat tissue, suggesting these small RNAs play a role in the development and maintenance of bovine subcutaneous fat tissue. Putative targets for miRNAn25 and miRNAn26 were predicted, and the 61 most significant target transcripts were related to lipid and fatty acid metabolism. Of interest, the canonical pathway and gene networks analyses revealed that PPARα/RXRα activation and LXR/RXR activation were important components of the gene interaction hierarchy results. In the present study, we explored the backfat miRNAome differences between cattle of different developmental stages, expanding the expression repertoire of bovine miRNAs that could contribute to further studies on the fat development of cattle. Predication of target genes analysis of miRNA25 and miRNA26 also showed potential gene networks that affect lipid and fatty acid metabolism. These results may help in the design of new intervention strategies to improve beef quality.

Discovery of Novel and Differentially Expressed MicroRNAs between Fetal and Adult Backfat in Cattle

PubMed Central

Sun, Jiajie; Zhou, Yang; Cai, Hanfang; Lan, Xianyong; Lei, Chuzhao; Zhao, Xin; Zhang, Chunlei; Chen, Hong

2014-01-01

The posttranscriptional gene regulation mediated by microRNAs (miRNAs) plays an important role in various species. Recently, a large number of miRNAs and their expression patterns have been identified. However, to date, limited miRNAs have been reported to modulate adipogenesis and lipid deposition in beef cattle. Total RNAs from Chinese Qinchuan bovine backfat at fetal and adult stages were used to construct small RNA libraries for Illumina next-generation sequencing. A total of 13,915,411 clean reads were obtained from a fetal library and 14,244,946 clean reads from an adult library. In total, 475 known and 36 novel miRNA candidates from backfat were identified. The nucleotide bias, base editing, and family of the known miRNAs were also analyzed. Based on stem-loop qPCR, 15 specific miRNAs were detected, and the results showed that bta-miRNAn25 and miRNAn26 were highly expressed in backfat tissue, suggesting these small RNAs play a role in the development and maintenance of bovine subcutaneous fat tissue. Putative targets for miRNAn25 and miRNAn26 were predicted, and the 61 most significant target transcripts were related to lipid and fatty acid metabolism. Of interest, the canonical pathway and gene networks analyses revealed that PPARα/RXRα activation and LXR/RXR activation were important components of the gene interaction hierarchy results. In the present study, we explored the backfat miRNAome differences between cattle of different developmental stages, expanding the expression repertoire of bovine miRNAs that could contribute to further studies on the fat development of cattle. Predication of target genes analysis of miRNA25 and miRNA26 also showed potential gene networks that affect lipid and fatty acid metabolism. These results may help in the design of new intervention strategies to improve beef quality. PMID:24587298

Site-Dependent Differences in DNA Methylation and Their Impact on Plant Establishment and Phosphorus Nutrition in Populus trichocarpa

PubMed Central

Schönberger, Brigitte; Chen, Xiaochao; Mager, Svenja

2016-01-01

The propagation via clonal stem cuttings is a frequent practice in tree plantations. Despite their clonal origin, the trees establish differently according to weather, temperature and nutrient availability, as well as the presence of various stresses. Here, clonal Populus trichocarpa (cv. Muhle Larson) cuttings from different sites were transferred into a common, fully nutrient supplied environment. Despite identical underlying genetics, stem cuttings derived from sites with lower phosphorus availability established worse, independent of phosphorus (P) level after transplantation. Differential growth of material from the sites was reflected in differences in the whole genome DNA methylome. Methylation differences were sequence context-dependent, but differentially methylated regions (DMRs) were apparently unrelated to P nutrition genes. Despite the undisputed negative general correlation of DNA promoter methylation with gene repression, only few of the top-ranked DMRs resulted in differential gene expression in roots or shoots. However, differential methylation was associated with site-dependent, different total amounts of microRNAs (miRNAs), with few miRNAs sequences directly targeted by differential methylation. Interestingly, in roots and shoots, the miRNA amount was dependent on the previous habitat and changed in roots in a habitat-dependent way under phosphate starvation conditions. Differentially methylated miRNAs, together with their target genes, showed P-dependent expression profiles, indicating miRNA expression differences as a P-related epigenetic modification in poplar. Together with differences in DNA methylation, such epigenetic mechanisms may explain habitat or seasonal memory in perennials and site-dependent growth performances. PMID:27992519

microRNAs of parasites: current status and future perspectives

USDA-ARS?s Scientific Manuscript database

MicroRNAs (miRNAs) are a class of endogenous non-coding small RNAs regulating gene expression in eukaryotes at the post-transcriptional level. The complex life cycles of parasites may require the ability to respond to environmental and developmental signals through miRNA-mediated gene expression. Ov...

Analysis of high iron rice lines reveals new miRNAs that target iron transporters in roots

PubMed Central

Paul, Soumitra; Gayen, Dipak; Datta, Swapan K.; Datta, Karabi

2016-01-01

The present study highlights the molecular regulation of iron transport in soyFER1-overexpressing transgenic rice. Accumulation of iron in three different seed developmental stages, milk, dough, and mature, has been examined. The transgenic seeds of the milk stage showed significant augmentation of iron and zinc levels compared with wild-type seeds, and similar results were observed throughout the dough and mature stages. To investigate the regulation of iron transport, the role of miRNAs was studied in roots of transgenic rice. Sequencing of small RNA libraries revealed 153 known and 41 novel miRNAs in roots. Among them, 59 known and 14 novel miRNAs were found to be significantly expressed. miR166, miR399, and miR408 were identified as playing a vital role in iron uptake in roots of transgenic plants . Most importantly, four putative novel miRNAs, namely miR11, miR26, miR30, and miR31, were found to be down-regulated in roots of transgenic plants. For all these four novel miRNAs, natural resistance-associated macrophage protein 4 (NRAMP4), encoding a metal transporter, was predicted as a target gene. It is hypothesized that the NRAMP4 transporter is activated in roots of transgenic plants due to the lower abundance of its corresponding putative novel miRNAs. The relative transcript level of the NRAMP4 transcript was increased from 0.107 in the wild type to 65.24 and 55.39 in transgenic plants, which demonstrates the elevated amount of iron transport in transgenic plants. In addition, up-regulation of OsYSL15, OsFRO2, and OsIRT1 in roots also facilitates iron loading in transgenic seeds. PMID:27729476

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

ARMOUR - A Rice miRNA: mRNA Interaction Resource.

PubMed

Sanan-Mishra, Neeti; Tripathi, Anita; Goswami, Kavita; Shukla, Rohit N; Vasudevan, Madavan; Goswami, Hitesh

2018-01-01

ARMOUR was developed as A Rice miRNA:mRNA interaction resource. This informative and interactive database includes the experimentally validated expression profiles of miRNAs under different developmental and abiotic stress conditions across seven Indian rice cultivars. This comprehensive database covers 689 known and 1664 predicted novel miRNAs and their expression profiles in more than 38 different tissues or conditions along with their predicted/known target transcripts. The understanding of miRNA:mRNA interactome in regulation of functional cellular machinery is supported by the sequence information of the mature and hairpin structures. ARMOUR provides flexibility to users in querying the database using multiple ways like known gene identifiers, gene ontology identifiers, KEGG identifiers and also allows on the fly fold change analysis and sequence search query with inbuilt BLAST algorithm. ARMOUR database provides a cohesive platform for novel and mature miRNAs and their expression in different experimental conditions and allows searching for their interacting mRNA targets, GO annotation and their involvement in various biological pathways. The ARMOUR database includes a provision for adding more experimental data from users, with an aim to develop it as a platform for sharing and comparing experimental data contributed by research groups working on rice.

MicroRNAs (miRNAs) as biomarker(s) for prognosis and diagnosis of gastrointestinal (GI) cancers.

PubMed

Macha, Muzafar A; Seshacharyulu, Parthasarathy; Krishn, Shiv Ram; Pai, Priya; Rachagani, Satyanarayana; Jain, Maneesh; Batra, Surinder K

2014-01-01

Gastrointestinal (GI) cancers remain one of the most common malignancies and are the second common cause of cancer deaths worldwide. The limited effectiveness of therapy for patients with advanced stage and recurrent disease is a reflection of an incomplete understanding of the molecular basis of GI carcinogenesis. Major advancements have improved our understanding of pathology and pathogenesis of GI cancers, but high mortality rates, unfavorable prognosis and lack of clinical predictive biomarkers provide an impetus to investigate new sensitive and specific diagnostic and prognostic markers for GI cancers. MicroRNAs (miRNAs) are short (19-24 nucleotides) noncoding RNA molecules that regulate gene expression at the posttranscriptional level thus playing an important role in modulating various biological processes including, but not limited to developmental processes, proliferation, apoptosis, metabolism, differentiation, epithelial-mechenchymal transition and are involved in the initiation and progression of various human cancers. Unique miRNA expression profiles have been observed in various cancer types at different stages, suggesting their potential as diagnostic and prognostic biomarkers. Due to their tumor-specific and tissue-specific expression profiles, stability, robust clinical assays for detection in serum as well as in formalin-fixed tissue samples, miRNAs have emerged as attractive candidates for diagnostic and prognostic applications. This review summarizes recent research supporting the utility of miRNAs as novel diagnostic and prognostic tools for GI cancers.

MicroRNAs (miRNAs) as Biomarker(s) for Prognosis and Diagnosis of Gastrointestinal (GI) Cancers

PubMed Central

Macha, Muzafar A.; Seshacharyulu, Parthasarathy; Krishn, Shiv Ram; Pai, Priya; Rachagani, Satyanarayana; Jain, Maneesh; Batra, Surinder K.

2014-01-01

Gastrointestinal (GI) cancers remain one of the most common malignancies and are the second common cause of cancer deaths worldwide. The limited effectiveness of therapy for patients with advanced stage and recurrent disease is a reflection of an incomplete understanding of the molecular basis of GI carcinogenesis. Major advancements have improved our understanding of pathology and pathogenesis of GI cancers, but high mortality rates, unfavorable prognosis and lack of clinical predictive biomarkers provide an impetus to investigate new sensitive and specific diagnostic and prognostic markers for GI cancers. MicroRNAs (miRNAs) are short (19–24 nucleotides) noncoding RNA molecules that regulate gene expression at the posttranscriptional level thus playing an important role in modulating various biological processes including, but not limited, to developmental processes, proliferation, apoptosis, metabolism, differentiation, epithelial-mechenchymal transition and are involved in the initiation and progression of various human cancers. Unique miRNA expression profiles have been observed in various cancer types at different stages, suggesting their potential as diagnostic and prognostic biomarkers. Due to their tumor-specific and tissue-specific expression profiles, stability, robust clinical assays for detection in serum as well as in formalin-fixed tissue samples, miRNAs have emerged as attractive candidates for diagnostic and prognostic applications. This review summarizes recent research supporting the utility of miRNAs as novel diagnostic and prognostic tools for GI cancers. PMID:24479799

MicroRNAs in B-cell lymphomas: how a complex biology gets more complex.

PubMed

Musilova, K; Mraz, M

2015-05-01

MicroRNAs (miRNAs) represent important regulators of gene expression besides transcriptional control. miRNA regulation can be involved in the cell developmental fate decisions, but can also have more subtle roles in buffering stochastic fluctuations in gene expression. They participate in pathways fundamental to B-cell development like B-cell receptor (BCR) signalling, B-cell migration/adhesion, cell-cell interactions in immune niches, and the production and class-switching of immunoglobulins. miRNAs influence B-cell maturation, generation of pre-, marginal zone, follicular, B1, plasma and memory B cells. In this review, we discuss miRNAs with essential functions in malignant B-cell development (such as miR-150, miR-155, miR-21, miR-34a, miR-17-92 and miR-15-16). We also put these miRNAs in the context of normal B-cell differentiation, as this is intimately connected to neoplastic B-cell development. We review miRNAs' role in the most common B-cell malignancies, including chronic lymphocytic leukaemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and mantle cell lymphoma (MCL). We focus on miR-contribution to the regulation of important signalling pathways (such as NF-κB, PI3K/AKT and TGF-β), BCR signalling and its modulators (such as PTEN, SHIP-1, ZAP-70, GAB1 and BTK), anti- and pro-apoptotic proteins (such as BCL2, MCL1, TCL1, BIM, p53 and SIRT1) and transcription factors (such as MYC, MYB, PU.1, FOXP1 and BCL6). We also discuss the association of miRNAs' expression levels with the patients' survival and response to therapy, summarizing their potential use as predictive and prognostic markers. Importantly, the targeting of miRNAs (like use of anti-miR-155 or miR-34a mimic) could provide a novel therapeutic approach as evidenced by tumour regression in xenograft mouse models and initial promising data from clinical trials.

Aberrant expression of miR-218 and miR-204 in human mesial temporal lobe epilepsy and hippocampal sclerosis-convergence on axonal guidance.

PubMed

Kaalund, Sanne S; Venø, Morten T; Bak, Mads; Møller, Rikke S; Laursen, Henning; Madsen, Flemming; Broholm, Helle; Quistorff, Bjørn; Uldall, Peter; Tommerup, Niels; Kauppinen, Sakari; Sabers, Anne; Fluiter, Kees; Møller, Lisbeth B; Nossent, Anne Y; Silahtaroglu, Asli; Kjems, Jørgen; Aronica, Eleonora; Tümer, Zeynep

2014-12-01

Mesial temporal lobe epilepsy (MTLE) is one of the most common types of the intractable epilepsies and is most often associated with hippocampal sclerosis (HS), which is characterized by pronounced loss of hippocampal pyramidal neurons. microRNAs (miRNAs) have been shown to be dysregulated in epilepsy and neurodegenerative diseases, and we hypothesized that miRNAs could be involved in the pathogenesis of MTLE and HS. miRNA expression was quantified in hippocampal specimens from human patients using miRNA microarray and quantitative real-time polymerase chain reaction RT-PCR, and by RNA-seq on fetal brain specimens from domestic pigs. In situ hybridization was used to show the spatial distribution of miRNAs in the human hippocampus. The potential effect of miRNAs on targets genes was investigated using the dual luciferase reporter gene assay. miRNA expression profiling showed that 25 miRNAs were up-regulated and 5 were down-regulated in hippocampus biopsies of MTLE/HS patients compared to controls. We showed that miR-204 and miR-218 were significantly down-regulated in MTLE and HS, and both were expressed in neurons in all subfields of normal hippocampus. Moreover, miR-204 and miR-218 showed strong changes in expression during fetal development of the hippocampus in pigs, and we identified four target genes, involved in axonal guidance and synaptic plasticity, ROBO1, GRM1, SLC1A2, and GNAI2, as bona fide targets of miR-218. GRM1 was also shown to be a direct target of miR-204. miR-204 and miR-218 are developmentally regulated in the hippocampus and may contribute to the molecular mechanisms underlying the pathogenesis of MTLE and HS. Wiley Periodicals, Inc. © 2014 International League Against Epilepsy.

MicroRNA Dysregulation, Gene Networks, and Risk for Schizophrenia in 22q11.2 Deletion Syndrome

PubMed Central

Merico, Daniele; Costain, Gregory; Butcher, Nancy J.; Warnica, William; Ogura, Lucas; Alfred, Simon E.; Brzustowicz, Linda M.; Bassett, Anne S.

2014-01-01

The role of microRNAs (miRNAs) in the etiology of schizophrenia is increasingly recognized. Microdeletions at chromosome 22q11.2 are recurrent structural variants that impart a high risk for schizophrenia and are found in up to 1% of all patients with schizophrenia. The 22q11.2 deletion region overlaps gene DGCR8, encoding a subunit of the miRNA microprocessor complex. We identified miRNAs overlapped by the 22q11.2 microdeletion and for the first time investigated their predicted target genes, and those implicated by DGCR8, to identify targets that may be involved in the risk for schizophrenia. The 22q11.2 region encompasses seven validated or putative miRNA genes. Employing two standard prediction tools, we generated sets of predicted target genes. Functional enrichment profiles of the 22q11.2 region miRNA target genes suggested a role in neuronal processes and broader developmental pathways. We then constructed a protein interaction network of schizophrenia candidate genes and interaction partners relevant to brain function, independent of the 22q11.2 region miRNA mechanisms. We found that the predicted gene targets of the 22q11.2 deletion miRNAs, and targets of the genome-wide miRNAs predicted to be dysregulated by DGCR8 hemizygosity, were significantly represented in this schizophrenia network. The findings provide new insights into the pathway from 22q11.2 deletion to expression of schizophrenia, and suggest that hemizygosity of the 22q11.2 region may have downstream effects implicating genes elsewhere in the genome that are relevant to the general schizophrenia population. These data also provide further support for the notion that robust genetic findings in schizophrenia may converge on a reasonable number of final pathways. PMID:25484875

Temporal analysis of reciprocal miRNA-mRNA expression patterns predicts regulatory networks during differentiation in human skeletal muscle cells

PubMed Central

Sjögren, Rasmus J. O.; Egan, Brendan; Katayama, Mutsumi; Zierath, Juleen R.

2014-01-01

microRNAs (miRNAs) are short noncoding RNAs that regulate gene expression through posttranscriptional repression of target genes. miRNAs exert a fundamental level of control over many developmental processes, but their role in the differentiation and development of skeletal muscle from myogenic progenitor cells in humans remains incompletely understood. Using primary cultures established from human skeletal muscle satellite cells, we performed microarray profiling of miRNA expression during differentiation of myoblasts (day 0) into myotubes at 48 h intervals (day 2, 4, 6, 8, and 10). Based on a time-course analysis, we identified 44 miRNAs with altered expression [false discovery rate (FDR) < 5%, fold change > ±1.2] during differentiation, including the marked upregulation of the canonical myogenic miRNAs miR-1, miR-133a, miR-133b, and miR-206. Microarray profiling of mRNA expression at day 0, 4, and 10 identified 842 and 949 genes differentially expressed (FDR < 10%) at day 4 and 10, respectively. At day 10, 42% of altered transcripts demonstrated reciprocal expression patterns in relation to the directional change of their in silico predicted regulatory miRNAs based on analysis using Ingenuity Pathway Analysis microRNA Target Filter. Bioinformatic analysis predicted networks of regulation during differentiation including myomiRs miR-1/206 and miR-133a/b, miRNAs previously established in differentiation including miR-26 and miR-30, and novel miRNAs regulated during differentiation of human skeletal muscle cells such as miR-138-5p and miR-20a. These reciprocal expression patterns may represent new regulatory nodes in human skeletal muscle cell differentiation. This analysis serves as a reference point for future studies of human skeletal muscle differentiation and development in healthy and disease states. PMID:25547110

Functional Genomic Analysis of the let-7 Regulatory Network in Caenorhabditis elegans

PubMed Central

Zisoulis, Dimitrios G.; Lovci, Michael T.; Melnik-Martinez, Katya V.; Yeo, Gene W.; Pasquinelli, Amy E.

2013-01-01

The let-7 microRNA (miRNA) regulates cellular differentiation across many animal species. Loss of let-7 activity causes abnormal development in Caenorhabditis elegans and unchecked cellular proliferation in human cells, which contributes to tumorigenesis. These defects are due to improper expression of protein-coding genes normally under let-7 regulation. While some direct targets of let-7 have been identified, the genome-wide effect of let-7 insufficiency in a developing animal has not been fully investigated. Here we report the results of molecular and genetic assays aimed at determining the global network of genes regulated by let-7 in C. elegans. By screening for mis-regulated genes that also contribute to let-7 mutant phenotypes, we derived a list of physiologically relevant potential targets of let-7 regulation. Twenty new suppressors of the rupturing vulva or extra seam cell division phenotypes characteristic of let-7 mutants emerged. Three of these genes, opt-2, prmt-1, and T27D12.1, were found to associate with Argonaute in a let-7–dependent manner and are likely novel direct targets of this miRNA. Overall, a complex network of genes with various activities is subject to let-7 regulation to coordinate developmental timing across tissues during worm development. PMID:23516374

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.

MicroRNA Signaling in Embryo Development

PubMed Central

Gross, Nicole; Khatib, Hasan

2017-01-01

Expression of microRNAs (miRNAs) is essential for embryonic development and serves important roles in gametogenesis. miRNAs are secreted into the extracellular environment by the embryo during the preimplantation stage of development. Several cell types secrete miRNAs into biological fluids in the extracellular environment. These fluid-derived miRNAs have been shown to circulate the body. Stable transport is dependent on proper packaging of the miRNAs into extracellular vesicles (EVs), including exosomes. These vesicles, which also contain RNA, DNA and proteins, are on the forefront of research on cell-to-cell communication. Interestingly, EVs have been identified in many reproductive fluids, such as uterine fluid, where their miRNA content is proposed to serve as a mechanism of crosstalk between the mother and conceptus. Here, we review the role of miRNAs in molecular signaling and discuss their transport during early embryo development and implantation. PMID:28906477

Coordinated regulation of Arabidopsis microRNA biogenesis and red light signaling through Dicer-like 1 and phytochrome-interacting factor 4

PubMed Central

Sun, Zhenfei; Li, Min; Zhou, Ying; Guo, Tongtong; Liu, Yin; Zhang, Hui

2018-01-01

Light and microRNAs (miRNAs) are key external and internal signals for plant development, respectively. However, the relationship between the light signaling and miRNA biogenesis pathways remains unknown. Here we found that miRNA processer proteins DCL1 and HYL1 interact with a basic helix-loop-helix (bHLH) transcription factor, phytochrome-interacting factor 4 (PIF4), which mediates the destabilization of DCL1 during dark-to-red-light transition. PIF4 acts as a transcription factor for some miRNA genes and is necessary for the proper accumulation of miRNAs. DCL1, HYL1, and mature miRNAs play roles in the regulation of plant hypocotyl growth. These results uncovered a previously unknown crosstalk between miRNA biogenesis and red light signaling through the PIF4-dependent regulation of miRNA transcription and processing to affect red-light-directed plant photomorphogenesis. PMID:29522510

Sensitive and label-free detection of miRNA-145 by triplex formation.

PubMed

Aviñó, Anna; Huertas, César S; Lechuga, Laura M; Eritja, Ramon

2016-01-01

The development of new strategies for detecting microRNAs (miRNAs) has become a crucial step in the diagnostic field. miRNA profiles depend greatly on the sample and the analytical platform employed, leading sometimes to contradictory results. In this work, we study the use of modified parallel tail-clamps to detect a miRNA sequence involved in tumor suppression by triplex formation. Thermal denaturing curves and circular dichroism (CD) measurements have been performed to confirm that parallel clamps carrying 8-aminoguanine form the most stable triplex structures with their target miRNA. The modified tail-clamps have been tested as bioreceptors in a surface plasmon resonance (SPR) biosensor for the detection of miRNA-145. The detection limit was improved 2.4 times demonstrating that a stable triplex structure is formed between target miRNA and 8-aminoguanine tail-clamp bioreceptor. This new approach is an essential step toward the label-free and reliable detection of miRNA signatures for diagnostic purposes.

MiRAR-miRNA Activity Reporter for Living Cells.

PubMed

Turk, Matthew A; Chung, Christina Z; Manni, Emad; Zukowski, Stephanie A; Engineer, Anish; Badakhshi, Yasaman; Bi, Yumin; Heinemann, Ilka U

2018-06-19

microRNA (miRNA) activity and regulation are of increasing interest as new therapeutic targets. Traditional approaches to assess miRNA levels in cells rely on RNA sequencing or quantitative PCR. While useful, these approaches are based on RNA extraction and cannot be applied in real-time to observe miRNA activity with single-cell resolution. We developed a green fluorescence protein (GFP)-based reporter system that allows for a direct, real-time readout of changes in miRNA activity in live cells. The miRNA activity reporter (MiRAR) consists of GFP fused to a 3′ untranslated region containing specific miRNA binding sites, resulting in miRNA activity-dependent GFP expression. Using qPCR, we verified the inverse relationship of GFP fluorescence and miRNA levels. We demonstrated that this novel optogenetic reporter system quantifies cellular levels of the tumor suppressor miRNA let-7 in real-time in single Human embryonic kidney 293 (HEK 293) cells. Our data shows that the MiRAR can be applied to detect changes in miRNA levels upon disruption of miRNA degradation pathways. We further show that the reporter could be adapted to monitor another disease-relevant miRNA, miR-122. With trivial modifications, this approach could be applied across the miRNome for quantification of many specific miRNA in cell cultures, tissues, or transgenic animal models.

Determining causal miRNAs and their signaling cascade in diseases using an influence diffusion model.

PubMed

Nalluri, Joseph J; Rana, Pratip; Barh, Debmalya; Azevedo, Vasco; Dinh, Thang N; Vladimirov, Vladimir; Ghosh, Preetam

2017-08-15

In recent studies, miRNAs have been found to be extremely influential in many of the essential biological processes. They exhibit a self-regulatory mechanism through which they act as positive/negative regulators of expression of genes and other miRNAs. This has direct implications in the regulation of various pathophysiological conditions, signaling pathways and different types of cancers. Studying miRNA-disease associations has been an extensive area of research; however deciphering miRNA-miRNA network regulatory patterns in several diseases remains a challenge. In this study, we use information diffusion theory to quantify the influence diffusion in a miRNA-miRNA regulation network across multiple disease categories. Our proposed methodology determines the critical disease specific miRNAs which play a causal role in their signaling cascade and hence may regulate disease progression. We extensively validate our framework using existing computational tools from the literature. Furthermore, we implement our framework on a comprehensive miRNA expression data set for alcohol dependence and identify the causal miRNAs for alcohol-dependency in patients which were validated by the phase-shift in their expression scores towards the early stages of the disease. Finally, our computational framework for identifying causal miRNAs implicated in diseases is available as a free online tool for the greater scientific community.

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

Plant microRNAs in larval food regulate honeybee caste development

PubMed Central

Zhou, Zhen; Kong, Yan; Liang, Hongwei; Lin, Zheguang; Luo, Jun; Zheng, Huoqing; Wan, Ping; Zhang, Junfeng; Zen, Ke; Chen, Jiong; Hu, Fuliang; Zhang, Chen-Yu; Ren, Jie

2017-01-01

The major environmental determinants of honeybee caste development come from larval nutrients: royal jelly stimulates the differentiation of larvae into queens, whereas beebread leads to worker bee fate. However, these determinants are not fully characterized. Here we report that plant RNAs, particularly miRNAs, which are more enriched in beebread than in royal jelly, delay development and decrease body and ovary size in honeybees, thereby preventing larval differentiation into queens and inducing development into worker bees. Mechanistic studies reveal that amTOR, a stimulatory gene in caste differentiation, is the direct target of miR162a. Interestingly, the same effect also exists in non-social Drosophila. When such plant RNAs and miRNAs are fed to Drosophila larvae, they cause extended developmental times and reductions in body weight and length, ovary size and fecundity. This study identifies an uncharacterized function of plant miRNAs that fine-tunes honeybee caste development, offering hints for understanding cross-kingdom interaction and co-evolution. PMID:28859085

Plant microRNAs in larval food regulate honeybee caste development.

PubMed

Zhu, Kegan; Liu, Minghui; Fu, Zheng; Zhou, Zhen; Kong, Yan; Liang, Hongwei; Lin, Zheguang; Luo, Jun; Zheng, Huoqing; Wan, Ping; Zhang, Junfeng; Zen, Ke; Chen, Jiong; Hu, Fuliang; Zhang, Chen-Yu; Ren, Jie; Chen, Xi

2017-08-01

The major environmental determinants of honeybee caste development come from larval nutrients: royal jelly stimulates the differentiation of larvae into queens, whereas beebread leads to worker bee fate. However, these determinants are not fully characterized. Here we report that plant RNAs, particularly miRNAs, which are more enriched in beebread than in royal jelly, delay development and decrease body and ovary size in honeybees, thereby preventing larval differentiation into queens and inducing development into worker bees. Mechanistic studies reveal that amTOR, a stimulatory gene in caste differentiation, is the direct target of miR162a. Interestingly, the same effect also exists in non-social Drosophila. When such plant RNAs and miRNAs are fed to Drosophila larvae, they cause extended developmental times and reductions in body weight and length, ovary size and fecundity. This study identifies an uncharacterized function of plant miRNAs that fine-tunes honeybee caste development, offering hints for understanding cross-kingdom interaction and co-evolution.

Ribosomal protein RPS-14 modulates let-7 microRNA function in Caenorhabditis elegans

PubMed Central

Chan, Shih-Peng; Slack, Frank J.

2009-01-01

The let-7 microRNA (miRNA) regulates developmental timing at the larval-to-adult transition in Caenorhabditis elegans. Dysregulation of let-7 results in irregular hypodermal and vulval development. Disrupted let-7 function is also a feature of human lung cancer. However, little is known about the mechanism and co-factors of let-7. Here we demonstrate that ribosomal protein RPS-14 is able to modulate let-7 function in C. elegans. The RPS-14 protein co-immunoprecipitated with the nematode Argonaute homolog, ALG-1. Reduction of rps-14 gene expression by RNAi suppressed the aberrant vulva and hypodermis development phenotypes of let-7(n2853) mutant animals and the mis-regulation of a reporter bearing the lin-41 3′UTR, a well established let-7 target. Our results indicate an interactive relationship between let-7 miRNA function and ribosomal protein RPS-14 in regulation of terminal differentiation that may help in understanding the mechanism of translational control by miRNAs. PMID:19627982

Novel microRNA-like viral small regulatory RNAs arising during human hepatitis A virus infection.

PubMed

Shi, Jiandong; Sun, Jing; Wang, Bin; Wu, Meini; Zhang, Jing; Duan, Zhiqing; Wang, Haixuan; Hu, Ningzhu; Hu, Yunzhang

2014-10-01

MicroRNAs (miRNAs), including host miRNAs and viral miRNAs, play vital roles in regulating host-virus interactions. DNA viruses encode miRNAs that regulate the viral life cycle. However, it is generally believed that cytoplasmic RNA viruses do not encode miRNAs, owing to inaccessible cellular miRNA processing machinery. Here, we provide a comprehensive genome-wide analysis and identification of miRNAs that were derived from hepatitis A virus (HAV; Hu/China/H2/1982), which is a typical cytoplasmic RNA virus. Using deep-sequencing and in silico approaches, we identified 2 novel virally encoded miRNAs, named hav-miR-1-5p and hav-miR-2-5p. Both of the novel virally encoded miRNAs were clearly detected in infected cells. Analysis of Dicer enzyme silencing demonstrated that HAV-derived miRNA biogenesis is Dicer dependent. Furthermore, we confirmed that HAV mature miRNAs were generated from viral miRNA precursors (pre-miRNAs) in host cells. Notably, naturally derived HAV miRNAs were biologically and functionally active and induced post-transcriptional gene silencing (PTGS). Genomic location analysis revealed novel miRNAs located in the coding region of the viral genome. Overall, our results show that HAV naturally generates functional miRNA-like small regulatory RNAs during infection. This is the first report of miRNAs derived from the coding region of genomic RNA of a cytoplasmic RNA virus. These observations demonstrate that a cytoplasmic RNA virus can naturally generate functional miRNAs, as DNA viruses do. These findings also contribute to improved understanding of host-RNA virus interactions mediated by RNA virus-derived miRNAs. © FASEB.

The effect of dys-1 mutation on miRNA expression profile in Caenorhabditis elegans during Shenzhou-8 mission

NASA Astrophysics Data System (ADS)

Xu, Dan; Sun, Yeqing; Gao, Ying; Xing, Yanfang

microRNAs (miRNAs) is reported to be sensitive to radiation exposure and altered gravity, involved in a variety of biological processes through negative regulation of gene expression. Dystrophin-like dys-1 gene is expressed and required in muscle tissue, which plays a vital role in mechanical transduction when gravity varies. In the present study, we investigated the effect of dys-1 mutation on miRNA expression profile in Caenorhabditis elegans (C. elegans) under space radiation associated with microgravity (R+M) and radiation alone (R) environment during Shenzhou-8 mission. We performed miRNA microarray analysis in dys-1 mutant and wide-type (WT) of dauer larvae and found that 27 miRNAs changed in abundance after spaceflight. Compared with WT, there was different miRNA expression pattern in different treatments in dys-1 mutant. Cel-miR-796 and miR-124 were reversely expressed under R+M and R environment in WT and dys-1 mutant, respectively, indicating they might be affected by microgravity. Mutation of dys-1 remarkably reduced the number of altered miRNAs under space environment, resulting in the decrease of genes in biological categories of “body morphogenesis”, “behavior”, “cell adhesion” and so on. Particularly, we found that those genes controlling regulation of locomotion in WT were lost in dys-1 mutant, while genes in positive regulation of developmental process only existed in dys-1 mutant. miR-796 was predicted to target genes ace-1 and dyc-1 that are functionally linked to dys-1. Integration analysis of miRNA and mRNA expression profile revealed that miR-56 and miR-124 were involved in behavior and locomotion by regulating different target genes under space environment, among which nep-11, deb-1, C07H4.1 and F11H8.2 might be associated with neuromuscular system. Our findings suggest that dys-1 could cause alteration of miRNAs and target genes, involved in regulating the response of C. elegans to space microgravity in neuromuscular system. This research will provide new insight for better understanding of the mechanism in microgravity-induced muscular dystrophy.

Application of small RNA technology for improved control of parasitic helminths.

PubMed

Britton, Collette; Winter, Alan D; Marks, Neil D; Gu, Henry; McNeilly, Tom N; Gillan, Victoria; Devaney, Eileen

2015-08-15

Over the last decade microRNAs (miRNAs) and small interfering RNAs (siRNAs) have emerged as important regulators of post-transcriptional gene expression. miRNAs are short, non-coding RNAs that regulate a variety of processes including cancer, organ development and immune function. This class of small RNAs bind with partial complementarity to their target mRNA sequences, most often in the 3'UTR, to negatively regulate gene expression. In parasitic helminths, miRNAs are being increasingly studied for their potential roles in development and host-parasite interactions. The availability of genome data, combined with small RNA sequencing, has paved the way to profile miRNAs expressed at particular developmental stages for many parasitic helminths. While some miRNAs are conserved across species, others appear to be unique to specific parasites, suggesting important roles in adaptation and survival in the host environment. Some miRNAs are released from parasites, in exosomes or in protein complexes, and the potential effects of these on host immune function are being increasingly studied. In addition, release of miRNAs from schistosome and filarial parasites into host plasma can be exploited for the development of specific and sensitive diagnostic biomarkers of infection. Interfering with miRNA function, as well as silencing key components of the pathways they regulate, will progress our understanding of parasite development and provide a novel approach to therapeutic control. RNA interference (RNAi) by siRNAs has proven to be inconsistent in parasitic nematodes. However, the recent successes reported for schistosome and liver fluke RNAi, encourage further efforts to enhance delivery of RNA and improve in vitro culture systems and assays to monitor phenotypic effects in nematodes. These improvements are important for the establishment of reliable functional genomic platforms for novel drug and vaccine development. In this review we focus on the important roles of miRNAs and siRNAs in post-transcriptional gene regulation in veterinary parasitic helminths and the potential value of these in parasite diagnosis and control. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

MicroRNA-Dependent Transcriptional Silencing of Transposable Elements in Drosophila Follicle Cells.

PubMed

Mugat, Bruno; Akkouche, Abdou; Serrano, Vincent; Armenise, Claudia; Li, Blaise; Brun, Christine; Fulga, Tudor A; Van Vactor, David; Pélisson, Alain; Chambeyron, Séverine

2015-05-01

RNA interference-related silencing mechanisms concern very diverse and distinct biological processes, from gene regulation (via the microRNA pathway) to defense against molecular parasites (through the small interfering RNA and the Piwi-interacting RNA pathways). Small non-coding RNAs serve as specificity factors that guide effector proteins to ribonucleic acid targets via base-pairing interactions, to achieve transcriptional or post-transcriptional regulation. Because of the small sequence complementarity required for microRNA-dependent post-transcriptional regulation, thousands of microRNA (miRNA) putative targets have been annotated in Drosophila. In Drosophila somatic ovarian cells, genomic parasites, such as transposable elements (TEs), are transcriptionally repressed by chromatin changes induced by Piwi-interacting RNAs (piRNAs) that prevent them from invading the germinal genome. Here we show, for the first time, that a functional miRNA pathway is required for the piRNA-mediated transcriptional silencing of TEs in this tissue. Global miRNA depletion, caused by tissue- and stage-specific knock down of drosha (involved in miRNA biogenesis), AGO1 or gawky (both responsible for miRNA activity), resulted in loss of TE-derived piRNAs and chromatin-mediated transcriptional de-silencing of TEs. This specific TE de-repression was also observed upon individual titration (by expression of the complementary miRNA sponge) of two miRNAs (miR-14 and miR-34) as well as in a miR-14 loss-of-function mutant background. Interestingly, the miRNA defects differentially affected TE- and 3' UTR-derived piRNAs. To our knowledge, this is the first indication of possible differences in the biogenesis or stability of TE- and 3' UTR-derived piRNAs. This work is one of the examples of detectable phenotypes caused by loss of individual miRNAs in Drosophila and the first genetic evidence that miRNAs have a role in the maintenance of genome stability via piRNA-mediated TE repression.

miRNAs mediate SnRK1-dependent energy signaling in Arabidopsis

PubMed Central

Confraria, Ana; Martinho, Cláudia; Elias, Alexandre; Rubio-Somoza, Ignacio; Baena-González, Elena

2013-01-01

The SnRK1 protein kinase, the plant ortholog of mammalian AMPK and yeast Snf1, is activated by the energy depletion caused by adverse environmental conditions. Upon activation, SnRK1 triggers extensive transcriptional changes to restore homeostasis and promote stress tolerance and survival partly through the inhibition of anabolism and the activation of catabolism. Despite the identification of a few bZIP transcription factors as downstream effectors, the mechanisms underlying gene regulation, and in particular gene repression by SnRK1, remain mostly unknown. microRNAs (miRNAs) are 20–24 nt RNAs that regulate gene expression post-transcriptionally by driving the cleavage and/or translation attenuation of complementary mRNA targets. In addition to their role in plant development, mounting evidence implicates miRNAs in the response to environmental stress. Given the involvement of miRNAs in stress responses and the fact that some of the SnRK1-regulated genes are miRNA targets, we postulated that miRNAs drive part of the transcriptional reprogramming triggered by SnRK1. By comparing the transcriptional response to energy deprivation between WT and dcl1-9, a mutant deficient in miRNA biogenesis, we identified 831 starvation genes misregulated in the dcl1-9 mutant, out of which 155 are validated or predicted miRNA targets. Functional clustering analysis revealed that the main cellular processes potentially co-regulated by SnRK1 and miRNAs are translation and organelle function and uncover TCP transcription factors as one of the most highly enriched functional clusters. TCP repression during energy deprivation was impaired in miR319 knockdown (MIM319) plants, demonstrating the involvement of miR319 in the stress-dependent regulation of TCPs. Altogether, our data indicates that miRNAs are components of the SnRK1 signaling cascade contributing to the regulation of specific mRNA targets and possibly tuning down particular cellular processes during the stress response. PMID:23802004

MicroRNAs-Dependent Regulation of PPARs in Metabolic Diseases and Cancers

PubMed Central

Portius, Dorothea; Sobolewski, Cyril

2017-01-01

Peroxisome proliferator-activated receptors (PPARs) are a family of ligand-dependent nuclear receptors, which control the transcription of genes involved in energy homeostasis and inflammation and cell proliferation/differentiation. Alterations of PPARs' expression and/or activity are commonly associated with metabolic disorders occurring with obesity, type 2 diabetes, and fatty liver disease, as well as with inflammation and cancer. Emerging evidence now indicates that microRNAs (miRNAs), a family of small noncoding RNAs, which fine-tune gene expression, play a significant role in the pathophysiological mechanisms regulating the expression and activity of PPARs. Herein, the regulation of PPARs by miRNAs is reviewed in the context of metabolic disorders, inflammation, and cancer. The reciprocal control of miRNAs expression by PPARs, as well as the therapeutic potential of modulating PPAR expression/activity by pharmacological compounds targeting miRNA, is also discussed. PMID:28167956

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

microRNA profiles and functions in mosquitoes

PubMed Central

Zhou, Shuisen; Wang, Jingwen; Hu, Wei

2018-01-01

Mosquitoes are incriminated as vectors for many crippling diseases, including malaria, West Nile fever, Dengue fever, and other neglected tropical diseases (NTDs). microRNAs (miRNAs) can interact with multiple target genes to elicit biological functions in the mosquitoes. However, characterization and function of individual miRNAs and their potential targets have not been fully determined to date. We conducted a systematic review of published literature following PRISMA guidelines. We summarize the information about miRNAs in mosquitoes to better understand their metabolism, development, and responses to microorganisms. Depending on the study, we found that miRNAs were dysregulated in a species-, sex-, stage-, and tissue/organ-specific manner. Aberrant miRNA expressions were observed in development, metabolism, host-pathogen interactions, and insecticide resistance. Of note, many miRNAs were down-regulated upon pathogen infection. The experimental studies have expanded the identification of miRNA target from the 3′ untranslated regions (UTRs) of mRNAs of mosquitoes to the 5′ UTRs of mRNAs of the virus. In addition, we discuss current trends in mosquito miRNA research and offer suggestions for future studies. PMID:29718912

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.

The expansion of the metazoan microRNA repertoire

PubMed Central

Hertel, Jana; Lindemeyer, Manuela; Missal, Kristin; Fried, Claudia; Tanzer, Andrea; Flamm, Christoph; Hofacker, Ivo L; Stadler, Peter F

2006-01-01

Background MicroRNAs have been identified as crucial regulators in both animals and plants. Here we report on a comprehensive comparative study of all known miRNA families in animals. We expand the MicroRNA Registry 6.0 by more than 1000 new homologs of miRNA precursors whose expression has been verified in at least one species. Using this uniform data basis we analyze their evolutionary history in terms of individual gene phylogenies and in terms of preservation of genomic nearness across species. This allows us to reliably identify microRNA clusters that are derived from a common transcript. Results We identify three episodes of microRNA innovation that correspond to major developmental innovations: A class of about 20 miRNAs is common to protostomes and deuterostomes and might be related to the advent of bilaterians. A second large wave of innovations maps to the branch leading to the vertebrates. The third significant outburst of miRNA innovation coincides with placental (eutherian) mammals. In addition, we observe the expected expansion of the microRNA inventory due to genome duplications in early vertebrates and in an ancestral teleost. The non-local duplications in the vertebrate ancestor are predated by local (tandem) duplications leading to the formation of about a dozen ancient microRNA clusters. Conclusion Our results suggest that microRNA innovation is an ongoing process. Major expansions of the metazoan miRNA repertoire coincide with the advent of bilaterians, vertebrates, and (placental) mammals. PMID:16480513

MicroRNA miR-30 family regulates non-attachment growth of breast cancer cells

PubMed Central

2013-01-01

Background A subset of breast cancer cells displays increased ability to self-renew and reproduce breast cancer heterogeneity. The characterization of these so-called putative breast tumor-initiating cells (BT-ICs) may open the road for novel therapeutic strategies. As microRNAs (miRNAs) control developmental programs in stem cells, BT-ICs may also rely on specific miRNA profiles for their sustained activity. To explore the notion that miRNAs may have a role in sustaining BT-ICs, we performed a comprehensive profiling of miRNA expression in a model of putative BT-ICs enriched by non-attachment growth conditions. Results We found breast cancer cells grown under non-attachment conditions display a unique pattern of miRNA expression, highlighted by a marked low expression of miR-30 family members relative to parental cells. We further show that miR-30a regulates non-attachment growth. A target screening revealed that miR-30 family redundantly modulates the expression of apoptosis and proliferation-related genes. At least one of these targets, the anti-apoptotic protein AVEN, was able to partially revert the effect of miR-30a overexpression. Finally, overexpression of miR-30a in vivo was associated with reduced breast tumor progression. Conclusions miR30-family regulates the growth of breast cancer cells in non-attachment conditions. This is the first analysis of target prediction in a whole family of microRNAs potentially involved in survival of putative BT-ICs. PMID:23445407

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.

Identification of MicroRNAs in Helicoverpa armigera and Spodoptera litura Based on Deep Sequencing and Homology Analysis

PubMed Central

Ge, Xie; Zhang, Yong; Jiang, Jianhao; Zhong, Yi; Yang, Xiaonan; Li, Zhiqian; Huang, Yongping; Tan, Anjiang

2013-01-01

The current identification of microRNAs (miRNAs) in insects is largely dependent on genome sequences. However, the lack of available genome sequences inhibits the identification of miRNAs in various insect species. In this study, we used a miRNA database of the silkworm Bombyx mori as a reference to identify miRNAs in Helicoverpa armigera and Spodoptera litura using deep sequencing and homology analysis. Because all three species belong to the Lepidoptera, the experiment produced reliable results. Our study identified 97 and 91 conserved miRNAs in H. armigera and S. litura, respectively. Using the genome of B. mori and BAC sequences of H. armigera as references, 1 novel miRNA and 8 novel miRNA candidates were identified in H. armigera, and 4 novel miRNA candidates were identified in S. litura. An evolutionary analysis revealed that most of the identified miRNAs were insect-specific, and more than 20 miRNAs were Lepidoptera-specific. The investigation of the expression patterns of miR-2a, miR-34, miR-2796-3p and miR-11 revealed their potential roles in insect development. miRNA target prediction revealed that conserved miRNA target sites exist in various genes in the 3 species. Conserved miRNA target sites for the Hsp90 gene among the 3 species were validated in the mammalian 293T cell line using a dual-luciferase reporter assay. Our study provides a new approach with which to identify miRNAs in insects lacking genome information and contributes to the functional analysis of insect miRNAs. PMID:23289012

Maternal whole blood cell miRNA-340 is elevated in gestational diabetes and inversely regulated by glucose and insulin.

PubMed

Stirm, Laura; Huypens, Peter; Sass, Steffen; Batra, Richa; Fritsche, Louise; Brucker, Sara; Abele, Harald; Hennige, Anita M; Theis, Fabian; Beckers, Johannes; Hrabě de Angelis, Martin; Fritsche, Andreas; Häring, Hans-Ulrich; Staiger, Harald

2018-01-22

The number of pregnancies complicated by gestational diabetes (GDM) is increasing worldwide. To identify novel characteristics of GDM, we studied miRNA profiles of maternal and fetal whole blood cells (WBCs) from GDM and normal glucose tolerant (NGT) pregnant women matched for body mass index and maternal age. After adjustment for maternal weight gain and pregnancy week, we identified 29 mature micro-RNAs (miRNAs) up-regulated in GDM, one of which, i.e., miRNA-340, was validated by qPCR. mRNA and protein expression of PAIP1, a miRNA-340 target gene, was found down-regulated in GDM women, accordingly. In lymphocytes derived from the mothers' blood and treated in vitro, insulin increased and glucose reduced miRNA-340 expression. In fetal cord blood samples, no associations of miRNA-340 with maternal GDM were observed. Our results provide evidence for insulin-induced epigenetic, i.e., miRNA-dependent, programming of maternal WBCs in GDM.

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.

MiR-277/4989 regulate transcriptional landscape during juvenile to adult transition in the parasitic helminth Schistosoma mansoni

PubMed Central

van Dongen, Stijn; Collins, Julie; Quintais, Leonor; Ribeiro, Diogo M.; Sessler, Florian; Hunt, Martin; Rinaldi, Gabriel; Collins, James J.; Enright, Anton J.; Berriman, Matthew

2017-01-01

Schistosomes are parasitic helminths that cause schistosomiasis, a disease affecting circa 200 million people, primarily in underprivileged regions of the world. Schistosoma mansoni is the most experimentally tractable schistosome species due to its ease of propagation in the laboratory and the high quality of its genome assembly and annotation. Although there is growing interest in microRNAs (miRNAs) in trematodes, little is known about the role these molecules play in the context of developmental processes. We use the completely unaware “miRNA-blind” bioinformatics tool Sylamer to analyse the 3’-UTRs of transcripts differentially expressed between the juvenile and adult stages. We show that the miR-277/4989 family target sequence is the only one significantly enriched in the transition from juvenile to adult worms. Further, we describe a novel miRNA, sma-miR-4989 showing that its proximal genomic location to sma-miR-277 suggests that they form a miRNA cluster, and we propose hairpin folds for both miRNAs compatible with the miRNA pathway. In addition, we found that expression of sma-miR-277/4989 miRNAs are up-regulated in adults while their predicted targets are characterised by significant down-regulation in paired adult worms but remain largely undisturbed in immature “virgin” females. Finally, we show that sma-miR-4989 is expressed in tegumental cells located proximal to the oesophagus gland and also distributed throughout the male worms’ body. Our results indicate that sma-miR-277/4989 might play a dominant role in post-transcriptional regulation during development of juvenile worms and suggest an important role in the sexual development of female schistosomes. PMID:28542189

miRCat2: accurate prediction of plant and animal microRNAs from next-generation sequencing datasets

PubMed Central

Paicu, Claudia; Mohorianu, Irina; Stocks, Matthew; Xu, Ping; Coince, Aurore; Billmeier, Martina; Dalmay, Tamas; Moulton, Vincent; Moxon, Simon

2017-01-01

Abstract Motivation MicroRNAs are a class of ∼21–22 nt small RNAs which are excised from a stable hairpin-like secondary structure. They have important gene regulatory functions and are involved in many pathways including developmental timing, organogenesis and development in eukaryotes. There are several computational tools for miRNA detection from next-generation sequencing datasets. However, many of these tools suffer from high false positive and false negative rates. Here we present a novel miRNA prediction algorithm, miRCat2. miRCat2 incorporates a new entropy-based approach to detect miRNA loci, which is designed to cope with the high sequencing depth of current next-generation sequencing datasets. It has a user-friendly interface and produces graphical representations of the hairpin structure and plots depicting the alignment of sequences on the secondary structure. Results We test miRCat2 on a number of animal and plant datasets and present a comparative analysis with miRCat, miRDeep2, miRPlant and miReap. We also use mutants in the miRNA biogenesis pathway to evaluate the predictions of these tools. Results indicate that miRCat2 has an improved accuracy compared with other methods tested. Moreover, miRCat2 predicts several new miRNAs that are differentially expressed in wild-type versus mutants in the miRNA biogenesis pathway. Availability and Implementation miRCat2 is part of the UEA small RNA Workbench and is freely available from http://srna-workbench.cmp.uea.ac.uk/. Contact v.moulton@uea.ac.uk or s.moxon@uea.ac.uk Supplementary information Supplementary data are available at Bioinformatics online. PMID:28407097

ADAR2 induces reproducible changes in sequence and abundance of mature microRNAs in the mouse brain

PubMed Central

Vesely, Cornelia; Tauber, Stefanie; Sedlazeck, Fritz J.; Tajaddod, Mansoureh; von Haeseler, Arndt; Jantsch, Michael F.

2014-01-01

Adenosine deaminases that act on RNA (ADARs) deaminate adenosines to inosines in double-stranded RNAs including miRNA precursors. A to I editing is widespread and required for normal life. By comparing deep sequencing data of brain miRNAs from wild-type and ADAR2 deficient mouse strains, we detect editing sites and altered miRNA processing at high sensitivity. We detect 48 novel editing events in miRNAs. Some editing events reach frequencies of up to 80%. About half of all editing events depend on ADAR2 while some miRNAs are preferentially edited by ADAR1. Sixty-four percent of all editing events are located within the seed region of mature miRNAs. For the highly edited miR-3099, we experimentally prove retargeting of the edited miRNA to novel 3′ UTRs. We show further that an abundant editing event in miR-497 promotes processing by Drosha of the corresponding pri-miRNA. We also detect reproducible changes in the abundance of specific miRNAs in ADAR2-deficient mice that occur independent of adjacent A to I editing events. This indicates that ADAR2 binding but not editing of miRNA precursors may influence their processing. Correlating with changes in miRNA abundance we find misregulation of putative targets of these miRNAs in the presence or absence of ADAR2. PMID:25260591

Detection and quantification of extracellular microRNAs in murine biofluids

PubMed Central

2014-01-01

Background MicroRNAs (miRNAs) are short RNA molecules which regulate gene expression in eukaryotic cells, and are abundant and stable in biofluids such as blood serum and plasma. As such, there has been heightened interest in the utility of extracellular miRNAs as minimally invasive biomarkers for diagnosis and monitoring of a wide range of human pathologies. However, quantification of extracellular miRNAs is subject to a number of specific challenges, including the relatively low RNA content of biofluids, the possibility of contamination with serum proteins (including RNases and PCR inhibitors), hemolysis, platelet contamination/activation, a lack of well-established reference miRNAs and the biochemical properties of miRNAs themselves. Protocols for the detection and quantification of miRNAs in biofluids are therefore of high interest. Results The following protocol was validated by quantifying miRNA abundance in C57 (wild-type) and dystrophin-deficient (mdx) mice. Important differences in miRNA abundance were observed depending on whether blood was taken from the jugular or tail vein. Furthermore, efficiency of miRNA recovery was reduced when sample volumes greater than 50 μl were used. Conclusions Here we describe robust and novel procedures to harvest murine serum/plasma, extract biofluid RNA, amplify specific miRNAs by RT-qPCR and analyze the resulting data, enabling the determination of relative and absolute miRNA abundance in extracellular biofluids with high accuracy, specificity and sensitivity. PMID:24629058

The Arabidopsis miR396 mediates pathogen-associated molecular pattern-triggered immune responses against fungal pathogens

PubMed Central

Soto-Suárez, Mauricio; Baldrich, Patricia; Weigel, Detlef; Rubio-Somoza, Ignacio; San Segundo, Blanca

2017-01-01

MicroRNAs (miRNAs) play a pivotal role in regulating gene expression during plant development. Although a substantial fraction of plant miRNAs has proven responsive to pathogen infection, their role in disease resistance remains largely unknown, especially during fungal infections. In this study, we screened Arabidopsis thaliana lines in which miRNA activity has been reduced using artificial miRNA target mimics (MIM lines) for their response to fungal pathogens. Reduced activity of miR396 (MIM396 plants) was found to confer broad resistance to necrotrophic and hemibiotrophic fungal pathogens. MiR396 levels gradually decreased during fungal infection, thus, enabling its GRF (GROWTH-REGULATING FACTOR) transcription factor target genes to trigger host reprogramming. Pathogen resistance in MIM396 plants is based on a superactivation of defense responses consistent with a priming event during pathogen infection. Notably, low levels of miR396 are not translated in developmental defects in absence of pathogen challenge. Our findings support a role of miR396 in regulating plant immunity, and broaden our knowledge about the molecular players and processes that sustain defense priming. That miR396 modulates innate immunity without growth costs also suggests fine-tuning of miR396 levels as an effective biotechnological means for protection against pathogen infection. PMID:28332603

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.

[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.

Dicer-dependent endothelial microRNAs are necessary for postnatal angiogenesis.

PubMed

Suárez, Yajaira; Fernández-Hernando, Carlos; Yu, Jun; Gerber, Scott A; Harrison, Kenneth D; Pober, Jordan S; Iruela-Arispe, M Luisa; Merkenschlager, Matthias; Sessa, William C

2008-09-16

Posttranscriptional gene regulation by microRNAs (miRNAs) is important for many aspects of development, homeostasis, and disease. Here, we show that reduction of endothelial miRNAs by cell-specific inactivation of Dicer, the terminal endonuclease responsible for the generation of miRNAs, reduces postnatal angiogenic response to a variety of stimuli, including exogenous VEGF, tumors, limb ischemia, and wound healing. Furthermore, VEGF regulated the expression of several miRNAs, including the up-regulation of components of the c-Myc oncogenic cluster miR-17-92. Transfection of endothelial cells with components of the miR-17-92 cluster, induced by VEGF treatment, rescued the induced expression of thrombospondin-1 and the defect in endothelial cell proliferation and morphogenesis initiated by the loss of Dicer. Thus, endothelial miRNAs regulate postnatal angiogenesis and VEGF induces the expression of miRNAs implicated in the regulation of an integrated angiogenic response.

The extent of sequence complementarity correlates with the potency of cellular miRNA-mediated restriction of HIV-1

PubMed Central

Houzet, Laurent; Klase, Zachary; Yeung, Man Lung; Wu, Annie; Le, Shu-Yun; Quiñones, Mariam; Jeang, Kuan-Teh

2012-01-01

MicroRNAs (miRNAs) are 22-nt non-coding RNAs involved in the regulation of cellular gene expression and potential cellular defense against viral infection. Using in silico analyses, we predicted target sites for 22 human miRNAs in the HIV genome. Transfection experiments using synthetic miRNAs showed that five of these miRNAs capably decreased HIV replication. Using one of these five miRNAs, human miR-326 as an example, we demonstrated that the degree of complementarity between the predicted viral sequence and cellular miR-326 correlates, in a Dicer-dependent manner, with the potency of miRNA-mediated restriction of viral replication. Antagomirs to miR-326 that knocked down this cell endogenous miRNA increased HIV-1 replication in cells, suggesting that miR-326 is physiologically functional in moderating HIV-1 replication in human cells. PMID:23042677

IsomiR expression profiles in human lymphoblastoid cell lines exhibit population and gender dependencies

PubMed Central

Loher, Phillipe; Londin, Eric R.; Rigoutsos, Isidore

2014-01-01

For many years it was believed that each mature microRNA (miRNA) existed as a single entity with fixed endpoints and a ‘static’ and unchangeable primary sequence. However, recent evidence suggests that mature miRNAs are more ‘dynamic’ and that each miRNA precursor arm gives rise to multiple isoforms, the isomiRs. Here we report on our identification of numerous and abundant isomiRs in the lymphoblastoid cell lines (LCLs) of 452 men and women from five different population groups. Unexpectedly, we find that these isomiRs exhibit an expression profile that is population-dependent and gender-dependent. This is important as it indicates that the LCLs of each gender/population combination have their own unique collection of mature miRNA transcripts. Moreover, each identified isomiR has its own characteristic abundance that remains consistent across biological replicates indicating that these are not degradation products. The primary sequences of identified isomiRs differ from the known miRBase miRNA either at their 5´-endpoint (leads to a different ‘seed’ sequence and suggests a different targetome), their 3´-endpoint, or both simultaneously. Our analysis of Argonaute PAR-CLIP data from LCLs supports the association of many of these newly identified isomiRs with the Argonaute silencing complex and thus their functional roles through participation in the RNA interference pathway. PMID:25229428

IsomiR expression profiles in human lymphoblastoid cell lines exhibit population and gender dependencies.

PubMed

Loher, Phillipe; Londin, Eric R; Rigoutsos, Isidore

2014-09-30

For many years it was believed that each mature microRNA (miRNA) existed as a single entity with fixed endpoints and a 'static' and unchangeable primary sequence. However, recent evidence suggests that mature miRNAs are more 'dynamic' and that each miRNA precursor arm gives rise to multiple isoforms, the isomiRs. Here we report on our identification of numerous and abundant isomiRs in the lymphoblastoid cell lines (LCLs) of 452 men and women from five different population groups. Unexpectedly, we find that these isomiRs exhibit an expression profile that is population-dependent and gender-dependent. This is important as it indicates that the LCLs of each gender/population combination have their own unique collection of mature miRNA transcripts. Moreover, each identified isomiR has its own characteristic abundance that remains consistent across biological replicates indicating that these are not degradation products. The primary sequences of identified isomiRs differ from the known miRBase miRNA either at their 5´-endpoint (leads to a different 'seed' sequence and suggests a different targetome), their 3´-endpoint, or both simultaneously. Our analysis of Argonaute PAR-CLIP data from LCLs supports the association of many of these newly identified isomiRs with the Argonaute silencing complex and thus their functional roles through participation in the RNA interference pathway.

High-Throughput Profiling of Caenorhabditis elegans Starvation-Responsive microRNAs

PubMed Central

Garcia-Segura, Laura; Abreu-Goodger, Cei; Hernandez-Mendoza, Armando; Dimitrova Dinkova, Tzvetanka D.; Padilla-Noriega, Luis; Perez-Andrade, Martha Elva; Miranda-Rios, Juan

2015-01-01

MicroRNAs (miRNAs) are non-coding RNAs of ~22 nucleotides in length that regulate gene expression by interfering with the stability and translation of mRNAs. Their expression is regulated during development, under a wide variety of stress conditions and in several pathological processes. In nature, animals often face feast or famine conditions. We observed that subjecting early L4 larvae from Caenorhabditis elegans to a 12-hr starvation period produced worms that are thinner and shorter than well-fed animals, with a decreased lipid accumulation, diminished progeny, reduced gonad size, and an increased lifespan. Our objective was to identify which of the 302 known miRNAs of C. elegans changed their expression under starvation conditions as compared to well-fed worms by means of deep sequencing in early L4 larvae. Our results indicate that 13 miRNAs (miR-34-3p, the family of miR-35-3p to miR-41-3p, miR-39-5p, miR-41-5p, miR-240-5p, miR-246-3p and miR-4813-5p) were upregulated, while 2 miRNAs (let-7-3p and miR-85-5p) were downregulated in 12-hr starved vs. well-fed early L4 larvae. Some of the predicted targets of the miRNAs that changed their expression in starvation conditions are involved in metabolic or developmental process. In particular, miRNAs of the miR-35 family were upregulated 6–20 fold upon starvation. Additionally, we showed that the expression of gld-1, important in oogenesis, a validated target of miR-35-3p, was downregulated when the expression of miR-35-3p was upregulated. The expression of another reported target, the cell cycle regulator lin-23, was unchanged during starvation. This study represents a starting point for a more comprehensive understanding of the role of miRNAs during starvation in C. elegans. PMID:26554708

High-Throughput Profiling of Caenorhabditis elegans Starvation-Responsive microRNAs.

PubMed

Garcia-Segura, Laura; Abreu-Goodger, Cei; Hernandez-Mendoza, Armando; Dimitrova Dinkova, Tzvetanka D; Padilla-Noriega, Luis; Perez-Andrade, Martha Elva; Miranda-Rios, Juan

2015-01-01

MicroRNAs (miRNAs) are non-coding RNAs of ~22 nucleotides in length that regulate gene expression by interfering with the stability and translation of mRNAs. Their expression is regulated during development, under a wide variety of stress conditions and in several pathological processes. In nature, animals often face feast or famine conditions. We observed that subjecting early L4 larvae from Caenorhabditis elegans to a 12-hr starvation period produced worms that are thinner and shorter than well-fed animals, with a decreased lipid accumulation, diminished progeny, reduced gonad size, and an increased lifespan. Our objective was to identify which of the 302 known miRNAs of C. elegans changed their expression under starvation conditions as compared to well-fed worms by means of deep sequencing in early L4 larvae. Our results indicate that 13 miRNAs (miR-34-3p, the family of miR-35-3p to miR-41-3p, miR-39-5p, miR-41-5p, miR-240-5p, miR-246-3p and miR-4813-5p) were upregulated, while 2 miRNAs (let-7-3p and miR-85-5p) were downregulated in 12-hr starved vs. well-fed early L4 larvae. Some of the predicted targets of the miRNAs that changed their expression in starvation conditions are involved in metabolic or developmental process. In particular, miRNAs of the miR-35 family were upregulated 6-20 fold upon starvation. Additionally, we showed that the expression of gld-1, important in oogenesis, a validated target of miR-35-3p, was downregulated when the expression of miR-35-3p was upregulated. The expression of another reported target, the cell cycle regulator lin-23, was unchanged during starvation. This study represents a starting point for a more comprehensive understanding of the role of miRNAs during starvation in C. elegans.

A pilot study of urinary microRNA as a biomarker for urothelial cancer

PubMed Central

Snowdon, Jaime; Boag, Sandy; Feilotter, Harriet; Izard, Jason; Siemens, D. Robert

2013-01-01

Objective: MicroRNAs (miRNAs) are part of a class of small ribonucleic acid (RNAs). They are important regulatory molecules, involved in several cell processes, such as developmental timing, stem cell division and apoptosis. Dysregulated miRNAs have been identified in several human malignancies, including bladder cancer tissue samples, and may confer a “tumour signature” that can be exploited for diagnostic purposes. We report on a prospective pilot study investigating the diagnostic capability of miRNAs in the urine of patients with urothelial cancer. Methods: Voided urine samples were collected from patients with urothelial carcinoma just prior to bladder tumour resection, as well as age-matched healthy control patients. Pathology demonstrated both low- and high-grade cancer. Total RNA was isolated and quantitative reverse transcriptase-polymerase chain reaction was performed on the RNA extracts using primers for 4 miRNAs shown previously to be dysregulated in solid urothelial carcinomas with RNU6B as the endogenous control. Standard urine cytology was performed on all samples in a blinded fashion. Results: Two miRNAs of interest were dysregulated in the urine from cancer patients with miR-125b showing an average 10.42-fold decrease (p < 0.01) and miR-126 showing an average 2.70-fold increase (p = 0.30) in the cancer samples compared to the normal controls. The sensitivity and specificity of the cytology on the same urine samples were 50% and 80%, respectively. Using these 2 miRNAs only, a decision-tree prediction model was generated for a validation cohort of patients yielding a specificity of 100% and a sensitivity of 80%. Discussion: This preliminary study of candidate urinary miRNA in patients with low- and high-grade urothelial cancer demonstrated a significantly improved diagnostic accuracy over cytology. These results provide rationale for further studies on discovery and validation of candidate miRNAs in voided urine and may potentially lead to the development of a non-invasive and sensitive test for bladder cancer diagnosis and prognosis. PMID:22630336

Synchronization of developmental processes and defense signaling by growth regulating transcription factors.

PubMed

Liu, Jinyi; Rice, J Hollis; Chen, Nana; Baum, Thomas J; Hewezi, Tarek

2014-01-01

Growth regulating factors (GRFs) are a conserved class of transcription factor in seed plants. GRFs are involved in various aspects of tissue differentiation and organ development. The implication of GRFs in biotic stress response has also been recently reported, suggesting a role of these transcription factors in coordinating the interaction between developmental processes and defense dynamics. However, the molecular mechanisms by which GRFs mediate the overlaps between defense signaling and developmental pathways are elusive. Here, we report large scale identification of putative target candidates of Arabidopsis GRF1 and GRF3 by comparing mRNA profiles of the grf1/grf2/grf3 triple mutant and those of the transgenic plants overexpressing miR396-resistant version of GRF1 or GRF3. We identified 1,098 and 600 genes as putative targets of GRF1 and GRF3, respectively. Functional classification of the potential target candidates revealed that GRF1 and GRF3 contribute to the regulation of various biological processes associated with defense response and disease resistance. GRF1 and GRF3 participate specifically in the regulation of defense-related transcription factors, cell-wall modifications, cytokinin biosynthesis and signaling, and secondary metabolites accumulation. GRF1 and GRF3 seem to fine-tune the crosstalk between miRNA signaling networks by regulating the expression of several miRNA target genes. In addition, our data suggest that GRF1 and GRF3 may function as negative regulators of gene expression through their association with other transcription factors. Collectively, our data provide new insights into how GRF1 and GRF3 might coordinate the interactions between defense signaling and plant growth and developmental pathways.

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

microRNA regulation of T lymphocyte immunity: modulation of molecular networks responsible for T cell activation, differentiation and development

PubMed Central

Podshivalova, Katie; Salomon, Daniel R.

2014-01-01

MicroRNAs (miRNA) are a class of small non-coding RNAs that constitute an essential and evolutionarily conserved mechanism for post-transcriptional gene regulation. Multiple miRNAs have been described to play key roles in T lymphocyte development, differentiation and function. In this review we highlight the current literature regarding the differential expression of miRNAs in various models of mouse and human T cell biology and emphasize mechanistic understandings of miRNA regulation of thymocyte development, T cell activation, and differentiation into effector and memory subsets. We describe the participation of miRNAs in complex regulatory circuits shaping T cell proteomes in a context-dependent manner. It is striking that some miRNAs regulate multiple processes, while others only appear in limited functional contexts. It is also evident that the expression and function of specific miRNAs can differ between mouse and human systems. Ultimately, it is not always correct to simplify the complex events of T cell biology into a model driven by only one or two master regulator miRNAs. In reality, T cell activation and differentiation involves the expression of multiple miRNAs with many mRNA targets and thus, the true extent of miRNA regulation of T cell biology is likely far more vast than currently appreciated. PMID:24099302

Functional screening identifies miRNAs inducing cardiac regeneration.

PubMed

Eulalio, Ana; Mano, Miguel; Dal Ferro, Matteo; Zentilin, Lorena; Sinagra, Gianfranco; Zacchigna, Serena; Giacca, Mauro

2012-12-20

In mammals, enlargement of the heart during embryonic development is primarily dependent on the increase in cardiomyocyte numbers. Shortly after birth, however, cardiomyocytes stop proliferating and further growth of the myocardium occurs through hypertrophic enlargement of the existing myocytes. As a consequence of the minimal renewal of cardiomyocytes during adult life, repair of cardiac damage through myocardial regeneration is very limited. Here we show that the exogenous administration of selected microRNAs (miRNAs) markedly stimulates cardiomyocyte proliferation and promotes cardiac repair. We performed a high-content microscopy, high-throughput functional screening for human miRNAs that promoted neonatal cardiomyocyte proliferation using a whole-genome miRNA library. Forty miRNAs strongly increased both DNA synthesis and cytokinesis in neonatal mouse and rat cardiomyocytes. Two of these miRNAs (hsa-miR-590 and hsa-miR-199a) were further selected for testing and were shown to promote cell cycle re-entry of adult cardiomyocytes ex vivo and to promote cardiomyocyte proliferation in both neonatal and adult animals. After myocardial infarction in mice, these miRNAs stimulated marked cardiac regeneration and almost complete recovery of cardiac functional parameters. The miRNAs identified hold great promise for the treatment of cardiac pathologies consequent to cardiomyocyte loss.

Replication of Many Human Viruses Is Refractory to Inhibition by Endogenous Cellular MicroRNAs

PubMed Central

Bogerd, Hal P.; Skalsky, Rebecca L.; Kennedy, Edward M.; Furuse, Yuki; Whisnant, Adam W.; Flores, Omar; Schultz, Kimberly L. W.; Putnam, Nicole; Barrows, Nicholas J.; Sherry, Barbara; Scholle, Frank; Garcia-Blanco, Mariano A.; Griffin, Diane E.

2014-01-01

ABSTRACT The issue of whether viruses are subject to restriction by endogenous microRNAs (miRNAs) and/or by virus-induced small interfering RNAs (siRNAs) in infected human somatic cells has been controversial. Here, we address this question in two ways. First, using deep sequencing, we demonstrate that infection of human cells by the RNA virus dengue virus (DENV) or West Nile virus (WNV) does not result in the production of any virus-derived siRNAs or viral miRNAs. Second, to more globally assess the potential of small regulatory RNAs to inhibit virus replication, we used gene editing to derive human cell lines that lack a functional Dicer enzyme and that therefore are unable to produce miRNAs or siRNAs. Infection of these cells with a wide range of viruses, including DENV, WNV, yellow fever virus, Sindbis virus, Venezuelan equine encephalitis virus, measles virus, influenza A virus, reovirus, vesicular stomatitis virus, human immunodeficiency virus type 1, or herpes simplex virus 1 (HSV-1), failed to reveal any enhancement in the replication of any of these viruses, although HSV-1, which encodes at least eight Dicer-dependent viral miRNAs, did replicate somewhat more slowly in the absence of Dicer. We conclude that most, and perhaps all, human viruses have evolved to be resistant to inhibition by endogenous human miRNAs during productive replication and that dependence on a cellular miRNA, as seen with hepatitis C virus, is rare. How viruses have evolved to avoid inhibition by endogenous cellular miRNAs, which are generally highly conserved during metazoan evolution, remains to be determined. IMPORTANCE Eukaryotic cells express a wide range of small regulatory RNAs, including miRNAs, that have the potential to inhibit the expression of mRNAs that show sequence complementarity. Indeed, previous work has suggested that endogenous miRNAs have the potential to inhibit viral gene expression and replication. Here, we demonstrate that the replication of a wide range of pathogenic viruses is not enhanced in human cells engineered to be unable to produce miRNAs, indicating that viruses have evolved to be resistant to inhibition by miRNAs. This result is important, as it implies that manipulation of miRNA levels is not likely to prove useful in inhibiting virus replication. It also focuses attention on the question of how viruses have evolved to resist inhibition by miRNAs and whether virus mutants that have lost this resistance might prove useful, for example, in the development of attenuated virus vaccines. PMID:24807715

Let-7 represses Nr6a1 and a mid-gestation developmental program in adult fibroblasts

PubMed Central

Gurtan, Allan M.; Ravi, Arvind; Rahl, Peter B.; Bosson, Andrew D.; JnBaptiste, Courtney K.; Bhutkar, Arjun; Whittaker, Charles A.; Young, Richard A.; Sharp, Phillip A.

2013-01-01

MicroRNAs (miRNAs) are critical to proliferation, differentiation, and development. Here, we characterize gene expression in murine Dicer-null adult mesenchymal stem cell lines, a fibroblast cell type. Loss of Dicer leads to derepression of let-7 targets at levels that exceed 10-fold to 100-fold with increases in transcription. Direct and indirect targets of this miRNA belong to a mid-gestation embryonic program that encompasses known oncofetal genes as well as oncogenes not previously associated with an embryonic state. Surprisingly, this mid-gestation program represents a distinct period that occurs between the pluripotent state of the inner cell mass at embryonic day 3.5 (E3.5) and the induction of let-7 upon differentiation at E10.5. Within this mid-gestation program, we characterize the let-7 target Nr6a1, an embryonic transcriptional repressor that regulates gene expression in adult fibroblasts following miRNA loss. In total, let-7 is required for the continual suppression of embryonic gene expression in adult cells, a mechanism that may underlie its tumor-suppressive function. PMID:23630078

microRNA-mediated R gene regulation: molecular scabbards for double-edged swords.

PubMed

Deng, Yingtian; Liu, Minglei; Li, Xiaofei; Li, Feng

2018-02-01

Plant resistance (R) proteins are immune receptors that recognize pathogen effectors and trigger rapid defense responses, namely effector-triggered immunity. R protein-mediated pathogen resistance is usually race specific. During plant-pathogen coevolution, plant genomes accumulated large numbers of R genes. Even though plant R genes provide important natural resources for breeding disease-resistant crops, their presence in the plant genome comes at a cost. Misregulation of R genes leads to developmental defects, such as stunted growth and reduced fertility. In the past decade, many microRNAs (miRNAs) have been identified to target various R genes in plant genomes. miRNAs reduce R gene levels under normal conditions and allow induction of R gene expression under various stresses. For these reasons, we consider R genes to be double-edged "swords" and miRNAs as molecular "scabbards". In the present review, we summarize the contributions and potential problems of these "swords" and discuss the features and production of the "scabbards", as well as the mechanisms used to pull the "sword" from the "scabbard" when needed.

miRNome Expression Analysis Reveals New Players on Leprosy Immune Physiopathology

PubMed Central

Salgado, Claudio Guedes; Pinto, Pablo; Bouth, Raquel Carvalho; Gobbo, Angélica Rita; Messias, Ana Caroline Cunha; Sandoval, Tatiana Vinasco; dos Santos, André Mauricio Ribeiro; Moreira, Fabiano Cordeiro; Vidal, Amanda Ferreira; Goulart, Luiz Ricardo; Barreto, Josafá Gonçalves; da Silva, Moisés Batista; Frade, Marco Andrey Cipriani; Spencer, John Stewart; Santos, Sidney; Ribeiro-dos-Santos, Ândrea

2018-01-01

Leprosy remains as a public health problem and its physiopathology is still not fully understood. MicroRNAs (miRNA) are small RNA non-coding that can interfere with mRNA to regulate gene expression. A few studies using DNA chip microarrays have explored the expression of miRNA in leprosy patients using a predetermined set of genes as targets, providing interesting findings regarding the regulation of immune genes. However, using a predetermined set of genes restricted the possibility of finding new miRNAs that might be involved in different mechanisms of disease. Thus, we examined the miRNome of tuberculoid (TT) and lepromatous (LL) patients using both blood and lesional biopsies from classical leprosy patients (LP) who visited the Dr. Marcello Candia Reference Unit in Sanitary Dermatology in the State of Pará and compared them with healthy subjects. Using a set of tools to correlate significantly differentially expressed miRNAs with their gene targets, we identified possible interactions and networks of miRNAs that might be involved in leprosy immunophysiopathology. Using this approach, we showed that the leprosy miRNA profile in blood is distinct from that in lesional skin as well as that four main groups of genes are the targets of leprosy miRNA: (1) recognition and phagocytosis, with activation of immune effector cells, where the immunosuppressant profile of LL and immunoresponsive profile of TT are clearly affected by miRNA expression; (2) apoptosis, with supportive data for an antiapoptotic leprosy profile based on BCL2, MCL1, and CASP8 expression; (3) Schwann cells (SCs), demyelination and epithelial–mesenchymal transition (EMT), supporting a role for different developmental or differentiation gene families, such as Sox, Zeb, and Hox; and (4) loss of sensation and neuropathic pain, revealing that RHOA, ROCK1, SIGMAR1, and aquaporin-1 (AQP1) may be involved in the loss of sensation or leprosy pain, indicating possible new therapeutic targets. Additionally, AQP1 may also be involved in skin dryness and loss of elasticity, which are well known signs of leprosy but with unrecognized physiopathology. In sum, miRNA expression reveals new aspects of leprosy immunophysiopathology, especially on the regulation of the immune system, apoptosis, SC demyelination, EMT, and neuropathic pain. PMID:29593724

Sex specific expression and distribution of small RNAs in papaya.

PubMed

Aryal, Rishi; Jagadeeswaran, Guru; Zheng, Yun; Yu, Qingyi; Sunkar, Ramanjulu; Ming, Ray

2014-01-13

Regulatory function of small non-coding RNAs (sRNA) in response to environmental and developmental cues has been established. Additionally, sRNA, also plays an important role in maintaining the heterochromatin and centromere structures of the chromosome. Papaya, a trioecious species with recently evolved sex chromosomes, has emerged as an excellent model system to study sex determination and sex chromosome evolution in plants. However, role of small RNA in papaya sex determination is yet to be explored. We analyzed the high throughput sRNAs reads in the Illumina libraries prepared from male, female, and hermaphrodite flowers of papaya. Using the sRNA reads, we identified 29 miRNAs that were not previously reported from papaya. Including this and two previous studies, a total of 90 miRNAs has been identified in papaya. We analyzed the expression of these miRNAs in each sex types. A total of 65 miRNAs, including 31 conserved and 34 novel mirNA, were detected in at least one library. Fourteen of the 65 miRNAs were differentially expressed among different sex types. Most of the miRNA expressed higher in male flowers were related to the auxin signaling pathways, whereas the miRNAs expressed higher in female flowers were the potential regulators of the apical meristem identity genes. Aligning the sRNA reads identified the sRNA hotspots adjacent to the gaps of the X and Y chromosomes. The X and Y chromosomes sRNA hotspots has a 7.8 and 4.4 folds higher expression of sRNA, respectively, relative to the chromosome wide average. Approximately 75% of the reads aligned to the X chromosome hotspot was identical to that of the Y chromosome hotspot. By analyzing the large-scale sRNA sequences from three sex types, we identified the sRNA hotspots flanking the gaps of papaya X, Y, and Yh chromosome. The sRNAs expression patterns in these regions were reminiscent of the pericentromeric region indicating that the only remaining gap in each of these chromosomes is likely the centromere. We also identified 14 differentially expressed miRNAs in male, female and hermaphrodite flowers of papaya. Our results provide valuable information toward understanding the papaya sex determination.

miRNome Expression Analysis Reveals New Players on Leprosy Immune Physiopathology.

PubMed

Salgado, Claudio Guedes; Pinto, Pablo; Bouth, Raquel Carvalho; Gobbo, Angélica Rita; Messias, Ana Caroline Cunha; Sandoval, Tatiana Vinasco; Dos Santos, André Mauricio Ribeiro; Moreira, Fabiano Cordeiro; Vidal, Amanda Ferreira; Goulart, Luiz Ricardo; Barreto, Josafá Gonçalves; da Silva, Moisés Batista; Frade, Marco Andrey Cipriani; Spencer, John Stewart; Santos, Sidney; Ribeiro-Dos-Santos, Ândrea

2018-01-01

Leprosy remains as a public health problem and its physiopathology is still not fully understood. MicroRNAs (miRNA) are small RNA non-coding that can interfere with mRNA to regulate gene expression. A few studies using DNA chip microarrays have explored the expression of miRNA in leprosy patients using a predetermined set of genes as targets, providing interesting findings regarding the regulation of immune genes. However, using a predetermined set of genes restricted the possibility of finding new miRNAs that might be involved in different mechanisms of disease. Thus, we examined the miRNome of tuberculoid (TT) and lepromatous (LL) patients using both blood and lesional biopsies from classical leprosy patients (LP) who visited the Dr. Marcello Candia Reference Unit in Sanitary Dermatology in the State of Pará and compared them with healthy subjects. Using a set of tools to correlate significantly differentially expressed miRNAs with their gene targets, we identified possible interactions and networks of miRNAs that might be involved in leprosy immunophysiopathology. Using this approach, we showed that the leprosy miRNA profile in blood is distinct from that in lesional skin as well as that four main groups of genes are the targets of leprosy miRNA: (1) recognition and phagocytosis, with activation of immune effector cells, where the immunosuppressant profile of LL and immunoresponsive profile of TT are clearly affected by miRNA expression; (2) apoptosis, with supportive data for an antiapoptotic leprosy profile based on BCL2, MCL1 , and CASP8 expression; (3) Schwann cells (SCs), demyelination and epithelial-mesenchymal transition (EMT), supporting a role for different developmental or differentiation gene families, such as Sox, Zeb, and Hox; and (4) loss of sensation and neuropathic pain, revealing that RHOA, ROCK1, SIGMAR1 , and aquaporin-1 ( AQP1 ) may be involved in the loss of sensation or leprosy pain, indicating possible new therapeutic targets. Additionally, AQP1 may also be involved in skin dryness and loss of elasticity, which are well known signs of leprosy but with unrecognized physiopathology. In sum, miRNA expression reveals new aspects of leprosy immunophysiopathology, especially on the regulation of the immune system, apoptosis, SC demyelination, EMT, and neuropathic pain.

miR156a Mimic Represses the Epithelial-Mesenchymal Transition of Human Nasopharyngeal Cancer Cells by Targeting Junctional Adhesion Molecule A.

PubMed

Tian, Yunhong; Cai, Longmei; Tian, Yunming; Tu, Yinuo; Qiu, Huizhi; Xie, Guofeng; Huang, Donglan; Zheng, Ronghui; Zhang, Weijun

2016-01-01

MicroRNAs (miRNAs) have been documented as having an important role in the development of cancer. Broccoli is very popular in large groups of the population and has anticancer properties. Junctional adhesion molecule A (JAMA) is preferentially concentrated at tight junctions and influences cell morphology and migration. Epithelial-mesenchymal transition (EMT) is a developmental program associated with cancer progression and metastasis. In this study we aimed to investigate the role of miRNAs from broccoli in human nasopharyngeal cancer (NPC). We demonstrated that a total of 84 conserved miRNAs and 184 putative novel miRNAs were found in broccoli by sequencing technology. Among these, miR156a was expressed the most. In addition, synthetic miR156a mimic inhibited the EMT of NPC cells in vitro. Furthermore, it was confirmed that JAMA was the target of miR156a mimic as validated by 3' UTR luciferase reporter assays and western blotting. Knockdown of JAMA was consistent with the effects of miR156a mimic on the EMT of NPC, and the up-regulation of JAMA could partially restore EMT repressed by miR156a mimic. In conclusion, these results indicate that the miR156a mimic inhibits the EMT of NPC cells by targeting the 3' UTR of JAMA. These miRNA profiles of broccoli provide a fundamental basis for further research. Moreover, the discovery of miR156a may have clinical implications for the treatment of patients with NPC.

Plant growth retardation and conserved miRNAs are correlated to Hibiscus chlorotic ringspot virus infection.

PubMed

Gao, Ruimin; Wan, Zi Yi; Wong, Sek-Man

2013-01-01

Virus infection may cause a multiplicity of symptoms in their host including discoloration, distortion and growth retardation. Hibiscus chlorotic ringspot virus (HCRSV) infection was studied using kenaf (Hibiscus cannabinus L.), a non-wood fiber-producing crop in this study. Infection by HCRSV reduced the fiber yield and concomitant economic value of kenaf. We investigated kenaf growth retardation and fluctuations of four selected miRNAs after HCRSV infection. Vegetative growth (including plant height, leaf size and root development) was severely retarded. From the transverse and radial sections of the mock and HCRSV-infected kenaf stem, the vascular bundles of HCRSV-infected plants were severely disrupted. In addition, four conserved plant developmental and defence related microRNAs (miRNAs) (miR165, miR167, miR168 and miR171) and their respective target genes phabulosa (PHB), auxin response factor 8 (ARF8), argonaute 1 (AGO1) and scarecrow-like protein 1 (SCL1) displayed variation in expression levels after HCRSV infection. Compared with the mock inoculated kenaf plants, miR171 and miR168 and their targets SCL1 and AGO1 showed greater fluctuations after HCRSV infection. As HCRSV upregulates plant SO transcript in kenaf and upregulated AGO1 in HCRSV-infected plants, the expression level of AGO1 transcript was further investigated under sulfite oxidase (SO) overexpression or silencing condition. Interestingly, the four selected miRNAs were also up- or down-regulated upon overexpression or silencing of SO. Plant growth retardation and fluctuation of four conserved miRNAs are correlated to HCRSV infection.

Plant Growth Retardation and Conserved miRNAs Are Correlated to Hibiscus Chlorotic Ringspot Virus Infection

PubMed Central

Gao, Ruimin; Wan, Zi Yi; Wong, Sek-Man

2013-01-01

Virus infection may cause a multiplicity of symptoms in their host including discoloration, distortion and growth retardation. Hibiscus chlorotic ringspot virus (HCRSV) infection was studied using kenaf (Hibiscus cannabinus L.), a non-wood fiber-producing crop in this study. Infection by HCRSV reduced the fiber yield and concomitant economic value of kenaf. We investigated kenaf growth retardation and fluctuations of four selected miRNAs after HCRSV infection. Vegetative growth (including plant height, leaf size and root development) was severely retarded. From the transverse and radial sections of the mock and HCRSV-infected kenaf stem, the vascular bundles of HCRSV-infected plants were severely disrupted. In addition, four conserved plant developmental and defence related microRNAs (miRNAs) (miR165, miR167, miR168 and miR171) and their respective target genes phabulosa (PHB), auxin response factor 8 (ARF8), argonaute 1 (AGO1) and scarecrow-like protein 1 (SCL1) displayed variation in expression levels after HCRSV infection. Compared with the mock inoculated kenaf plants, miR171 and miR168 and their targets SCL1 and AGO1 showed greater fluctuations after HCRSV infection. As HCRSV upregulates plant SO transcript in kenaf and upregulated AGO1 in HCRSV-infected plants, the expression level of AGO1 transcript was further investigated under sulfite oxidase (SO) overexpression or silencing condition. Interestingly, the four selected miRNAs were also up- or down-regulated upon overexpression or silencing of SO. Plant growth retardation and fluctuation of four conserved miRNAs are correlated to HCRSV infection. PMID:24386476

Role of microRNAs in the regulation of innate immune cells under neuroinflammatory conditions.

PubMed

Cardoso, Ana L; Guedes, Joana R; de Lima, Maria C Pedroso

2016-02-01

MiRNAs are short, evolutionary conserved noncoding RNA molecules with the ability to control the magnitude of inflammation. The immunosuppressive nature of the brain is sustained by miRNA-dependent regulation of microglial cells, which become activated under neuroinflammatory conditions, such as brain injury and neurodegeneration. The pro-inflammatory and suppressive role of the most studied neuroimmune miRNAs, miR-155 and miR-146a, has been recently challenged. Although the molecular targets of these miRNAs remain unchanged across brain diseases, different kinetics of miRNA expression and degradation can produce different immune outcomes and change microglia phenotypes. Here, we discuss current knowledge regarding the implications of disruption of miRNA networks in neuroinflammation and in the pathophysiology of acute and chronic CNS diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparative characterization of microRNAs in Schistosoma japonicum schistosomula from Wistar rats and BALB/c mice.

PubMed

Han, Hongxiao; Peng, Jinbiao; Hong, Yang; Fu, Zhiqiang; Lu, Ke; Li, Hao; Zhu, Chuangang; Zhao, Qiuhua; Lin, Jiaojiao

2015-07-01

More than 40 kinds of mammals in China are known to be naturally infected with Schistosoma japonicum (S. japonicum) (Peng et al. Parasitol Res 106:967-76, 2010). Compared with permissive BALB/c mice, rats are less susceptible to S. japonicum infection and are considered to provide an unsuitable microenvironment for parasite growth and development. MicroRNAs (miRNAs), via the regulation of gene expression at the transcriptional and post-transcriptional levels, may be responsible for developmental differences between schistosomula in these two rodent hosts. Solexa deep-sequencing technology was used to identify differentially expressed miRNAs from schistosomula isolated from Wistar rats and BALB/c mice 10 days post-infection. The deep-sequencing analysis revealed that nearly 40 % of raw reads (10.37 and 10.84 million reads in schistosomula isolated from Wistar rats and BALB/c mice, respectively) can be mapped to selected mirs in miRBase or in species-specific genomes. Further analysis revealed that several miRNAs were differentially expressed in schistosomula isolated from these two rodents; 18 were downregulated (by <2-fold) and 23 were up-regulated (>2-fold) (expression levels in rats compare with those in mice). Additionally, three novel miRNAs were primarily predicted and identified. Among the 41 differentially expressed miRNAs, 4 miRNAs had been identified with specific functions in schistosome development or host-parasite interaction, such as sexual maturation (sja-miR-1, sja-miR-7-5p), embryo development (sja-miR-36-3p) in schistosome, and pathogenesis of schistosomiasis (sja-bantam). Then, the target genes were mapped, filtered, and correlated with a set of genes that were differentially expressed genes in schistosomula isolated from mice and rats, which we identified in a S. japonicum oligonucleotide microarray analysis in a previous study. Gene Ontology (GO) analysis of the predicted target genes of 13 differentially expressed miRNAs revealed that they were involved in some important biological pathways, such as metabolic processes, the regulation of protein catabolic processes, catalytic activity, oxidoreductase activity, and hydrolase activity. The study presented here includes the first identification of differentially expressed miRNAs between schistosomula in mice or rats. Therefore, we hypothesized that the differentially expressed miRNAs may affect the development, growth, and maturation of the schistosome in its life cycle. Our analysis suggested that some differentially expressed miRNAs may impact the survival and development of the parasite within a host. This study increases our understanding of schistosome development and host-parasite interactions.

The therapeutic potential of miRNAs in cardiac fibrosis: where do we stand?

PubMed

Wijnen, Wino J; Pinto, Yigal M; Creemers, Esther E

2013-12-01

Recent developments in basic and clinical science have turned the spotlight to miRNAs for their potential therapeutic efficacy. Since their discovery in 1993, it has become clear that miRNAs act as posttranscriptional regulators of protein expression. Their clinical potential was further highlighted by the results of miRNA-based interventions in small laboratory animals. More importantly, their therapeutic effectiveness has been shown recently in phase 2a clinical studies in patients with hepatitis C virus infection, where inhibition of miRNA-122 showed prolonged and dose-dependent viral suppression. A recent study surprisingly revealed the presence of plant-derived miRNAs in the blood of healthy humans. This finding opens up the possibility to explore miRNA-mediated therapeutics derived from (genetically modified) food. Having arrived at this point in our understanding of miRNAs, we provide an overview of current evidence and future potential of miRNA-based therapeutics, focusing on their application in cardiac fibrosis.

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

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.

Regulation of mouse stomach development and Barx1 expression by specific microRNAs

PubMed Central

Kim, Byeong-Moo; Woo, Janghee; Kanellopoulou, Chryssa; Shivdasani, Ramesh A.

2011-01-01

Although microRNAs (miRNAs) are postulated to fine-tune many developmental processes, their relationships with specific targets and tissues remain largely undefined. The mesenchymal transcription factor Barx1 controls spleen and stomach morphogenesis and is required to specify stomach-specific epithelium in adjacent endoderm. Barx1 expression is precisely regulated in space and time, with a sharp drop in stomach levels after epithelial specification. We tested the hypothesis that specific miRNAs mediate this marked decline in Barx1 levels. Depletion of the miRNA-processing enzyme Dicer in cultured stomach mesenchyme and conditional Dicer gene deletion in mice significantly increased Barx1 levels, disrupted stomach and intestine development and caused spleen agenesis. Computational and experimental studies identified miR-7a and miR-203 as candidate miRNAs that regulate Barx1 and are expressed in inverse proportion to it in the fetal mouse stomach. Through specific interactions with cognate sequences in the Barx1 3′ untranslated region, miR-7a and miR-203 repress Barx1 expression in stomach mesenchymal cells and its function in inducing gastric epithelium. These results indicate that miRNAs are required for proper digestive tract organogenesis and that miR-7a and miR-203 control expression of the stomach homeotic regulator Barx1. PMID:21307095

Oxidative stress, microRNAs and cytosolic calcium homeostasis.

PubMed

Magenta, Alessandra; Dellambra, Elena; Ciarapica, Roberta; Capogrossi, Maurizio C

2016-09-01

Reactive oxygen species increase cytosolic [Ca(2+)], (Cai), and also modulate the expression of some microRNAs (miRNAs), however the link among oxidative stress, miRNAs and Cai is poorly characterized. In this review we have focused on three groups of miRNAs: (a) miRNAs that are modulated both by ROS and Cai: miR-181a and miR-205; (b) miRNAs that are modulated by ROS and have an effect on Cai: miR-1, miR-21, miR-24, miR-25, miR-185 and miR-214; (c) miRNAs that modulate both ROS and Cai: miR-133; miR-145, miR-495, and we have analyzed their effects on cell signaling and cell function. Finally, in the last section we have examined the role of these miRNAs in the skin, under conditions associated with enhanced oxidative stress, i.e. skin aging, the response to ultraviolet light and two important skin diseases, psoriasis and atopic dermatitis. It is apparent that although some experimental evidence is already available on (a) the role of Cai in miRNAs expression and (b) on the ability of some miRNAs to modulate Cai-dependent intracellular signaling, these research lines are still largely unexplored and represent important areas of future studies. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

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

Global MicroRNA Profiling in Human Bone Marrow Skeletal-Stromal or Mesenchymal-Stem Cells Identified Candidates for Bone Regeneration.

PubMed

Chang, Chi-Chih; Venø, Morten T; Chen, Li; Ditzel, Nicholas; Le, Dang Q S; Dillschneider, Philipp; Kassem, Moustapha; Kjems, Jørgen

2018-02-07

Bone remodeling and regeneration are highly regulated multistep processes involving posttranscriptional regulation by microRNAs (miRNAs). Here, we performed a global profiling of differentially expressed miRNAs in bone-marrow-derived skeletal cells (BMSCs; also known as stromal or mesenchymal stem cells) during in vitro osteoblast differentiation. We functionally validated the regulatory effects of several miRNAs on osteoblast differentiation and identified 15 miRNAs, most significantly miR-222 and miR-423, as regulators of osteoblastogenesis. In addition, we tested the possible targeting of miRNAs for enhancing bone tissue regeneration. Scaffolds functionalized with miRNA nano-carriers enhanced osteoblastogenesis in 3D culture and retained this ability at least 2 weeks after storage. Additionally, anti-miR-222 enhanced in vivo ectopic bone formation through targeting the cell-cycle inhibitor CDKN1B (cyclin-dependent kinase inhibitor 1B). A number of additional miRNAs exerted additive osteoinductive effects on BMSC differentiation, suggesting that pools of miRNAs delivered locally from an implanted scaffold can provide a promising approach for enhanced bone regeneration. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Circulating microRNAs in breast cancer: novel diagnostic and prognostic biomarkers

PubMed Central

Hamam, Rimi; Hamam, Dana; Alsaleh, Khalid A; Kassem, Moustapha; Zaher, Waleed; Alfayez, Musaad; Aldahmash, Abdullah; Alajez, Nehad M

2017-01-01

Effective management of breast cancer depends on early diagnosis and proper monitoring of patients’ response to therapy. However, these goals are difficult to achieve because of the lack of sensitive and specific biomarkers for early detection and for disease monitoring. Accumulating evidence in the past several years has highlighted the potential use of peripheral blood circulating nucleic acids such as DNA, mRNA and micro (mi)RNA in breast cancer diagnosis, prognosis and for monitoring response to anticancer therapy. Among these, circulating miRNA is increasingly recognized as a promising biomarker, given the ease with which miRNAs can be isolated and their structural stability under different conditions of sample processing and isolation. In this review, we provide current state-of-the-art of miRNA biogenesis, function and discuss the advantages, limitations, as well as pitfalls of using circulating miRNAs as diagnostic, prognostic or predictive biomarkers in breast cancer management. PMID:28880270

Circulating microRNAs in breast cancer: novel diagnostic and prognostic biomarkers.

PubMed

Hamam, Rimi; Hamam, Dana; Alsaleh, Khalid A; Kassem, Moustapha; Zaher, Waleed; Alfayez, Musaad; Aldahmash, Abdullah; Alajez, Nehad M

2017-09-07

Effective management of breast cancer depends on early diagnosis and proper monitoring of patients' response to therapy. However, these goals are difficult to achieve because of the lack of sensitive and specific biomarkers for early detection and for disease monitoring. Accumulating evidence in the past several years has highlighted the potential use of peripheral blood circulating nucleic acids such as DNA, mRNA and micro (mi)RNA in breast cancer diagnosis, prognosis and for monitoring response to anticancer therapy. Among these, circulating miRNA is increasingly recognized as a promising biomarker, given the ease with which miRNAs can be isolated and their structural stability under different conditions of sample processing and isolation. In this review, we provide current state-of-the-art of miRNA biogenesis, function and discuss the advantages, limitations, as well as pitfalls of using circulating miRNAs as diagnostic, prognostic or predictive biomarkers in breast cancer management.

Precursor microRNA Programmed Silencing Complex Assembly Pathways in Mammals

PubMed Central

Liu, Xuhang; Jin, Dong-Yan; McManus, Michael T.; Mourelatos, Zissimos

2012-01-01

Summary Assembly of microRNA Ribonucleoproteins (miRNPs) or RNA-Induced Silencing Complexes (RISCs) is essential for the function of miRNAs and initiates from processing of precursor miRNAs (pre-miRNAs) by Dicer or by Ago2. Here, we report an in-vitro miRNP/RISC assembly assay programmed by pre-miRNAs from mammalian cell lysates. Combining in-vivo studies in Dicer Knock-Out cells reconstituted with wild type or catalytically inactive Dicer, we find that the miRNA Loading Complex (miRLC) is the primary machinery linking pre-miRNA processing to miRNA loading. We show that a miRNA Precursor Deposit Complex (miPDC) plays a crucial role in Dicer-independent miRNA biogenesis and promotes miRNP assembly of certain Dicer-dependent miRNAs. Furthermore, we find that 5′-uridine, 3′-mid base pairing and 5′-mid mismatches within pre-miRNAs promote their assembly into miPDC. Our studies provide a comprehensive view of miRNP/RISC assembly pathways in mammals and our assay provides a versatile platform for further mechanistic dissection of such pathways in mammals. PMID:22503104

Identification of heavy-ion radiation-induced microRNAs in rice

NASA Astrophysics Data System (ADS)

Zhang, Meng; Liang, Shujian; Hang, Xiaoming; Xiang, Yingxia; Cheng, Zhenlong; Li, Wenjian; Shi, Jinming; Huang, Lei; Sun, Yeqing

2011-03-01

MicroRNAs (miRNAs) are a family of small non-coding RNAs, which play significant roles in regulating development and stress responses in plant. As an excellent model organism for studying the effects of environmental stress, rice has been used to assess the damage of the space radiation environment for decades. Heavy-ions radiation show higher relative biological effectiveness compared to other cosmic-rays radiation. To identify the specific miRNAs that underlie biological effects of heavy-ion radiation, the germinated seeds of rice were exposed to 1 Gy, 10 Gy and 20 Gy dose of 12C heavy-ion radiation, respectively. Analysis of phenotype indicated that 20 Gy dose of heavy-ion radiation was the semi-lethal dose of rice seedling. The microarray of μparaflo™ chip was employed to monitor the expression profiles of miRNAs in rice (Oryza sativa) under 20 Gy dose of radiation stress. miR164a, miR164c, miR164d and miR156a-j were identified as heavy-ion radiation-induced miRNAs. miR164 and miR156 family were increased in all three exposed samples by using quantitative real-time PCR (qRT-RCP). As targets of miR156 and miR164, SQUAMOSA PROMOTER BINDING-LIKE (SPL) transcription factors and NAM/ATAF/CUC (NAC) transcription factors expression were down-regulated correlating with an up-regulated level of the regulated miRNAs. Since SPL transcription factors and NAC transcription factors regulated growth and development of plant, we used 2-dimension electrophoresis (2-DE) gel to analyze changes of functional proteins in 20 Gy exposed samples. It was evident that both the height and survival rates of seedlings were markedly decreased. The abundance of some developmentally regulated proteins was also changed. To our knowledge, this study is the first to report heavy-ion radiation stress responsive miRNAs in plant. Moreover, our findings are important to understand the molecular mechanism of space biology.

Cisplatin-resistant lung cancer cell-derived exosomes increase cisplatin resistance of recipient cells in exosomal miR-100-5p-dependent manner.

PubMed

Qin, Xiaobing; Yu, Shaorong; Zhou, Leilei; Shi, Meiqi; Hu, Yong; Xu, Xiaoyue; Shen, Bo; Liu, Siwen; Yan, Dali; Feng, Jifeng

2017-01-01

Exosomes derived from lung cancer cells confer cisplatin (DDP) resistance to other cancer cells. However, the underlying mechanism is still unknown. A549 resistance to DDP (A549/DDP) was established. Microarray was used to analyze microRNA (miRNA) expression profiles of A549 cells, A549/DDP cells, A549 exosomes, and A549/DDP exosomes. There was a strong correlation of miRNA profiles between exosomes and their maternal cells. A total of 11 miRNAs were significantly upregulated both in A549/DDP cells compared with A549 cells and in exosomes derived from A549/DDP cells in contrast to exosomes from A549 cells. A total of 31 downregulated miRNAs were also observed. miR-100-5p was the most prominent decreased miRNA in DDP-resistant exosomes compared with the corresponding sensitive ones. Downregulated miR-100-5p was proved to be involved in DDP resistance in A549 cells, and mammalian target of rapamycin (mTOR) expression was reverse regulated by miR-100-5p. Exosomes confer recipient cells' resistance to DDP in an exosomal miR-100-5p-dependent manner with mTOR as its potential target both in vitro and in vivo. Exosomes from DDP-resistant lung cancer cells A549 can alter other lung cancer cells' sensitivity to DDP in exosomal miR-100-5p-dependent manner. Our study provides new insights into the molecular mechanism of DDP resistance in lung cancer.

Systematic Identification, Characterization and Target Gene Analysis of microRNAs Involved in Osteoarthritis Subchondral Bone Pathogenesis.

PubMed

Prasadam, Indira; Batra, Jyotsna; Perry, Samuel; Gu, Wenyi; Crawford, Ross; Xiao, Yin

2016-07-01

This study aimed to identify the microRNAs associated with sclerotic status of subchondral bone in the pathogenesis of osteoarthritis (OA). Total RNA was extracted from non-sclerotic and sclerotic OA subchondral bone from patients undergoing knee replacement surgeries. miRCURY™ LNA miRNA chip and qRT-PCR were used to profile and validate differential microRNA expression. In addition, we further confirmed profiles of altered miRNAs in an OA rat meniscectomy animal model and their putative targets of the miRNAs were predicted using ingenuity (IPA) software. Finally, five short-listed miRNAs were reactivated by transient in vitro overexpression (miRNA mimics) in subchondral bone osteoblasts and their phenotypes were assessed. Functional screening identified 30 differentiated miRNAs in sclerotic subchondral bone compared to non-sclerotic bone of OA patients. Data integration resulted in confirmation of the eight miRNAs, with aberrant expression in independent human OA bone sample set. In silico analysis (IPA) identified 732 mRNA transcripts as putative targets of the eight altered miRNAs, of which twenty genes were validated to be differentially expressed in sclerotic compared to non-sclerotic bone samples. Out of eight dysregulated miRNA's, five of them showed consistent time-dependent downregulation in a rat OA model. Furthermore, synthetic miR-199a-3p, miR-199a-5p, miR-590-5p, and miR-211-5p mimics rescued the abnormal osteoarthritic subchondral bone osteoblast gene expression and mineralization. We have identified four novel miRNAs that play important roles in subchondral bone pathogenesis in OA. Additional studies are required to develop these miRNAs into therapeutic modalities for OA.

miRNAs as therapeutic targets in ischemic heart disease.

PubMed

Frost, Robert J A; van Rooij, Eva

2010-06-01

Ischemic heart disease is a form of congestive heart failure that is caused by insufficient blood supply to the heart, resulting in a loss of viable tissue. In response to the injury, the non-ischemic myocardium displays signs of secondary remodeling, like interstitial fibrosis and hypertrophy of cardiac myocytes. This remodeling process further deteriorates pump function and increases susceptibility to arrhythmias. MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression in a sequence-dependent manner. Recently, several groups identified miRNAs as crucial gene regulators in response to myocardial infarction (MI) and during post-MI remodeling. In this review, we discuss how modulation of these miRNAs represents a promising new therapeutic strategy to improve the clinical outcome in ischemic heart disease.

The role of micro-RNAs in hepatocellular carcinoma: from molecular biology to treatment.

PubMed

D'Anzeo, Marco; Faloppi, Luca; Scartozzi, Mario; Giampieri, Riccardo; Bianconi, Maristella; Del Prete, Michela; Silvestris, Nicola; Cascinu, Stefano

2014-05-19

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third leading cause of cancer deaths. microRNAs (miRNAs) are evolutionary conserved small non-coding RNA that negatively regulate gene expression and protein translation. Recent evidences have shown that they are involved in many biological processes, from development and cell-cycle regulation to apoptosis. miRNAs can behave as tumor suppressor or promoter of oncogenesis depending on the cellular function of their targets. Moreover, they are frequently dysregulated in HCC. In this review we summarize the latest findings of miRNAs regulation in HCC and their role as potentially diagnostic and prognostic biomarkers for HCC. We highlight development of miRNAs as potential therapeutic targets for HCC.

MiR-214 regulates the function of osteoblast under simulated microgravity by targeting ATF4

NASA Astrophysics Data System (ADS)

Li, Yingxian; Wang, Xiaogang; Li, Qi; Lv, Ke; Wan, Yumin; Li, Yinghui; Bai, Yanqiang

Background: MicroRNAs (miRNAs) are small fragments of single-stranded RNA containing 18-24 nucleotides, and are generated from endogenous transcripts. MicroRNAs function in post-transcriptional gene silencing by targeting the 3'-untranslated region (UTR) of mRNAs, resulting in translational repression. Growing evidence shows that microRNAs (miRNAs) regu-late various developmental and homeostatic events in vertebrates and invertebrates. Osteoblast differentiation is a key step in proper skeletal development and acquisition of bone mass; How-ever, the physiological role of non-coding small RNAs, especially miRNAs, in osteoblast dif-ferentiation remains elusive. Methods: To study the potential involvement of miRNAs in osteoblast differentiation under stimulated microgravity, we analyzed the expression of 20 bone relative miRNAs using real time PCR platform to find particularly miRNAs whose expression is altered during osteoblast differentiation. TargetScan, miRBase and Miranda were used to predict the target gene of candidate miRNA. To investigate whether ATF4 can be directly targeted by miR-214, we engineered luciferase reporters that have either the wild-type 3'UTRs of these genes, or the mutant UTRs with a 6 base pair (bp) deletion in the target sites. Lastly, to address the in vivo role of miR-214 in bone formation, tail suspension mice model was used to simulate the change of osteoblast function and bone loss. Results: Recent studies have sug-gested that miRNAs might play a role in osteoblast differentiation and bone formation. Here, we identify miR-214 in MC3T3-E1 cells, which is a primary mouse osteoblasts cell line, to promote osteoblast differentiation by repressing Activating Transcription Factor4 (ATF4) ex-pression at the posttranscriptional level. What is more, miR-214 was found to be transcribed in C2C12 cells during bone morphogenetic protein 2-induced (BMP2-induced) osteogenesis, and overexpression of miR-214 attenuated BMP2-induced osteoblastogenesis, whereas inhibition of miR-214 expression enhanced this progress. The levels of miR-214 increased dramatically in tail suspension mice. Conclusions: Thus, our studies show that miR-214 plays an important role in osteoblast differentiation by targeting ATF4 under stimulated microgravity induced bone loss and contributes to osteoporosis via its effect on osteoblasts.

Profiling of short RNAs during fleshy fruit development reveals stage-specific sRNAome expression patterns.

PubMed

Mohorianu, Irina; Schwach, Frank; Jing, Runchun; Lopez-Gomollon, Sara; Moxon, Simon; Szittya, Gyorgy; Sorefan, Karim; Moulton, Vincent; Dalmay, Tamas

2011-07-01

Plants feature a particularly diverse population of short (s)RNAs, the central component of all RNA silencing pathways. Next generation sequencing techniques enable deeper insights into this complex and highly conserved mechanism and allow identification and quantification of sRNAs. We employed deep sequencing to monitor the sRNAome of developing tomato fruits covering the period between closed flowers and ripened fruits by profiling sRNAs at 10 time-points. It is known that microRNAs (miRNAs) play an important role in development but very little information is available about the majority of sRNAs that are not miRNAs. Here we show distinctive patterns of sRNA expression that often coincide with stages of the developmental process such as flowering, early and late fruit maturation. Moreover, thousands of non-miRNA sRNAs are differentially expressed during fruit development and ripening. Some of these differentially expressed sRNAs derived from transposons but many derive from protein coding genes or regions that show homology to protein coding genes, several of which are known to play a role in flower and fruit development. These findings raise the possibility of a regulative role of these sRNAs during fruit onset and maturation in a crop species. We also identified six new miRNAs and experimentally validated two target mRNAs. These two mRNAs are targeted by the same miRNA but do not belong to the same gene family, which is rare for plant miRNAs. Expression pattern and putative function of these targets indicate a possible role in glutamate accumulation, which contributes to establishing the taste of the fruit. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

State-of-the-art on viral microRNAs in HPV infection and cancer development.

PubMed

Poltronieri, Palmiro; Sun, Binlian; Huang, Kai-Yao; Chang, Tzu-Hao; Lee, Tzong-Yi

2018-03-27

high-risk HPV subtypes are driving forces for human cancer development: HPV-16 and HPV-18 are responsible for most HPV-caused cancers. This review describes the present knowledge on HR-HPV genomes coding potential for viral miRNAs. HPV subtypes miRNA database, VIRmiRtar, has been constructed applying bioinformatics and a computational method, ViralMir, exploiting structural features, presence of hairpins, and validation by comparison with RNA sequencing datasets. Several miRNA candidates have been localised in the genomes of high-risk HPV subtypes. Among these, HPV-16 miR-1, miR-2 and miR-3. The database contains a list of host candidate gene targets that may be responsible for the oncogenesis in the various cellular environments. miRNA silencing therapies, based on specific cellular uptake of miRNA mimics and antagomiRs, directed towards HPV encoded miRNAs and/or microRNAs deregulated in the host cells, could be a valuable approach to support pharmaceutical interventions in the treatment of HPV dependent cancers. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

Dicer inactivation leads to progressive functional and structural degeneration of the mouse retina

PubMed Central

Damiani, Devid; Alexander, John J; O'Rourke, Jason R; McManus, Mike; Jadhav, Ashutosh P; Cepko, Constance L; Hauswirth, William W; Harfe, Brian D; Strettoi, Enrica

2009-01-01

MicroRNAs (miRNAs) are small, highly conserved molecules that have been shown to regulate the expression of genes by binding to specific target mRNAs. Dicer, an RNase III endonuclease, is essential for the production and function of mature miRNAs and removal of Dicer has been shown to disrupt many developmental processes. In this report, Dicer was removed specifically from the retina using a floxed Dicer conditional allele and the retinal Chx10Cre transgene. Retinal Dicer knockout mice displayed a reproducible inability to respond to light. In addition, morphological defects were observed with the formation of photoreceptor rosettes at P16 which progressed to more general cellular disorganization and widespread degeneration of retinal cell types as the animals aged. This was accompanied by concomitant decrease in both scotopic and photopic ERG responses. Interestingly, removing a single allele of Dicer resulted in ERG deficits throughout life but not to morphological abnormalities. Northern blot analysis of Dicer depleted retinas showed a decrease in several microRNAs. The observation that progressive retinal degeneration occurred upon removal of Dicer raises the possibility that miRNAs are involved in retinal neurodegenerative disorders. PMID:18463241

Aryl hydrocarbon receptor (AhR)-dependent regulation of pulmonary miRNA by chronic cigarette smoke exposure.

PubMed

Rogers, Sarah; de Souza, Angela Rico; Zago, Michela; Iu, Matthew; Guerrina, Necola; Gomez, Alvin; Matthews, Jason; Baglole, Carolyn J

2017-01-12

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor historically known for its toxic responses to man-made pollutants such as dioxin. More recently, the AhR has emerged as a suppressor of inflammation, oxidative stress and apoptosis from cigarette smoke by mechanisms that may involve the regulation of microRNA. However, little is known about the AhR regulation of miRNA expression in the lung in response to inhaled toxicants. Therefore, we exposed Ahr -/- and Ahr +/- mice to cigarette smoke for 4 weeks and evaluated lung miRNA expression by PCR array. There was a dramatic regulation of lung miRNA by the AhR in the absence of exogenous ligand. In response to cigarette smoke, there were more up-regulated miRNA in Ahr -/- mice compared to Ahr +/- mice, including the cancer-associated miRNA miR-96. There was no significant change in the expression of the AhR regulated proteins HuR and cyclooxygenase-2 (COX-2). There were significant increases in the anti-oxidant gene sulfiredoxin 1 (Srxn1) and FOXO3a- predicted targets of miR-96. Collectively, these data support a prominent role for the AhR in regulating lung miRNA expression. Further studies to elucidate a role for these miRNA may further uncover novel biological function for the AhR in respiratory health and disease.

MicroRNAs That Contribute to Coordinating the Immune Response in Drosophila melanogaster

PubMed Central

Atilano, Magda L.; Glittenberg, Marcus; Monteiro, Annabel; Copley, Richard R.; Ligoxygakis, Petros

2017-01-01

Small noncoding RNAs called microRNAs (miRNAs) have emerged as post-transcriptional regulators of gene expression related to host defenses. Here, we have used Drosophila melanogaster to explore the contribution of individual or clusters of miRNAs in countering systemic Candida albicans infection. From a total of 72 tested, we identify 6 miRNA allelic mutant backgrounds that modulate the survival response to infection and the ability to control pathogen number. These mutants also exhibit dysregulation of the Toll pathway target transcripts Drosomycin (Drs) and Immune-Induced Molecule 1 (IM1). These are characteristics of defects in Toll signaling, and consistent with this, we demonstrate dependency for one of the miRNA mutants on the NF-κΒ homolog Dif. We also quantify changes in the miRNA expression profile over time in response to three pathogen types, and identify 13 mature miRNA forms affected by pathogens that stimulate Toll signaling. To complement this, we provide a genome-wide map of potential NF-κB sites in proximity to miRNA genes. Finally, we demonstrate that systemic C. albicans infection contributes to a reduction in the total amount of branch-chained amino acids, which is miRNA-regulated. Overall, our data reveal a new layer of miRNA complexity regulating the fly response to systemic fungal infection. PMID:28706002

RILES, a novel method for temporal analysis of the in vivo regulation of miRNA expression

PubMed Central

Ezzine, Safia; Vassaux, Georges; Pitard, Bruno; Barteau, Benoit; Malinge, Jean-Marc; Midoux, Patrick; Pichon, Chantal; Baril, Patrick

2013-01-01

Novel methods are required to investigate the complexity of microRNA (miRNA) biology and particularly their dynamic regulation under physiopathological conditions. Herein, a novel plasmid-based RNAi-Inducible Luciferase Expression System (RILES) was engineered to monitor the activity of endogenous RNAi machinery. When RILES is transfected in a target cell, the miRNA of interest suppresses the expression of a transcriptional repressor and consequently switch-ON the expression of the luciferase reporter gene. Hence, miRNA expression in cells is signed by the emission of bioluminescence signals that can be monitored using standard bioluminescence equipment. We validated this approach by monitoring in mice the expression of myomiRs-133, −206 and −1 in skeletal muscles and miRNA-122 in liver. Bioluminescence experiments demonstrated robust qualitative and quantitative data that correlate with the miRNA expression pattern detected by quantitative RT-PCR (qPCR). We further demonstrated that the regulation of miRNA-206 expression during the development of muscular atrophy is individual-dependent, time-regulated and more complex than the information generated by qPCR. As RILES is simple and versatile, we believe that this methodology will contribute to a better understanding of miRNA biology and could serve as a rationale for the development of a novel generation of regulatable gene expression systems with potential therapeutic applications. PMID:24013565

RILES, a novel method for temporal analysis of the in vivo regulation of miRNA expression.

PubMed

Ezzine, Safia; Vassaux, Georges; Pitard, Bruno; Barteau, Benoit; Malinge, Jean-Marc; Midoux, Patrick; Pichon, Chantal; Baril, Patrick

2013-11-01

Novel methods are required to investigate the complexity of microRNA (miRNA) biology and particularly their dynamic regulation under physiopathological conditions. Herein, a novel plasmid-based RNAi-Inducible Luciferase Expression System (RILES) was engineered to monitor the activity of endogenous RNAi machinery. When RILES is transfected in a target cell, the miRNA of interest suppresses the expression of a transcriptional repressor and consequently switch-ON the expression of the luciferase reporter gene. Hence, miRNA expression in cells is signed by the emission of bioluminescence signals that can be monitored using standard bioluminescence equipment. We validated this approach by monitoring in mice the expression of myomiRs-133, -206 and -1 in skeletal muscles and miRNA-122 in liver. Bioluminescence experiments demonstrated robust qualitative and quantitative data that correlate with the miRNA expression pattern detected by quantitative RT-PCR (qPCR). We further demonstrated that the regulation of miRNA-206 expression during the development of muscular atrophy is individual-dependent, time-regulated and more complex than the information generated by qPCR. As RILES is simple and versatile, we believe that this methodology will contribute to a better understanding of miRNA biology and could serve as a rationale for the development of a novel generation of regulatable gene expression systems with potential therapeutic applications.

In silico MCMV Silencing Concludes Potential Host-Derived miRNAs in Maize

PubMed Central

Iqbal, Muhammad Shahzad; Jabbar, Basit; Sharif, Muhammad Nauman; Ali, Qurban; Husnain, Tayyab; Nasir, Idrees A.

2017-01-01

Maize Chlorotic Mottle Virus (MCMV) is a deleterious pathogen which causes Maize Lethal Necrosis Disease (MLND) that results in substantial yield loss of Maize crop worldwide. The positive-sense RNA genome of MCMV (4.4 kb) encodes six proteins: P32 (32 kDa protein), RNA dependent RNA polymerases (P50 and P111), P31 (31 kDa protein), P7 (7 kDa protein), coat protein (25 kDa). P31, P7 and coat protein are encoded from sgRNA1, located at the 3′end of the genome and sgRNA2 is located at the extremity of the 3′genome end. The objective of this study is to locate the possible attachment sites of Zea mays derived miRNAs in the genome of MCMV using four diverse miRNA target prediction algorithms. In total, 321 mature miRNAs were retrieved from miRBase (miRNA database) and were tested for hybridization of MCMV genome. These algorithms considered the parameters of seed pairing, minimum free energy, target site accessibility, multiple target sites, pattern recognition and folding energy for attachment. Out of 321 miRNAs only 10 maize miRNAs are predicted for silencing of MCMV genome. The results of this study can hence act as the first step towards the development of MCMV resistant transgenic Maize plants through expression of the selected miRNAs. PMID:28400775

Importin 8 regulates the transport of mature microRNAs into the cell nucleus.

PubMed

Wei, Yao; Li, Limin; Wang, Dong; Zhang, Chen-Yu; Zen, Ke

2014-04-11

Mature microRNAs (miRNAs), ∼ 22-nucleotide noncoding RNAs regulating target gene expression at the post-transcriptional level, have been recently shown to be transported into the nucleus where they modulate the biogenesis of other miRNAs or their own expression. However, the mechanism that governs the transport of mature miRNAs from cytoplasm to nucleus remains unknown. Here, we report that importin 8 (IPO8), a member of the karyopherin β (also named the protein import receptor importin β) family, plays a critical role in mediating the cytoplasm-to-nucleus transport of mature miRNAs. Specifically knocking down IPO8 but not other karyopherin β family proteins via siRNA significantly decreases the nuclear transport of various known nucleus-enriched miRNAs without affecting their total cellular levels. IPO8-mediated nuclear transport of mature miRNAs is also dependent on the association of IPO8 with the Argonaute 2 (Ago2) complex. Cross-immunoprecipitation and Western blot analysis show that IPO8 is physically associated with Ago2. Knocking down IPO8 via siRNA markedly decreases the nuclear transport of Ago2 but does not affect the total cellular Ago2 level. Furthermore, dissociating the binding of miRNAs with Ago2 by trypaflavine strongly reduces the IPO8-mediated nuclear transport of miRNAs.

MicroRNAs That Contribute to Coordinating the Immune Response in Drosophila melanogaster.

PubMed

Atilano, Magda L; Glittenberg, Marcus; Monteiro, Annabel; Copley, Richard R; Ligoxygakis, Petros

2017-09-01

Small noncoding RNAs called microRNAs (miRNAs) have emerged as post-transcriptional regulators of gene expression related to host defenses. Here, we have used Drosophila melanogaster to explore the contribution of individual or clusters of miRNAs in countering systemic Candida albicans infection. From a total of 72 tested, we identify 6 miRNA allelic mutant backgrounds that modulate the survival response to infection and the ability to control pathogen number. These mutants also exhibit dysregulation of the Toll pathway target transcripts Drosomycin ( Drs ) and Immune-Induced Molecule 1 ( IM1 ). These are characteristics of defects in Toll signaling, and consistent with this, we demonstrate dependency for one of the miRNA mutants on the NF-κΒ homolog Dif. We also quantify changes in the miRNA expression profile over time in response to three pathogen types, and identify 13 mature miRNA forms affected by pathogens that stimulate Toll signaling. To complement this, we provide a genome-wide map of potential NF-κB sites in proximity to miRNA genes. Finally, we demonstrate that systemic C. albicans infection contributes to a reduction in the total amount of branch-chained amino acids, which is miRNA-regulated. Overall, our data reveal a new layer of miRNA complexity regulating the fly response to systemic fungal infection. Copyright © 2017 Atilano et al.

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

Aneurysm miRNA Signature Differs, Depending on Disease Localization and Morphology

PubMed Central

Busch, Albert; Busch, Martin; Scholz, Claus-Jürgen; Kellersmann, Richard; Otto, Christoph; Chernogubova, Ekaterina; Maegdefessel, Lars; Zernecke, Alma; Lorenz, Udo

2016-01-01

Limited comprehension of aneurysm pathology has led to inconclusive results from clinical trials. miRNAs are key regulators of post-translational gene modification and are useful tools in elucidating key features of aneurysm pathogenesis in distinct entities of abdominal and popliteal aneurysms. Here, surgically harvested specimens from 19 abdominal aortic aneurysm (AAA) and 8 popliteal artery aneurysm (PAA) patients were analyzed for miRNA expression and histologically classified regarding extracellular matrix (ECM) remodeling and inflammation. DIANA-based computational target prediction and pathway enrichment analysis verified our results, as well as previous ones. miRNA-362, -19b-1, -194, -769, -21 and -550 were significantly down-regulated in AAA samples depending on degree of inflammation. Similar or inverse regulation was found for miR-769, 19b-1 and miR-550, -21, whereas miR-194 and -362 were unaltered in PAA. In situ hybridization verified higher expression of miR-550 and -21 in PAA compared to AAA and computational analysis for target genes and pathway enrichment affirmed signal transduction, cell-cell-interaction and cell degradation pathways, in line with previous results. Despite the vague role of miRNAs for potential diagnostic and treatment purposes, the number of candidates from tissue signature studies is increasing. Tissue morphology influences subsequent research, yet comparison of distinct entities of aneurysm disease can unravel core pathways. PMID:26771601

Design and implementation of a synthetic pre-miR switch for controlling miRNA biogenesis in mammals

PubMed Central

Atanasov, Janina; Groher, Florian

2017-01-01

Abstract Synthetic RNA-based systems have increasingly been used for the regulation of eukaryotic gene expression. Due to their structural properties, riboregulators provide a convenient basis for the development of ligand-dependent controllable systems. Here, we demonstrate reversible conditional control of miRNA biogenesis with an aptamer domain as a sensing unit connected to a natural miRNA precursor for the first time. For the design of the pre-miR switch, we replaced the natural terminal loop with the TetR aptamer. Thus, the TetR aptamer was positioned close to the Dicer cleavage sites, which allowed sterical control over pre-miR processing by Dicer. Our design proved to be highly versatile, allowing us to regulate the biogenesis of three structurally different miRNAs: miR-126, -34a and -199a. Dicer cleavage was inhibited up to 143-fold via co-expression of the TetR protein, yet could be completely restored upon addition of doxycycline. Moreover, we showed the functionality of the pre-miR switches for gene regulation through the interaction of the respective miRNA with its specific target sequence. Our designed device is capable of robust and reversible control of miRNA abundance. Thus, we offer a novel investigational tool for functional miRNA analysis. PMID:29036355

Identification of Four Oxidative Stress-Responsive MicroRNAs, miR-34a-5p, miR-1915-3p, miR-638, and miR-150-3p, in Hepatocellular Carcinoma.

PubMed

Wan, Yong; Cui, Ruixia; Gu, Jingxian; Zhang, Xing; Xiang, Xiaohong; Liu, Chang; Qu, Kai; Lin, Ting

2017-01-01

Increasing evidence suggests that oxidative stress plays an essential role during carcinogenesis. However, the underlying mechanism between oxidative stress and carcinogenesis remains unknown. Recently, microRNAs (miRNAs) are revealed to be involved in oxidative stress response and carcinogenesis. This study aims to identify miRNAs in hepatocellular carcinoma (HCC) cells which might involve in oxidative stress response. An integrated analysis of miRNA expression signature was performed by employing robust rank aggregation (RRA) method, and four miRNAs (miR-34a-5p, miR-1915-3p, miR-638, and miR-150-3p) were identified as the oxidative stress-responsive miRNAs. Pathway enrichment analysis suggested that these four miRNAs played an important role in antiapoptosis process. Our data also revealed miR-34a-5p and miR-1915-3p, but not miR-150-3p and miR-638, were regulated by p53 in HCC cell lines under oxidative stress. In addition, clinical investigation revealed that these four miRNAs might be involved in oxidative stress response by targeting oxidative stress-related genes in HCC tissues. Kaplan-Meier analysis showed that these four miRNAs were associated with patients' overall survival. In conclusion, we identified four oxidative stress-responsive miRNAs, which were regulated by p53-dependent (miR-34a-5p and miR-1915-3p) and p53-independent pathway (miR-150-3p and miR-638). These four miRNAs may offer new strategy for HCC diagnosis and prognosis.

MicroRNA-dependent regulation of transcription in non-small cell lung cancer.

PubMed

Molina-Pinelo, Sonia; Gutiérrez, Gabriel; Pastor, Maria Dolores; Hergueta, Marta; Moreno-Bueno, Gema; García-Carbonero, Rocío; Nogal, Ana; Suárez, Rocío; Salinas, Ana; Pozo-Rodríguez, Francisco; Lopez-Rios, Fernando; Agulló-Ortuño, Maria Teresa; Ferrer, Irene; Perpiñá, Asunción; Palacios, José; Carnero, Amancio; Paz-Ares, Luis

2014-01-01

Squamous cell lung cancer (SCC) and adenocarcinoma are the most common histological subtypes of non-small cell lung cancer (NSCLC), and have been traditionally managed in the clinic as a single entity. Increasing evidence, however, illustrates the biological diversity of these two histological subgroups of lung cancer, and supports the need to improve our understanding of the molecular basis beyond the different phenotypes if we aim to develop more specific and individualized targeted therapy. The purpose of this study was to identify microRNA (miRNA)-dependent transcriptional regulation differences between SCC and adenocarcinoma histological lung cancer subtypes. In this work, paired miRNA (667 miRNAs by TaqMan Low Density Arrays (TLDA)) and mRNA profiling (Whole Genome 44 K array G112A, Agilent) was performed in tumor samples of 44 NSCLC patients. Nine miRNAs and 56 mRNAs were found to be differentially expressed in SCC versus adenocarcinoma samples. Eleven of these 56 mRNA were predicted as targets of the miRNAs identified to be differently expressed in these two histological conditions. Of them, 6 miRNAs (miR-149, miR-205, miR-375, miR-378, miR-422a and miR-708) and 9 target genes (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B, MUC1) were validated by quantitative PCR in an independent cohort of 41 lung cancer patients. Furthermore, the inverse correlation between mRNAs and microRNAs expression was also validated. These results suggest miRNA-dependent transcriptional regulation differences play an important role in determining key hallmarks of NSCLC, and may provide new biomarkers for personalized treatment strategies.

MicroRNA-Dependent Regulation of Transcription in Non-Small Cell Lung Cancer

PubMed Central

Molina-Pinelo, Sonia; Gutiérrez, Gabriel; Pastor, Maria Dolores; Hergueta, Marta; Moreno-Bueno, Gema; García-Carbonero, Rocío; Nogal, Ana; Suárez, Rocío; Salinas, Ana; Pozo-Rodríguez, Francisco; Lopez-Rios, Fernando; Agulló-Ortuño, Maria Teresa; Ferrer, Irene; Perpiñá, Asunción; Palacios, José; Carnero, Amancio; Paz-Ares, Luis

2014-01-01

Squamous cell lung cancer (SCC) and adenocarcinoma are the most common histological subtypes of non-small cell lung cancer (NSCLC), and have been traditionally managed in the clinic as a single entity. Increasing evidence, however, illustrates the biological diversity of these two histological subgroups of lung cancer, and supports the need to improve our understanding of the molecular basis beyond the different phenotypes if we aim to develop more specific and individualized targeted therapy. The purpose of this study was to identify microRNA (miRNA)-dependent transcriptional regulation differences between SCC and adenocarcinoma histological lung cancer subtypes. In this work, paired miRNA (667 miRNAs by TaqMan Low Density Arrays (TLDA)) and mRNA profiling (Whole Genome 44 K array G112A, Agilent) was performed in tumor samples of 44 NSCLC patients. Nine miRNAs and 56 mRNAs were found to be differentially expressed in SCC versus adenocarcinoma samples. Eleven of these 56 mRNA were predicted as targets of the miRNAs identified to be differently expressed in these two histological conditions. Of them, 6 miRNAs (miR-149, miR-205, miR-375, miR-378, miR-422a and miR-708) and 9 target genes (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B, MUC1) were validated by quantitative PCR in an independent cohort of 41 lung cancer patients. Furthermore, the inverse correlation between mRNAs and microRNAs expression was also validated. These results suggest miRNA-dependent transcriptional regulation differences play an important role in determining key hallmarks of NSCLC, and may provide new biomarkers for personalized treatment strategies. PMID:24625834

Time-Dependent Expression Profiles of microRNAs and mRNAs in Rat Milk Whey

PubMed Central

Izumi, Hirohisa; Kosaka, Nobuyoshi; Shimizu, Takashi; Sekine, Kazunori; Ochiya, Takahiro; Takase, Mitsunori

2014-01-01

Functional RNAs, such as microRNA (miRNA) and mRNA, are present in milk, but their roles are unknown. To clarify the roles of milk RNAs, further studies using experimental animals such as rats are needed. However, it is unclear whether rat milk also contains functional RNAs and what their time dependent expression profiles are. Thus, we prepared total RNA from whey isolated from rat milk collected on days 2, 9, and 16 postpartum and analyzed using microarrays and quantitative PCR. The concentration of RNA in colostrum whey (day 2) was markedly higher than that in mature milk whey (days 9 and 16). Microarray analysis detected 161 miRNAs and 10,948 mRNA transcripts. Most of the miRNAs and mRNA transcripts were common to all tested milks. Finally, we selected some immune- and development-related miRNAs and mRNAs, and analysed them by quantitative PCR (in equal sample volumes) to determine their time-dependent changes in expression in detail. Some were significantly more highly expressed in colostrum whey than in mature milk whey, but some were expressed equally. And mRNA expression levels of some cytokines and hormones did not reflect the protein levels. It is still unknown whether RNAs in milk play biological roles in neonates. However, our data will help guide future in vivo studies using experimental animals such as rats. PMID:24533154

microRNA modulation of circadian clock period and entrainment

PubMed Central

Cheng, Hai-Ying M.; Papp, Joseph W.; Varlamova, Olga; Dziema, Heather; Russell, Brandon; Curfman, John P.; Nakazawa, Takanobu; Shimizu, Kimiko; Okamura, Hitoshi; Impey, Soren; Obrietan, Karl

2007-01-01

microRNAs (miRNAs) are a class of small, non-coding, RNAs that regulate the stability or translation of mRNA transcripts. Although recent work has implicated miRNAs in development and in disease, the expression and function of miRNAs in the adult mammalian nervous system has not been extensively characterized. Here, we examine the role of two brain-specific miRNAs, miR-219 and miR-132, in modulating the circadian clock located in the suprachiasmatic nucleus. miR-219 is a target of the CLOCK/BMAL1 complex, exhibits robust circadian rhythms of expression and the in vivo knockdown of miR-219 lengthens the circadian period. miR-132 is induced by photic entrainment cues via a MAPK/CREB-dependent mechanism, modulates clock gene expression, and attenuates the entraining effects of light. Collectively, these data reveal miRNAs as clock- and light-regulated genes and provide a mechanistic examination of their roles as effectors of pacemaker activity and entrainment. PMID:17553428

Integrating mRNA and miRNA Weighted Gene Co-Expression Networks with eQTLs in the Nucleus Accumbens of Subjects with Alcohol Dependence

PubMed Central

Blevins, Tana; Aliev, Fazil; Adkins, Amy; Hack, Laura; Bigdeli, Tim; D. van der Vaart, Andrew; Web, Bradley Todd; Bacanu, Silviu-Alin; Kalsi, Gursharan; Kendler, Kenneth S.; Miles, Michael F.; Dick, Danielle; Riley, Brien P.; Dumur, Catherine; Vladimirov, Vladimir I.

2015-01-01

Alcohol consumption is known to lead to gene expression changes in the brain. After performing weighted gene co-expression network analyses (WGCNA) on genome-wide mRNA and microRNA (miRNA) expression in Nucleus Accumbens (NAc) of subjects with alcohol dependence (AD; N = 18) and of matched controls (N = 18), six mRNA and three miRNA modules significantly correlated with AD were identified (Bonferoni-adj. p≤ 0.05). Cell-type-specific transcriptome analyses revealed two of the mRNA modules to be enriched for neuronal specific marker genes and downregulated in AD, whereas the remaining four mRNA modules were enriched for astrocyte and microglial specific marker genes and upregulated in AD. Gene set enrichment analysis demonstrated that neuronal specific modules were enriched for genes involved in oxidative phosphorylation, mitochondrial dysfunction and MAPK signaling. Glial-specific modules were predominantly enriched for genes involved in processes related to immune functions, i.e. cytokine signaling (all adj. p≤ 0.05). In mRNA and miRNA modules, 461 and 25 candidate hub genes were identified, respectively. In contrast to the expected biological functions of miRNAs, correlation analyses between mRNA and miRNA hub genes revealed a higher number of positive than negative correlations (χ2 test p≤ 0.0001). Integration of hub gene expression with genome-wide genotypic data resulted in 591 mRNA cis-eQTLs and 62 miRNA cis-eQTLs. mRNA cis-eQTLs were significantly enriched for AD diagnosis and AD symptom counts (adj. p = 0.014 and p = 0.024, respectively) in AD GWAS signals in a large, independent genetic sample from the Collaborative Study on Genetics of Alcohol (COGA). In conclusion, our study identified putative gene network hubs coordinating mRNA and miRNA co-expression changes in the NAc of AD subjects, and our genetic (cis-eQTL) analysis provides novel insights into the etiological mechanisms of AD. PMID:26381263

miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNA

PubMed Central

Hansen, Thomas B; Wiklund, Erik D; Bramsen, Jesper B; Villadsen, Sune B; Statham, Aaron L; Clark, Susan J; Kjems, Jørgen

2011-01-01

MicroRNAs (miRNAs) are ∼22 nt non-coding RNAs that typically bind to the 3′ UTR of target mRNAs in the cytoplasm, resulting in mRNA destabilization and translational repression. Here, we report that miRNAs can also regulate gene expression by targeting non-coding antisense transcripts in human cells. Specifically, we show that miR-671 directs cleavage of a circular antisense transcript of the Cerebellar Degeneration-Related protein 1 (CDR1) locus in an Ago2-slicer-dependent manner. The resulting downregulation of circular antisense has a concomitant decrease in CDR1 mRNA levels, independently of heterochromatin formation. This study provides the first evidence for non-coding antisense transcripts as functional miRNA targets, and a novel regulatory mechanism involving a positive correlation between mRNA and antisense circular RNA levels. PMID:21964070

The pro-differentiating role of miR-124: indicating the road to become a neuron.

PubMed

Maiorano, Nicola Antonio; Mallamaci, Antonello

2010-01-01

miRNAs are essential post-transcriptional modulators affecting cell identity and fate, with a central role in cellular and developmental processes. The brain-enriched neuronal specific miRNAs-124 has been identified as a promoter of neuronogenesis in various conditions, in vitro and in vivo, with a potential role in regulating also activities of post-mitotic neurons, such as synaptic plasticity and memory formation. In this point of view, we recapitulate the main experimental findings substantiating the positive correlation between miR-124 expression and neuronogenesis progression. Then, we describe the impact of miR-124 on the molecular network driving the profound changes which take place in differentiating neuronal cells. Finally, we consider the possibility of a post-transcriptional modulation of miR-124 biogenesis, which may finely regulate--in turn--the activities of miR-124 in neural precursor cells.

Deconstructing transcriptional heterogeneity in pluripotent stem cells

PubMed Central

Shalek, Alex K.; Satija, Rahul; DaleyKeyser, AJay; Li, Hu; Zhang, Jin; Pardee, Keith; Gennert, David; Trombetta, John J.; Ferrante, Thomas C.; Regev, Aviv; Daley, George Q.; Collins, James J.

2014-01-01

SUMMARY Pluripotent stem cells (PSCs) are capable of dynamic interconversion between distinct substates, but the regulatory circuits specifying these states and enabling transitions between them are not well understood. We set out to characterize transcriptional heterogeneity in PSCs by single-cell expression profiling under different chemical and genetic perturbations. Signaling factors and developmental regulators show highly variable expression, with expression states for some variable genes heritable through multiple cell divisions. Expression variability and population heterogeneity can be influenced by perturbation of signaling pathways and chromatin regulators. Strikingly, either removal of mature miRNAs or pharmacologic blockage of signaling pathways drives PSCs into a low-noise ground state characterized by a reconfigured pluripotency network, enhanced self-renewal, and a distinct chromatin state, an effect mediated by opposing miRNA families acting on the c-myc / Lin28 / let-7 axis. These data illuminate the nature of transcriptional heterogeneity in PSCs. PMID:25471879

Can microRNAs act as biomarkers of aging?

PubMed Central

Kashyap, Luv

2011-01-01

Aging can be defined as a progressive decline in physiological efficiency regulated by an extremely complex multifactorial process. The genetic makeup of an individual appears to dictate this rate of aging in a species specific manner. For decades now, scientists have tried to look for tiny signatures or signs which might help us predict this rate of aging. MicroRNAs (miRNAs) are a unique class of short, non-coding RNAs that mediate the post-transcriptional regulation of gene expression ranging from developmental processes to disease induction or amelioration. Recently, they have also been implicated to have a role in aging in C.elegans. Based on the fact that there is a considerable similarity between aging in C.elegans and humans, these recent findings might suggest a possible role of miRNAs as bio-markers of aging. This mini-review brushes through the possibilities towards this direction. PMID:21383908

Can microRNAs act as biomarkers of aging?

PubMed

Kashyap, Luv

2011-02-07

Aging can be defined as a progressive decline in physiological efficiency regulated by an extremely complex multifactorial process. The genetic makeup of an individual appears to dictate this rate of aging in a species specific manner. For decades now, scientists have tried to look for tiny signatures or signs which might help us predict this rate of aging. MicroRNAs (miRNAs) are a unique class of short, non-coding RNAs that mediate the post-transcriptional regulation of gene expression ranging from developmental processes to disease induction or amelioration. Recently, they have also been implicated to have a role in aging in C.elegans. Based on the fact that there is a considerable similarity between aging in C.elegans and humans, these recent findings might suggest a possible role of miRNAs as bio-markers of aging. This mini-review brushes through the possibilities towards this direction.

Differentiation-associated microRNAs antagonize the Rb–E2F pathway to restrict proliferation

PubMed Central

Marzi, Matteo J.; Puggioni, Eleonora M. R.; Dall'Olio, Valentina; Bucci, Gabriele; Bernard, Loris; Bianchi, Fabrizio; Crescenzi, Marco

2012-01-01

The cancer-associated loss of microRNA (miRNA) expression leads to a proliferative advantage and aggressive behavior through largely unknown mechanisms. Here, we exploit a model system that recapitulates physiological terminal differentiation and its reversal upon oncogene expression to analyze coordinated mRNA/miRNA responses. The cell cycle reentry of myotubes, forced by the E1A oncogene, was associated with a pattern of mRNA/miRNA modulation that was largely reciprocal to that induced during the differentiation of myoblasts into myotubes. The E1A-induced mRNA response was preponderantly Retinoblastoma protein (Rb)-dependent. Conversely, the miRNA response was mostly Rb-independent and exerted through tissue-specific factors and Myc. A subset of these miRNAs (miR-1, miR-34, miR-22, miR-365, miR-29, miR-145, and Let-7) was shown to coordinately target Rb-dependent cell cycle and DNA replication mRNAs. Thus, a dual level of regulation—transcriptional regulation via Rb–E2F and posttranscriptional regulation via miRNAs—confers robustness to cell cycle control and provides a molecular basis to understand the role of miRNA subversion in cancer. PMID:23027903

Aryl hydrocarbon receptor (AhR)-dependent regulation of pulmonary miRNA by chronic cigarette smoke exposure

PubMed Central

Rogers, Sarah; de Souza, Angela Rico; Zago, Michela; Iu, Matthew; Guerrina, Necola; Gomez, Alvin; Matthews, Jason; Baglole, Carolyn J.

2017-01-01

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor historically known for its toxic responses to man-made pollutants such as dioxin. More recently, the AhR has emerged as a suppressor of inflammation, oxidative stress and apoptosis from cigarette smoke by mechanisms that may involve the regulation of microRNA. However, little is known about the AhR regulation of miRNA expression in the lung in response to inhaled toxicants. Therefore, we exposed Ahr−/− and Ahr+/− mice to cigarette smoke for 4 weeks and evaluated lung miRNA expression by PCR array. There was a dramatic regulation of lung miRNA by the AhR in the absence of exogenous ligand. In response to cigarette smoke, there were more up-regulated miRNA in Ahr−/− mice compared to Ahr+/− mice, including the cancer-associated miRNA miR-96. There was no significant change in the expression of the AhR regulated proteins HuR and cyclooxygenase-2 (COX-2). There were significant increases in the anti-oxidant gene sulfiredoxin 1 (Srxn1) and FOXO3a- predicted targets of miR-96. Collectively, these data support a prominent role for the AhR in regulating lung miRNA expression. Further studies to elucidate a role for these miRNA may further uncover novel biological function for the AhR in respiratory health and disease. PMID:28079158

Identification of microRNAs involved in lipid biosynthesis and seed size in developing sea buckthorn seeds using high-throughput sequencing.

PubMed

Ding, Jian; Ruan, Chengjiang; Guan, Ying; Krishna, Priti

2018-03-05

Sea buckthorn is a plant of medicinal and nutritional importance owing in part to the high levels of essential fatty acids, linoleic (up to 42%) and α-linolenic (up to 39%) acids in the seed oil. Sea buckthorn can produce seeds either via the sexual pathway or by apomixis. The seed development and maturation programs are critically dependent on miRNAs. To understand miRNA-mediated regulation of sea buckthorn seed development, eight small RNA libraries were constructed for deep sequencing from developing seeds of a low oil content line 'SJ1' and a high oil content line 'XE3'. High-throughput sequencing identified 137 known miRNA from 27 families and 264 novel miRNAs. The potential targets of the identified miRNAs were predicted based on sequence homology. Nineteen (four known and 15 novel) and 22 (six known and 16 novel) miRNAs were found to be involved in lipid biosynthesis and seed size, respectively. An integrated analysis of mRNA and miRNA transcriptome and qRT-PCR identified some key miRNAs and their targets (miR164d-ARF2, miR168b-Δ9D, novelmiRNA-108-ACC, novelmiRNA-23-GPD1, novelmiRNA-58-DGAT1, and novelmiRNA-191-DGAT2) potentially involved in seed size and lipid biosynthesis of sea buckthorn seed. These results indicate the potential importance of miRNAs in regulating lipid biosynthesis and seed size in sea buckthorn.

Enoxacin Elevates MicroRNA Levels in Rat Frontal Cortex and Prevents Learned Helplessness

PubMed Central

Smalheiser, Neil R.; Zhang, Hui; Dwivedi, Yogesh

2014-01-01

Major depressive disorder (MDD) is a major public health concern. Despite tremendous advancement, the pathogenic mechanisms associated with MDD are still unclear. Moreover, a significant number of MDD subjects do not respond to the currently available medication. MicroRNAs (miRNAs) are a class of small non-coding RNAs that control gene expression by modulating translation, mRNA degradation or stability of mRNA targets. The role of miRNAs in disease pathophysiology is emerging rapidly. Recently, we reported that miRNA expression is down-regulated in frontal cortex of depressed suicide subjects, and that rats exposed to repeated inescapable shock show differential miRNA changes depending on whether they exhibited normal adaptive responses or learned helpless (LH) behavior. Enoxacin, a fluoroquinolone used clinically as an anti-bacterial compound, enhances the production of miRNAs in vitro and in peripheral tissues in vivo, but has not yet been tested as an experimental tool to study the relation of miRNA expression to neural functions or behavior. Treatment of rats with 10 or 25 mg/kg enoxacin for 1 week increased the expression of miRNAs in frontal cortex and decreased the proportion of rats exhibiting LH behavior following inescapable shock. Further studies are warranted to learn whether enoxacin may ameliorate depressive behavior in other rodent paradigms and in human clinical situations, and if so whether its mechanism is due to upregulation of miRNAs. PMID:24575053

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

Cha, Min-Ji; Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752; Jang, Jin-Kyung

Highlights: •CaMKIIδ mediates H{sub 2}O{sub 2}-induced Ca{sup 2+} overload in cardiomyocytes. •miR-145 can inhibit Ca{sup 2+} overload. •A luciferase assay confirms that miR-145 functions as a CaMKIIδ-targeting miRNA. •Overexpression of miR-145 regulates CaMKIIδ-related genes and ameliorates apoptosis. -- Abstract: A change in intracellular free calcium (Ca{sup 2+}) is a common signaling mechanism of reperfusion-induced cardiomyocyte death. Calcium/calmodulin dependent protein kinase II (CaMKII) is a critical regulator of Ca{sup 2+} signaling and mediates signaling pathways responsible for functions in the heart including hypertrophy, apoptosis, arrhythmia, and heart disease. MicroRNAs (miRNA) are involved in the regulation of cell response, including survival, proliferation,more » apoptosis, and development. However, the roles of miRNAs in Ca{sup 2+}-mediated apoptosis of cardiomyocytes are uncertain. Here, we determined the potential role of miRNA in the regulation of CaMKII dependent apoptosis and explored its underlying mechanism. To determine the potential roles of miRNAs in H{sub 2}O{sub 2}-mediated Ca{sup 2+} overload, we selected and tested 6 putative miRNAs that targeted CaMKIIδ, and showed that miR-145 represses CaMKIIδ protein expression and Ca{sup 2+} overload. We confirmed CaMKIIδ as a direct downstream target of miR-145. Furthermore, miR-145 regulates Ca{sup 2+}-related signals and ameliorates apoptosis. This study demonstrates that miR-145 regulates reactive oxygen species (ROS)-induced Ca{sup 2+} overload in cardiomyocytes. Thus, miR-145 affects ROS-mediated gene regulation and cellular injury responses.« less

Blockage of epithelial to mesenchymal transition and upregulation of let 7b are critically involved in ursolic acid induced apoptosis in malignant mesothelioma cell

PubMed Central

Sohn, Eun Jung; Won, Gunho; Lee, Jihyun; Yoon, Sang Wook; Lee, Ilho; Kim, Hee Jeong; Kim, Sung-Hoon

2016-01-01

Malignant pleural mesothelioma (MPN), which is caused by asbestos exposure, is one of aggressive lung tumors. In the present study, we elucidated the anti-tumor mechanism of ursolic acid in malignant mesotheliomas. Ursolic acid significantly exerted cytotoxicity in a time and dose dependent manner in H28, H2452 and MSTO-211H mesothelioma cells and inhibited cell proliferation by colony formation assay in a dose-dependent fashion. Also, ursolic acid treatment accumulated the sub-G1 population, attenuated the expression of procapase 9, cyclin D1, pAKT, p-glycogen synthase kinase 3-alpha/beta (pGSK3α/β), β-catenin and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) and also cleaved caspase 3 and poly (ADP-ribose) polymerase (PARP) in mesothelioma cells. Furthermore, ursolic acid treatment blocked epithelial and mesenchymal transition (EMT) molecules by activating E-cadherin as an epithelial marker and attenuating Vimentin, and Twist as mesenchymal molecules. Interestingly, miRNA array revealed that 23 miRNAs (>2 folds) including let-7b and miRNA3613-5p, miRNA134 and miRNA196b were significantly upregulated while 33 miRNAs were downregulated in ursolic acid treated H2452 cells. Furthermore, overexpression of let 7b using let-7b mimics enhanced the antitumor effect of ursolic acid to attenuate the expression of procaspases 3, pro-PARP, pAKT, β-catenin and Twist and increase sub-G1 accumulation in H2452 mesothelioma cells. Overall, our findings suggest that ursolic acid induces apoptosis via inhibition of EMT and activation of let7b in mesothelioma cells as a potent chemotherapeutic agent for treatment of malignant mesotheliomas. PMID:28090191

Precursor microRNA-programmed silencing complex assembly pathways in mammals.

PubMed

Liu, Xuhang; Jin, Dong-Yan; McManus, Michael T; Mourelatos, Zissimos

2012-05-25

Assembly of microRNA ribonucleoproteins (miRNPs) or RNA-induced silencing complexes (RISCs) is essential for the function of miRNAs and initiates from processing of precursor miRNAs (pre-miRNAs) by Dicer or by Ago2. Here, we report an in vitro miRNP/RISC assembly assay programmed by pre-miRNAs from mammalian cell lysates. Combining in vivo studies in Dicer Knockout cells reconstituted with wild-type or catalytically inactive Dicer, we find that the miRNA loading complex (miRLC) is the primary machinery linking pre-miRNA processing to miRNA loading. We show that a miRNA precursor deposit complex (miPDC) plays a crucial role in Dicer-independent miRNA biogenesis and promotes miRNP assembly of certain Dicer-dependent miRNAs. Furthermore, we find that 5'-uridine, 3'-mid base pairing, and 5'-mid mismatches within pre-miRNAs promote their assembly into miPDC. Our studies provide a comprehensive view of miRNP/RISC assembly pathways in mammals, and our assay provides a versatile platform for further mechanistic dissection of such pathways in mammals. Copyright © 2012 Elsevier Inc. All rights reserved.

S6K2-mediated regulation of TRBP as a determinant of miRNA expression in human primary lymphatic endothelial cells

PubMed Central

Warner, Matthew J.; Bridge, Katherine S.; Hewitson, James P.; Hodgkinson, Michael R.; Heyam, Alex; Massa, Bailey C.; Haslam, Jessica C.; Chatzifrangkeskou, Maria; Evans, Gareth J.O.; Plevin, Michael J.; Sharp, Tyson V.; Lagos, Dimitris

2016-01-01

MicroRNAs (miRNAs) are short non-coding RNAs that silence mRNAs. They are generated following transcription and cleavage by the DROSHA/DGCR8 and DICER/TRBP/PACT complexes. Although it is known that components of the miRNA biogenesis machinery can be phosphorylated, it remains poorly understood how these events become engaged during physiological cellular activation. We demonstrate that S6 kinases can phosphorylate the extended C-terminal domain of TRBP and interact with TRBP in situ in primary cells. TRBP serines 283/286 are essential for S6K-mediated TRBP phosphorylation, optimal expression of TRBP, and the S6K-TRBP interaction in human primary cells. We demonstrate the functional relevance of this interaction in primary human dermal lymphatic endothelial cells (HDLECs). Angiopoietin-1 (ANG1) can augment miRNA biogenesis in HDLECs through enhancing TRBP phosphorylation and expression in an S6K2-dependent manner. We propose that the S6K2/TRBP node controls miRNA biogenesis in HDLECs and provides a molecular link between the mTOR pathway and the miRNA biogenesis machinery. PMID:27407113

A genome-wide identification of the miRNAome in response to salinity stress in date palm (Phoenix dactylifera L.).

PubMed

Yaish, Mahmoud W; Sunkar, Ramanjulu; Zheng, Yun; Ji, Bo; Al-Yahyai, Rashid; Farooq, Sardar A

2015-01-01

Although date palm is relatively salt-tolerant, little is known about the underlying molecular mechanisms that contribute to its salt tolerance. Only recently, investigators have uncovered microRNA-mediated post-transcriptional gene regulation, which is critical for typical plant development and adaptation to stress conditions such as salinity. To identify conserved and novel miRNAs in date palm and to characterize miRNAs that could play a role in salt tolerance, we have generated sRNA libraries from the leaves and roots of NaCl-treated and untreated seedlings of date palm. Deep sequencing of these four sRNA libraries yielded approximately 251 million reads. The bioinformatics analysis has identified 153 homologs of conserved miRNAs, 89 miRNA variants, and 180 putative novel miRNAs in date palm. Expression profiles under salinity revealed differential regulation of some miRNAs in date palm. In leaves, 54 of the identified miRNAs were significantly affected and the majority (70%) of them were upregulated, whereas in roots, 25 of the identified miRNAs were significantly affected and 76% of them were upregulated by the salinity stress. The salt-responsiveness of some of these miRNAs was further validated using semi-quantitative PCR (qPCR). Some of the predicted targets for the identified miRNA include genes with known functions in plant salt tolerance, such as potassium channel AKT2-like proteins, vacuolar protein sorting-associated protein, calcium-dependent and mitogen-activated proteins. As one of the first cultivated trees in the world that can tolerate a wide range of abiotic stresses, date palm contains a large population of conserved and non-conserved miRNAs that function at the post-transcriptional level. This study provided insights into miRNA-mediated gene expression that are important for adaptation to salinity in date palms.

Developmental exposure to lead (Pb) alters the expression of the human tau gene and its products in a transgenic animal model

PubMed Central

Dash, M.; Eid, A.; Subaiea, G.; Chang, J.; Deeb, R.; Masoud, A.; Renehan, W.E.; Adem, A.; Zawia, N.H.

2016-01-01

Tauopathies are a class of neurodegenerative diseases associated with the pathological aggregationof the tau protein in the human brain. The best known of these illnesses is Alzheimer's disease (AD); a disease where the microtubule associated protein tau (MAPT) becomes hyperphosphorylated (lowering its binding affinity to microtubules) and aggregates within neurons in the form of neurofibrillary tangles (NFTs). In this paper we examine whether environmental factors play a significant role in tau pathogenesis. Our studies were conducted in a double mutant mouse model that expressed the human tau gene and lacked the gene for murine tau. The human tau mouse model was tested for the transgene's ability to respond to an environmental toxicant. Pups were developmentally exposed to lead (Pb) from postnatal day (PND) 1-20 with 0.2% Pb acetate. Mice were then sacrificed at PND 20, 30, 40 and 60. Protein and mRNA levels for tau and CDK5 as well as tau phosphorylation at Ser396 were determined. In addition, the potential role of miRNA in tau expression was investigated by measuring levels of miR-34c, a miRNA that targets the mRNA for human tau, at PND20 and 50. The expression of the human tau transgene was altered by developmental exposure to Pb. This exposure also altered the expression of miR-34c. Our findings are the first of their kind to test the responsiveness of the human tau gene to an environmental toxicant and to examine an epigenetic mechanism that may be involved in the regulation of this gene's expression. PMID:27293183

Aging-induced dysregulation of dicer1-dependent microRNA expression impairs angiogenic capacity of rat cerebromicrovascular endothelial cells.

PubMed

Ungvari, Zoltan; Tucsek, Zsuzsanna; Sosnowska, Danuta; Toth, Peter; Gautam, Tripti; Podlutsky, Andrej; Csiszar, Agnes; Losonczy, Gyorgy; Valcarcel-Ares, M Noa; Sonntag, William E; Csiszar, Anna

2013-08-01

Age-related impairment of angiogenesis is likely to play a central role in cerebromicrovascular rarefaction and development of vascular cognitive impairment, but the underlying mechanisms remain elusive. To test the hypothesis that dysregulation of Dicer1 (ribonuclease III, a key enzyme of the microRNA [miRNA] machinery) impairs endothelial angiogenic capacity in aging, primary cerebromicrovascular endothelial cells (CMVECs) were isolated from young (3 months old) and aged (24 months old) Fischer 344 × Brown Norway rats. We found an age-related downregulation of Dicer1 expression both in CMVECs and in small cerebral vessels isolated from aged rats. In aged CMVECs, Dicer1 expression was increased by treatment with polyethylene glycol-catalase. Compared with young cells, aged CMVECs exhibited altered miRNA expression profile, which was associated with impaired proliferation, adhesion to vitronectin, collagen and fibronectin, cellular migration (measured by a wound-healing assay using electric cell-substrate impedance sensing technology), and impaired ability to form capillary-like structures. Overexpression of Dicer1 in aged CMVECs partially restored miRNA expression profile and significantly improved angiogenic processes. In young CMVECs, downregulation of Dicer1 (siRNA) resulted in altered miRNA expression profile associated with impaired proliferation, adhesion, migration, and tube formation, mimicking the aging phenotype. Collectively, we found that Dicer1 is essential for normal endothelial angiogenic processes, suggesting that age-related dysregulation of Dicer1-dependent miRNA expression may be a potential mechanism underlying impaired angiogenesis and cerebromicrovascular rarefaction in aging.

Regulation of MicroRNAs-Mediated Autophagic Flux: A New Regulatory Avenue for Neurodegenerative Diseases With Focus on Prion Diseases

PubMed Central

Shah, Syed Zahid Ali; Zhao, Deming; Hussain, Tariq; Sabir, Naveed; Yang, Lifeng

2018-01-01

Prion diseases are fatal neurological disorders affecting various mammalian species including humans. Lack of proper diagnostic tools and non-availability of therapeutic remedies are hindering the control strategies for prion diseases. MicroRNAs (miRNAs) are abundant endogenous short non-coding essential RNA molecules that negatively regulate the target genes after transcription. Several biological processes depend on miRNAs, and altered profiles of these miRNAs are potential biomarkers for various neurodegenerative diseases, including prion diseases. Autophagic flux degrades the misfolded prion proteins to reduce chronic endoplasmic reticulum stress and enhance cell survival. Recent evidence suggests that specific miRNAs target and regulate the autophagic mechanism, which is critical for alleviating cellular stress. miRNAs-mediated regulation of these specific proteins involved in the autophagy represents a new target with highly significant therapeutic prospects. Here, we will briefly describe the biology of miRNAs, the use of miRNAs as potential biomarkers with their credibility, the regulatory mechanism of miRNAs in major neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and prion diseases, degradation pathways for aggregated prion proteins, the role of autophagy in prion diseases. Finally, we will discuss the miRNAs-modulated autophagic flux in neurodegenerative diseases and employ them as potential therapeutic intervention strategy in prion diseases. PMID:29867448

An Atlas of Soybean Small RNAs Identifies Phased siRNAs from Hundreds of Coding Genes[W

PubMed Central

Kakrana, Atul; Huang, Kun; Zhai, Jixian; Yan, Zhe; Valdés-López, Oswaldo; Prince, Silvas; Musket, Theresa A.; Stacey, Gary

2014-01-01

Small RNAs are ubiquitous, versatile repressors and include (1) microRNAs (miRNAs), processed from mRNA forming stem-loops; and (2) small interfering RNAs (siRNAs), the latter derived in plants by a process typically requiring an RNA-dependent RNA polymerase. We constructed and analyzed an expression atlas of soybean (Glycine max) small RNAs, identifying over 500 loci generating 21-nucleotide phased siRNAs (phasiRNAs; from PHAS loci), of which 483 overlapped annotated protein-coding genes. Via the integration of miRNAs with parallel analysis of RNA end (PARE) data, 20 miRNA triggers of 127 PHAS loci were detected. The primary class of PHAS loci (208 or 41% of the total) corresponded to NB-LRR genes; some of these small RNAs preferentially accumulate in nodules. Among the PHAS loci, novel representatives of TAS3 and noncanonical phasing patterns were also observed. A noncoding PHAS locus, triggered by miR4392, accumulated preferentially in anthers; the phasiRNAs are predicted to target transposable elements, with their peak abundance during soybean reproductive development. Thus, phasiRNAs show tremendous diversity in dicots. We identified novel miRNAs and assessed the veracity of soybean miRNAs registered in miRBase, substantially improving the soybean miRNA annotation, facilitating an improvement of miRBase annotations and identifying at high stringency novel miRNAs and their targets. PMID:25465409

miRNAmeConverter: an R/bioconductor package for translating mature miRNA names to different miRBase versions.

PubMed

Haunsberger, Stefan J; Connolly, Niamh M C; Prehn, Jochen H M

2017-02-15

The miRBase database is the central and official repository for miRNAs and the current release is miRBase version 21.0. Name changes in different miRBase releases cause inconsistencies in miRNA names from version to version. When working with only a small number of miRNAs the translation can be done manually. However, with large sets of miRNAs, the necessary correction of such inconsistencies becomes burdensome and error-prone. We developed miRNAmeConverter , available as a Bioconductor R package and web interface that addresses the challenges associated with mature miRNA name inconsistencies. The main algorithm implemented enables high-throughput automatic translation of species-independent mature miRNA names to user selected miRBase versions. The web interface enables users less familiar with R to translate miRNA names given in form of a list or embedded in text and download of the results. The miRNAmeConverter R package is open source under the Artistic-2.0 license. It is freely available from Bioconductor ( http://bioconductor.org/packages/miRNAmeConverter ). The web interface is based on R Shiny and can be accessed under the URL http://www.systemsmedicineireland.ie/tools/mirna-name-converter/ . The database that miRNAmeConverter depends on is provided by the annotation package miRBaseVersions.db and can be downloaded from Bioconductor ( http://bioconductor.org/packages/miRBaseVersions.db ). Minimum R version 3.3.0 is required. stefanhaunsberger@rcsi.ie. 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

Assessment of Radiation Risk by Circulating microRNAs

NASA Astrophysics Data System (ADS)

Wang, Jufang

2016-07-01

Highly energized particles delivered by galactic cosmic rays as well as solar particle events are one of the most severe detrimental factors to the health of crews during long-term space missions. Researches related to the assessment of radiation risk have been carried out with ground-based accelerator facilities all around the world. Circulating microRNAs (miRNAs) in blood have the advantages of specificity and stability, which could be used as disease biomarkers and potential bio-dosimeters to monitor the radiation risk. Based on this backgroud, circulating miRNAs were isolated from blood after Kunming mice were whole-body exposed to 300MeV/u carbon ion beam which were generated by the Heavy Ion Research Facility in Lanzhou (HIRFL), and the levels of miRNA expression were detected by miRNA PCR array. It was found that more than one hundred of circulating miRNAs were responded to carbon ion irradiation. Among these radiosensitive miRNAs, most of them were closely associated with immune system and hematopoietic system. The miRNA levels changed more than 2-fold were further verified by qRT-PCR analysis following exposure to X rays and iron ion beam. Some miRNAs such as let-7a, miR-34a, miR-223 and miR-150 showed obvious radio-sensitivity and dose-dependent effect, demonstrating that they were potential biomarkers of radiation and could be used as ideal bio-dosimeters. Those findings indicate that with the properties of high radio-sensitivity and time-saving quantification method by standard PCR assay, circulating miRNAs may become potential biomarkers for radiation detection in space exploration.

Analysis of MicroRNA Expression in Newborns with Differential Birth Weight Using Newborn Screening Cards

PubMed Central

Rodil-Garcia, Patricia; Arellanes-Licea, Elvira del Carmen; Montoya-Contreras, Angélica; Salazar-Olivo, Luis A.

2017-01-01

Birth weight is an early predictor for metabolic diseases and microRNAs (miRNAs) are proposed as fetal programming participants. To evaluate the use of dried blood spots (DBS) on newborn screening cards (NSC) as a source of analyzable miRNAs, we optimized a commercial protocol to recover total miRNA from normal birth weight (NBW, n = 17–20), low birth weight (LBW, n = 17–20) and high birth weight (macrosomia, n = 17–20) newborns and analyzed the relative expression of selected miRNAs by stem-loop RT-qPCR. The possible role of miRNAs on the fetal programming of metabolic diseases was explored by bioinformatic tools. The optimized extraction of RNA resulted in a 1.2-fold enrichment of miRNAs respect to the commercial kit. miR-33b and miR-375 were overexpressed in macrosomia 9.8-fold (p < 0.001) and 1.7-fold, (p < 0.05), respectively and miR-454-3p was overexpressed in both LBW and macrosomia (19.7-fold, p < 0.001 and 10.8-fold, p < 0.001, respectively), as compared to NBW. Potential target genes for these miRNAs are associated to cyclic-guanosine monophosphate (cGMP)-dependent protein kinase (PKG), mitogen-activated protein kinase (MAPK), type 2 diabetes, transforming growth factor-β (TGF-β)and Forkhead box O protein (FoxO) pathways. In summary, we improved a protocol for analyzing miRNAs from NSC and provide the first evidence that birth weight modifies the expression of miRNAs associated to adult metabolic dysfunctions. Our work suggests archived NSC are an invaluable resource in the search for fetal programming biomarkers. PMID:29182561

Dramatic changes in 67 miRNAs during initiation of first wave of spermatogenesis in Mus musculus testis: global regulatory insights generated by miRNA-mRNA network analysis.

PubMed

Sree, Sreesha; Radhakrishnan, Karthika; Indu, Sivankutty; Kumar, Pradeep G

2014-09-01

We mapped global changes in miRNA and mRNA profiles spanning the first wave of spermatogenesis using prepubertal (Postnatal Day 8 [P8]), pubertal (P16), and adolescent (P24) Mus musculus testes and identified the differential expression of 67 miRNAs and 8226 mRNAs. These two data sets were integrated into miRNA-dependent regulatory networks based on miRWalk predictions. In a network representing the P8 to P16 transition, downregulation of four miRNAs and upregulation of 19 miRNAs were linked with 81 upregulated target mRNAs and 228 downregulated target mRNAs, respectively. Furthermore, during the P16 to P24 transition, two miRNAs were downregulated, and eight miRNAs were upregulated, which linked with 64 upregulated mRNAs and 389 downregulated mRNAs, respectively. Only three of the miRNAs present in the network (miR-34b-5p, miR-34c, and miR-449a) showed a progressive increase from P8 through P16 to P24, while the remaining miRNAs in the network showed statistically significant changes in their levels either during the P8 to P16 transition or during the P16 to P24 transition. Analysis of the chromosomal location of these differentially expressed miRNAs showed that 14 out of 25 miRNAs upregulated from P8 to P16, and 18 out of 40 miRNAs upregulated from P8 to P24 were X-linked. This is suggestive of their escape from meiotic sex chromosome inactivation and postmeiotic sex chromatin. This integrated network of miRNA-level and mRNA-level changes in mouse testis during the first wave of spermatogenesis is expected to build a base for evaluating the role of miRNA-mediated gene expression regulation in maturing mammalian testis. © 2014 by the Society for the Study of Reproduction, Inc.

Comparison of commercial exosome isolation kits for circulating exosomal microRNA profiling.

PubMed

Ding, Meng; Wang, Cheng; Lu, Xiaolan; Zhang, Cuiping; Zhou, Zhen; Chen, Xi; Zhang, Chen-Yu; Zen, Ke; Zhang, Chunni

2018-06-01

Circulating exosomal microRNAs (miRNAs) are valuable biomarker candidates; however, information on the characterization and mutual agreement of commercial kits for circulating exosomal miRNA profiling is scarce. Here, we analyzed the advantages and weaknesses of four commonly used commercial kits for exosomal miRNA profiling and their application to the sample of serum and/or plasma, respectively. NanoSight and Western blotting were conducted to evaluate the efficiency and purity of the isolated exosomes. In our conditions, the size distribution of the isolated particles was appropriate (40-150 nm), and ExoQuick™ Exosome Precipitation Solution (EXQ) generated a relatively high yield of exosomes. Nevertheless, albumin impurity was ubiquitous for all the four kits, and Total Exosome Isolation for serum or plasma (TEI) yielded a relatively pure isolation. We further performed Illumina sequencing combined with RT-qPCR to determine the ability of these kits for miRNA profiling. There was significant correlation of the exosomal miRNA profile and specific miRNAs between kits, but with differences depending on methods. exoRNeasy Serum/Plasma Midi Kit (EXR) and EXQ performed better in the specific exosomal miRNAs recovery. Intraassay CVs for specific miRNA measurement were 0.88-3.82, 1.19-3.77, 0-2.70, and 1.23-9.11% for EXR, TEI, EXQ, and RIBO™ Exosome Isolation Reagent (REI), respectively. In each kit, serum yielded a higher abundance of exosomes and exosomal miRNAs than plasma, yet with more albumin impurity. In conclusion, our data provide some valuable guidance for the methodology of disease biomarker identification of circulation exosomal miRNAs. Graphical abstract Circulating exosomal microRNAs (miRNAs) are valuable biomarker candidates; however, information on the characterization and mutual agreement of commercial kits for circulating exosomal miRNA profiling is scarce. In this study, we compared four commonly used commercially available kits for exosomal miRNAsextraction and analyzed the advantages and weaknesses of each kit and their application to the sample ofserum and/or plasma.

Developmental Decline in the MicroRNA 199a (miR-199a)/miR-214 Cluster in Human Fetal Lung Promotes Type II Cell Differentiation by Upregulating Key Transcription Factors.

PubMed

Mishra, Ritu; Benlhabib, Houda; Guo, Wei; Lerma Cervantes, Connie B; Mendelson, Carole R

2018-06-01

The major surfactant protein, SP-A (a product of the SFTPA gene), serves as a marker of type II pneumocyte differentiation and surfactant synthesis. SFTPA expression in cultured human fetal lung (HFL) epithelial cells is upregulated by hormones that increase cyclic AMP (cAMP) and activate TTF-1/NKX2.1 and NF-κB. To further define mechanisms for type II cell differentiation and induction of SP-A, we investigated roles of microRNAs (miRNAs). Using microarray to identify differentially expressed miRNAs in HFL epithelial cells during type II cell differentiation in culture, we observed that members of the miRNA 199a (miR-199a)/miR-214 cluster were significantly downregulated during differentiation. Validated and predicted targets of miR-199a-3p/miR-199a-5p and miR-214, which serve roles in type II cell differentiation (COX-2, NF-κB p50/p65, and CREB1), and the CREB1 target, C/EBPβ, were coordinately upregulated. Accordingly, overexpression of miR-199a-5p, miR-199a-3p, or miR-214 mimics in cultured HFL epithelial cells decreased COX-2, NF-κB p50/p65, CREB1, and C/EBPβ proteins, with an associated inhibition of SP-A expression. Interestingly, overexpression of the EMT factor, ZEB1, which declines during cAMP-induced type II cell differentiation, increased pri-miR-199a and reduced the expression of the targets NF-κB/p50 and COX-2. Collectively, these findings suggest that the developmental decline in miR-199a/miR-214 in HFL causes increased expression of critical targets that enhance type II cell differentiation and SP-A expression. Copyright © 2018 American Society for Microbiology.

Members of miR-169 family are induced by high salinity and transiently inhibit the NF-YA transcription factor.

PubMed

Zhao, Botao; Ge, Liangfa; Liang, Ruqiang; Li, Wei; Ruan, Kangcheng; Lin, Hongxuan; Jin, Youxin

2009-04-08

MicroRNAs (miRNAs) are endogenously expressed small RNAs with a length of about 21 nt. MiRNAs silence their target genes at the post-transcriptional level. In plants, miRNAs play various developmental and physiological roles by cleavaging mRNAs predominantly. Drought and high salinity are the most severe environmental abiotic stresses and cause crop losses all over the world. In this study, we identified miR-169g and miR-169n (o) as high salinity-responsive miRNAs in rice. MiR-169n and miR169o were in a miRNA cluster with a distance of 3707 base pairs (bp). The high degree of conservation and close phylogenic distance of pre-miR-169n and pre-miR-169o indicated that they were derived from a very recent tandem duplication evolutionary event. The existence of a cis-acting abscisic acid responsive element (ABRE) in the upstream region of miR-169n (o) suggested that miR-169n (o) may be regulated by ABA. In our previous study, we found that miR-169g was induced by the osmotic stress caused by drought via a dehydration-responsive element (DRE). Thus, our data showed that there were both overlapping and distinct responses of the miR-169 family to drought and salt stresses. We also showed that these miR-169 members selectively cleaved one of the NF-YA genes, Os03g29760, which is a CCAAT-box binding transcription factor and participates in transcriptional regulation of large number genes. Finally, we found one or more ath-miR-169 member that was also induced by high salinity. We identified members of the miR-169 family as salt-induced miRNAs and analyzed their evolution, gene organization, expression, transcriptional regulation motif and target gene. Our data also indicated that the salt-induction of some miR-169 members was a general property in plants.

Members of miR-169 family are induced by high salinity and transiently inhibit the NF-YA transcription factor

PubMed Central

Zhao, Botao; Ge, Liangfa; Liang, Ruqiang; Li, Wei; Ruan, Kangcheng; Lin, Hongxuan; Jin, Youxin

2009-01-01

Background MicroRNAs (miRNAs) are endogenously expressed small RNAs with a length of about 21 nt. MiRNAs silence their target genes at the post-transcriptional level. In plants, miRNAs play various developmental and physiological roles by cleavaging mRNAs predominantly. Drought and high salinity are the most severe environmental abiotic stresses and cause crop losses all over the world. Results In this study, we identified miR-169g and miR-169n (o) as high salinity-responsive miRNAs in rice. MiR-169n and miR169o were in a miRNA cluster with a distance of 3707 base pairs (bp). The high degree of conservation and close phylogenic distance of pre-miR-169n and pre-miR-169o indicated that they were derived from a very recent tandem duplication evolutionary event. The existence of a cis-acting abscisic acid responsive element (ABRE) in the upstream region of miR-169n (o) suggested that miR-169n (o) may be regulated by ABA. In our previous study, we found that miR-169g was induced by the osmotic stress caused by drought via a dehydration-responsive element (DRE). Thus, our data showed that there were both overlapping and distinct responses of the miR-169 family to drought and salt stresses. We also showed that these miR-169 members selectively cleaved one of the NF-YA genes, Os03g29760, which is a CCAAT-box binding transcription factor and participates in transcriptional regulation of large number genes. Finally, we found one or more ath-miR-169 member that was also induced by high salinity. Conclusion We identified members of the miR-169 family as salt-induced miRNAs and analyzed their evolution, gene organization, expression, transcriptional regulation motif and target gene. Our data also indicated that the salt-induction of some miR-169 members was a general property in plants. PMID:19351418

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy.

PubMed

Lemcke, Heiko; Voronina, Natalia; Steinhoff, Gustav; David, Robert

2017-06-19

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

miRTex: A Text Mining System for miRNA-Gene Relation Extraction

PubMed Central

Li, Gang; Ross, Karen E.; Arighi, Cecilia N.; Peng, Yifan; Wu, Cathy H.; Vijay-Shanker, K.

2015-01-01

MicroRNAs (miRNAs) regulate a wide range of cellular and developmental processes through gene expression suppression or mRNA degradation. Experimentally validated miRNA gene targets are often reported in the literature. In this paper, we describe miRTex, a text mining system that extracts miRNA-target relations, as well as miRNA-gene and gene-miRNA regulation relations. The system achieves good precision and recall when evaluated on a literature corpus of 150 abstracts with F-scores close to 0.90 on the three different types of relations. We conducted full-scale text mining using miRTex to process all the Medline abstracts and all the full-length articles in the PubMed Central Open Access Subset. The results for all the Medline abstracts are stored in a database for interactive query and file download via the website at http://proteininformationresource.org/mirtex. Using miRTex, we identified genes potentially regulated by miRNAs in Triple Negative Breast Cancer, as well as miRNA-gene relations that, in conjunction with kinase-substrate relations, regulate the response to abiotic stress in Arabidopsis thaliana. These two use cases demonstrate the usefulness of miRTex text mining in the analysis of miRNA-regulated biological processes. PMID:26407127

Integrated mRNA and microRNA transcriptome analyses reveal regulation of thermal acclimation in Gymnocypris przewalskii: A case study in Tibetan Schizothoracine fish

PubMed Central

Tian, Fei; Zhao, Kai

2017-01-01

Environmental acclimation is important episode in wildlife occupation of the high-altitude Tibetan Plateau (TP). Transcriptome-wide studies on thermal acclimation mechanism in fish species are rarely revealed in Tibetan Plateau fish at high altitude. Thus, we used mRNA and miRNA transcriptome sequencing to investigate regulation of thermal acclimation in larval Tibetan naked carp, Gymnocypris przewalskii. We first remodeled the regulation network of mRNA and miRNA in thermal acclimation, and then identified differential expression of miRNAs and target mRNAs enriched in metabolic and digestive pathways. Interestingly, we identified two candidate genes contributed to normal skeletal development. The altered expression of these gene groups could potentially be associated with the developmental issues of deformity and induced larval death. Our results have three important implications: first, these findings provide strong evidences to support our hypothesis that G. przewalskii possess ability to build heat-tolerance against the controversial issue. Second, this study shows that transcriptional and post-transcriptional regulations are extensively involved in thermal acclimation. Third, the integrated mRNA and microRNA transcriptome analyses provide a large number of valuable genetic resources for future studies on environmental stress response in G. przewalskii and as a case study in Tibetan Schizothoracine fish. PMID:29045433

MicroRNAs in Honey Bee Caste Determination

PubMed Central

Ashby, Regan; Forêt, Sylvain; Searle, Iain; Maleszka, Ryszard

2016-01-01

The cellular mechanisms employed by some organisms to produce contrasting morphological and reproductive phenotypes from the same genome remains one of the key unresolved issues in biology. Honeybees (Apis mellifera) use differential feeding and a haplodiploid sex determination system to generate three distinct organismal outcomes from the same genome. Here we investigate the honeybee female and male caste-specific microRNA and transcriptomic molecular signatures during a critical time of larval development. Both previously undetected and novel miRNAs have been discovered, expanding the inventory of these genomic regulators in invertebrates. We show significant differences in the microRNA and transcriptional profiles of diploid females relative to haploid drone males as well as between reproductively distinct females (queens and workers). Queens and drones show gene enrichment in physio-metabolic pathways, whereas workers show enrichment in processes associated with neuronal development, cell signalling and caste biased structural differences. Interestingly, predicted miRNA targets are primarily associated with non-physio-metabolic genes, especially neuronal targets, suggesting a mechanistic disjunction from DNA methylation that regulates physio-metabolic processes. Accordingly, miRNA targets are under-represented in methylated genes. Our data show how a common set of genetic elements are differentially harnessed by an organism, which may provide the remarkable level of developmental flexibility required. PMID:26739502

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

Activity-associated miRNA are packaged in Map1b-enriched exosomes released from depolarized neurons.

PubMed

Goldie, Belinda J; Dun, Matthew D; Lin, Minjie; Smith, Nathan D; Verrills, Nicole M; Dayas, Christopher V; Cairns, Murray J

2014-08-01

Rapid input-restricted change in gene expression is an important aspect of synaptic plasticity requiring complex mechanisms of post-transcriptional mRNA trafficking and regulation. Small non-coding miRNA are uniquely poised to support these functions by providing a nucleic-acid-based specificity component for universal-sequence-dependent RNA binding complexes. We investigated the subcellular distribution of these molecules in resting and potassium chloride depolarized human neuroblasts, and found both selective enrichment and depletion in neurites. Depolarization was associated with a neurite-restricted decrease in miRNA expression; a subset of these molecules was recovered from the depolarization medium in nuclease resistant extracellular exosomes. These vesicles were enriched with primate specific miRNA and the synaptic-plasticity-associated protein MAP1b. These findings further support a role for miRNA as neural plasticity regulators, as they are compartmentalized in neurons and undergo activity-associated redistribution or release into the extracellular matrix. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Zcchc11 Uridylates Mature miRNAs to Enhance Neonatal IGF-1 Expression, Growth, and Survival

PubMed Central

Kozlowski, Elyse; Matsuura, Kori Y.; Ferrari, Joseph D.; Morris, Samantha A.; Powers, John T.; Daley, George Q.; Quinton, Lee J.; Mizgerd, Joseph P.

2012-01-01

The Zcchc11 enzyme is implicated in microRNA (miRNA) regulation. It can uridylate let-7 precursors to decrease quantities of the mature miRNA in embryonic stem cell lines, suggested to mediate stem cell maintenance. It can uridylate mature miR-26 to relieve silencing activity without impacting miRNA content in cancer cell lines, suggested to mediate cytokine and growth factor expression. Broader roles of Zcchc11 in shaping or remodeling the miRNome or in directing biological or physiological processes remain entirely speculative. We generated Zcchc11-deficient mice to address these knowledge gaps. Zcchc11 deficiency had no impact on embryogenesis or fetal development, but it significantly decreased survival and growth immediately following birth, indicating a role for this enzyme in early postnatal fitness. Deep sequencing of small RNAs from neonatal livers revealed roles of this enzyme in miRNA sequence diversity. Zcchc11 deficiency diminished the lengths and terminal uridine frequencies for diverse mature miRNAs, but it had no influence on the quantities of any miRNAs. The expression of IGF-1, a liver-derived protein essential to early growth and survival, was enhanced by Zcchc11 expression in vitro, and miRNA silencing of IGF-1 was alleviated by uridylation events observed to be Zcchc11-dependent in the neonatal liver. In neonatal mice, Zcchc11 deficiency significantly decreased IGF-1 mRNA in the liver and IGF-1 protein in the blood. We conclude that the Zcchc11-mediated terminal uridylation of mature miRNAs is pervasive and physiologically significant, especially important in the neonatal period for fostering IGF-1 expression and enhancing postnatal growth and survival. We propose that the miRNA 3′ terminus is a regulatory node upon which multiple enzymes converge to direct silencing activity and tune gene expression. PMID:23209448

Identification of age- and disease-related alterations in circulating miRNAs in a mouse model of Alzheimer's disease

PubMed Central

Garza-Manero, Sylvia; Arias, Clorinda; Bermúdez-Rattoni, Federico; Vaca, Luis; Zepeda, Angélica

2015-01-01

Alzheimer's disease (AD) is a neurodegenerative disorder characterized clinically by the progressive decline of memory and cognition. Histopathologically, two main hallmarks have been identified in AD: amyloid-β peptide extracellular neuritic plaques and neurofibrillary tangles formed by posttranslational modified tau protein. A definitive diagnosis can only be achieved after the post mortem verification of the histological mentioned alterations. Therefore, the development of biomarkers that allow an early diagnosis and/or predict disease progression is imperative. The prospect of a blood-based biomarker is possible with the finding of circulating microRNAs (miRNAs), a class of small non-coding RNAs of 22–25 nucleotides length that regulate mRNA translation rate. miRNAs travel through blood and recent studies performed in potential AD cases suggest the possibility of finding pathology-associated differences in circulating miRNA levels that may serve to assist in early diagnosis of the disease. However, these studies analyzed samples at a single time-point, limiting the use of miRNAs as biomarkers in AD progression. In this study we evaluated miRNA levels in plasma samples at different time-points of the evolution of an AD-like pathology in a transgenic mouse model of the disease (3xTg-AD). We performed multiplex qRT-PCR and compared the plasmatic levels of 84 miRNAs previously associated to central nervous system development and disease. No significant differences were detected between WT and transgenic young mice. However, age-related significant changes in miRNA abundance were observed for both WT and transgenic mice, and some of these were specific for the 3xTg-AD. In agreement, variations in the levels of particular miRNAs were identified between WT and transgenic old mice thus suggesting that the age-dependent evolution of the AD-like pathology, rather than the presence and expression of the transgenes, modifies the circulating miRNA levels in the 3xTg-AD mice. PMID:25745387

Identification of age- and disease-related alterations in circulating miRNAs in a mouse model of Alzheimer's disease.

PubMed

Garza-Manero, Sylvia; Arias, Clorinda; Bermúdez-Rattoni, Federico; Vaca, Luis; Zepeda, Angélica

2015-01-01

Alzheimer's disease (AD) is a neurodegenerative disorder characterized clinically by the progressive decline of memory and cognition. Histopathologically, two main hallmarks have been identified in AD: amyloid-β peptide extracellular neuritic plaques and neurofibrillary tangles formed by posttranslational modified tau protein. A definitive diagnosis can only be achieved after the post mortem verification of the histological mentioned alterations. Therefore, the development of biomarkers that allow an early diagnosis and/or predict disease progression is imperative. The prospect of a blood-based biomarker is possible with the finding of circulating microRNAs (miRNAs), a class of small non-coding RNAs of 22-25 nucleotides length that regulate mRNA translation rate. miRNAs travel through blood and recent studies performed in potential AD cases suggest the possibility of finding pathology-associated differences in circulating miRNA levels that may serve to assist in early diagnosis of the disease. However, these studies analyzed samples at a single time-point, limiting the use of miRNAs as biomarkers in AD progression. In this study we evaluated miRNA levels in plasma samples at different time-points of the evolution of an AD-like pathology in a transgenic mouse model of the disease (3xTg-AD). We performed multiplex qRT-PCR and compared the plasmatic levels of 84 miRNAs previously associated to central nervous system development and disease. No significant differences were detected between WT and transgenic young mice. However, age-related significant changes in miRNA abundance were observed for both WT and transgenic mice, and some of these were specific for the 3xTg-AD. In agreement, variations in the levels of particular miRNAs were identified between WT and transgenic old mice thus suggesting that the age-dependent evolution of the AD-like pathology, rather than the presence and expression of the transgenes, modifies the circulating miRNA levels in the 3xTg-AD mice.

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.

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.

Microvesicles Derived from Adult Human Bone Marrow and Tissue Specific Mesenchymal Stem Cells Shuttle Selected Pattern of miRNAs

PubMed Central

Collino, Federica; Deregibus, Maria Chiara; Bruno, Stefania; Sterpone, Luca; Aghemo, Giulia; Viltono, Laura; Tetta, Ciro; Camussi, Giovanni

2010-01-01

Background Cell-derived microvesicles (MVs) have been described as a new mechanism of cell-to-cell communication. MVs after internalization within target cells may deliver genetic information. Human bone marrow derived mesenchymal stem cells (MSCs) and liver resident stem cells (HLSCs) were shown to release MVs shuttling functional mRNAs. The aim of the present study was to evaluate whether MVs derived from MSCs and HLSCs contained selected micro-RNAs (miRNAs). Methodology/Principal Findings MVs were isolated from MSCs and HLSCs. The presence in MVs of selected ribonucleoproteins involved in the traffic and stabilization of RNA was evaluated. We observed that MVs contained TIA, TIAR and HuR multifunctional proteins expressed in nuclei and stress granules, Stau1 and 2 implicated in the transport and stability of mRNA and Ago2 involved in miRNA transport and processing. RNA extracted from MVs and cells of origin was profiled for 365 known human mature miRNAs by real time PCR. Hierarchical clustering and similarity analysis of miRNAs showed 41 co-expressed miRNAs in MVs and cells. Some miRNAs were accumulated within MVs and absent in the cells after MV release; others were retained within the cells and not secreted in MVs. Gene ontology analysis of predicted and validated targets showed that the high expressed miRNAs in cells and MVs could be involved in multi-organ development, cell survival and differentiation. Few selected miRNAs shuttled by MVs were also associated with the immune system regulation. The highly expressed miRNAs in MVs were transferred to target cells after MV incorporation. Conclusions This study demonstrated that MVs contained ribonucleoproteins involved in the intracellular traffic of RNA and selected pattern of miRNAs, suggesting a dynamic regulation of RNA compartmentalization in MVs. The observation that MV-highly expressed miRNAs were transferred to target cells, rises the possibility that the biological effect of stem cells may, at least in part, depend on MV-shuttled miRNAs. Data generated from this study, stimulate further functional investigations on the predicted target genes and pathways involved in the biological effect of human adult stem cells. PMID:20668554

Exploiting Nanotechnology for the Development of MicroRNA-Based Cancer Therapeutics.

PubMed

Tyagi, Nikhil; Arora, Sumit; Deshmukh, Sachin K; Singh, Seema; Marimuthu, Saravanakumar; Singh, Ajay P

2016-01-01

MicroRNAs (miRNAs/miRs) represent a novel class of small non-coding RNAs that post-transcriptionally regulate gene expression by base pairing with complementary sequences in the 3' untranslated region (UTR) of target mRNAs. Functional studies suggest that miRNAs control almost every biological process, and their aberrant expression leads to a disease state, such as cancer. Differential expression of miRNAs in cancerous versus normal cells have generated enormous interest for the development of miRNA-based cancer cell-targeted therapeutics. Depending on the miRNA function and expression in cancer, two types of miRNA-based therapeutic strategies can be utilized that either restore or inhibit miRNA function through exogenous delivery of miRNAs mimics or inhibitors (anti-miRs). However, hydrophilic nature of miRNA mimics/anti-miRs, sensitivity to nuclease degradation in serum, poor penetration and reduced uptake by the tumor cells are chief hurdles in accomplishing their efficient in vivo delivery. To overcome these barriers, several nanotechnology-based systems are being developed and tested for delivery efficacy. This review summarizes the importance of miRNAs-based therapeutics in cancer, associated translational challenges and novel nanotechnology-assisted delivery systems that hold potential for next-generation miRNA-based cancer therapeutics.

Experimental design and data analysis of Ago-RIP-Seq experiments for the identification of microRNA targets.

PubMed

Tichy, Diana; Pickl, Julia Maria Anna; Benner, Axel; Sültmann, Holger

2017-03-31

The identification of microRNA (miRNA) target genes is crucial for understanding miRNA function. Many methods for the genome-wide miRNA target identification have been developed in recent years; however, they have several limitations including the dependence on low-confident prediction programs and artificial miRNA manipulations. Ago-RNA immunoprecipitation combined with high-throughput sequencing (Ago-RIP-Seq) is a promising alternative. However, appropriate statistical data analysis algorithms taking into account the experimental design and the inherent noise of such experiments are largely lacking.Here, we investigate the experimental design for Ago-RIP-Seq and examine biostatistical methods to identify de novo miRNA target genes. Statistical approaches considered are either based on a negative binomial model fit to the read count data or applied to transformed data using a normal distribution-based generalized linear model. We compare them by a real data simulation study using plasmode data sets and evaluate the suitability of the approaches to detect true miRNA targets by sensitivity and false discovery rates. Our results suggest that simple approaches like linear regression models on (appropriately) transformed read count data are preferable. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A transcriptome-wide study on the microRNA- and the Argonaute 1-enriched small RNA-mediated regulatory networks involved in plant leaf senescence.

PubMed

Qin, J; Ma, X; Yi, Z; Tang, Z; Meng, Y

2016-03-01

Leaf senescence is an important physiological process during the plant life cycle. However, systemic studies on the impact of microRNAs (miRNAs) on the expression of senescence-associated genes (SAGs) are lacking. Besides, whether other Argonaute 1 (AGO1)-enriched small RNAs (sRNAs) play regulatory roles in leaf senescence remains unclear. In this study, a total of 5,123 and 1,399 AGO1-enriched sRNAs, excluding miRNAs, were identified in Arabidopsis thaliana and rice (Oryza sativa), respectively. After retrieving SAGs from the Leaf Senescence Database, all of the AGO1-enriched sRNAs and the miRBase-registered miRNAs of these two plants were included for target identification. Supported by degradome signatures, 200 regulatory pairs involving 120 AGO1-enriched sRNAs and 40 SAGs, and 266 regulatory pairs involving 64 miRNAs and 42 SAGs were discovered in Arabidopsis. Moreover, 13 genes predicted to interact with some of the above-identified target genes at protein level were validated as regulated by 17 AGO1-enriched sRNAs and ten miRNAs in Arabidopsis. In rice, only one SAG was targeted by three AGO1-enriched sRNAs, and one SAG was targeted by miR395. However, five AGO1-enriched sRNAs were conserved between Arabidopsis and rice. Target genes conserved between the two plants were identified for three of the above five sRNAs, pointing to the conserved roles of these regulatory pairs in leaf senescence or other developmental procedures. Novel targets were discovered for three of the five AGO1-enriched sRNAs in rice, indicating species-specific functions of these sRNA-target pairs. These results could advance our understanding of the sRNA-involved molecular processes modulating leaf senescence. © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands.

Inhibition of glioblastoma cell invasion by hsa-miR-145-5p and hsa-miR-31-5p co-overexpression in human mesenchymal stem cells.

PubMed

Kurogi, Ryota; Nakamizo, Akira; Suzuki, Satoshi O; Mizoguchi, Masahiro; Yoshimoto, Koji; Amano, Toshiyuki; Amemiya, Takeo; Takagishi, So; Iihara, Koji

2018-03-09

OBJECTIVE Human bone marrow-derived mesenchymal stem cells (hMSCs) show tropism for brain tumors and may be a useful vehicle for drug or gene delivery to malignant gliomas. Recently, some microRNAs (miRNAs) have been shown to suppress the invasiveness of malignant gliomas. METHODS To test their potential to become vehicles for the delivery of miRNA to malignant gliomas, hMSCs were engineered so that hMSC secretion of miRNAs that inhibit glioma cell invasion was enabled without altering the hMSC tropism for glioma cells. RESULTS In coculture, hMSCs cotransfected with hsa-miR-145-5p and -31-5p miRNAs showed markedly reduced invasion by U87 glioma cells in a contact-dependent manner both in vitro and ex vivo, with invasion of hMSCs cotransfected with these 2 miRNAs by the U87 cells reduced to 60.7% compared with control cells. According to a Matrigel invasion assay, the tropism of the hMSCs for U87 cells was not affected. In glioma cell lines U251 and LN229, hMSCs exhibited tropism in vivo, and invasion of hMSCs cotransfected with hsa-miR-145-5p and -31-5p was also significantly less than that of control cells. When U87 cells were coimplanted into the striatum of organotypic rat brain slices with hMSCs cotransfected with hsa-miR-145 and -31-5p, the relative invasive area decreased by 37.1%; interestingly, these U87 cells showed a change to a rounded morphology that was apparent at the invasion front. Whole-genome microarray analysis of the expression levels of 58,341 genes revealed that the co-overexpression of hsa-miR-145-5p and -31-5p downregulated FSCN1 expression in U87 cells. CONCLUSIONS This study demonstrates that miRNA overexpression in hMSCs can alter the function of glioma cells via contact-dependent transfer. Co-overexpression of multiple miRNAs may be a useful and novel therapeutic strategy. The study results suggest that hMSCs can be applied as a delivery vehicle for miRNAs.

miR-191 and miR-135 are required for long-lasting spine remodelling associated with synaptic long-term depression

NASA Astrophysics Data System (ADS)

Hu, Zhonghua; Yu, Danni; Gu, Qin-Hua; Yang, Yanqin; Tu, Kang; Zhu, Jun; Li, Zheng

2014-02-01

Activity-dependent modification of dendritic spines, subcellular compartments accommodating postsynaptic specializations in the brain, is an important cellular mechanism for brain development, cognition and synaptic pathology of brain disorders. NMDA receptor-dependent long-term depression (NMDAR-LTD), a prototypic form of synaptic plasticity, is accompanied by prolonged remodelling of spines. The mechanisms underlying long-lasting spine remodelling in NMDAR-LTD, however, are largely unclear. Here we show that LTD induction causes global changes in miRNA transcriptomes affecting many cellular activities. Specifically, we show that expression changes of miR-191 and miR-135 are required for maintenance but not induction of spine restructuring. Moreover, we find that actin depolymerization and AMPA receptor exocytosis are regulated for extended periods of time by miRNAs to support long-lasting spine plasticity. These findings reveal a miRNA-mediated mechanism and a role for AMPA receptor exocytosis in long-lasting spine plasticity, and identify a number of candidate miRNAs involved in LTD.

Role of miRNAs in CD4 T cell plasticity during inflammation and tolerance

PubMed Central

Sethi, Apoorva; Kulkarni, Neeraja; Sonar, Sandip; Lal, Girdhari

2013-01-01

Gene expression is tightly regulated in a tuneable, cell-specific and time-dependent manner. Recent advancement in epigenetics and non-coding RNA (ncRNA) revolutionized the concept of gene regulation. In order to regulate the transcription, ncRNA can promptly response to the extracellular signals as compared to transcription factors present in the cells. microRNAs (miRNAs) are ncRNA (~22 bp) encoded in the genome, and present as intergenic or oriented antisense to neighboring genes. The strategic location of miRNA in coding genes helps in the coupled regulation of its expression with host genes. miRNA together with complex machinery called RNA-induced silencing complex (RISC) interacts with target mRNA and degrade the mRNA or inhibits the translation. CD4 T cells play an important role in the generation and maintenance of inflammation and tolerance. Cytokines and chemokines present in the inflamed microenvironment controls the differentiation and function of various subsets of CD4 T cells [Th1, Th2, Th17, and regulatory CD4 T cells (Tregs)]. Recent studies suggest that miRNAs play an important role in the development and function of all subsets of CD4 T cells. In current review, we focused on how various miRNAs are regulated by cell's extrinsic and intrinsic signaling, and how miRNAs affect the transdifferentiation of subsets of CD4 T cell and controls their plasticity during inflammation and tolerance. PMID:23386861

OmniSearch: a semantic search system based on the Ontology for MIcroRNA Target (OMIT) for microRNA-target gene interaction data.

PubMed

Huang, Jingshan; Gutierrez, Fernando; Strachan, Harrison J; Dou, Dejing; Huang, Weili; Smith, Barry; Blake, Judith A; Eilbeck, Karen; Natale, Darren A; Lin, Yu; Wu, Bin; Silva, Nisansa de; Wang, Xiaowei; Liu, Zixing; Borchert, Glen M; Tan, Ming; Ruttenberg, Alan

2016-01-01

As a special class of non-coding RNAs (ncRNAs), microRNAs (miRNAs) perform important roles in numerous biological and pathological processes. The realization of miRNA functions depends largely on how miRNAs regulate specific target genes. It is therefore critical to identify, analyze, and cross-reference miRNA-target interactions to better explore and delineate miRNA functions. Semantic technologies can help in this regard. We previously developed a miRNA domain-specific application ontology, Ontology for MIcroRNA Target (OMIT), whose goal was to serve as a foundation for semantic annotation, data integration, and semantic search in the miRNA field. In this paper we describe our continuing effort to develop the OMIT, and demonstrate its use within a semantic search system, OmniSearch, designed to facilitate knowledge capture of miRNA-target interaction data. Important changes in the current version OMIT are summarized as: (1) following a modularized ontology design (with 2559 terms imported from the NCRO ontology); (2) encoding all 1884 human miRNAs (vs. 300 in previous versions); and (3) setting up a GitHub project site along with an issue tracker for more effective community collaboration on the ontology development. The OMIT ontology is free and open to all users, accessible at: http://purl.obolibrary.org/obo/omit.owl. The OmniSearch system is also free and open to all users, accessible at: http://omnisearch.soc.southalabama.edu/index.php/Software.

MicroRNA and Transcriptomic Profiling Showed miRNA-Dependent Impairment of Systemic Regulation and Synthesis of Biomolecules in Rag2 KO Mice.

PubMed

Reza, Abu Musa Md Talimur; Choi, Yun-Jung; Kim, Jin-Hoi

2018-02-27

The Rag2 knockout (KO) mouse is a well-established immune-compromised animal model for biomedical research. A comparative study identified the deregulated expression of microRNAs (miRNAs) and messenger RNAs (mRNAs) in Rag2 KO mice. However, the interaction between deregulated genes and miRNAs in the alteration of systemic (cardiac, renal, hepatic, nervous, and hematopoietic) regulations and the synthesis of biomolecules (such as l-tryptophan, serotonin, melatonin, dopamine, alcohol, noradrenaline, putrescine, and acetate) are unclear. In this study, we analyzed both miRNA and mRNA expression microarray data from Rag2 KO and wild type mice to investigate the possible role of miRNAs in systemic regulation and biomolecule synthesis. A notable finding obtained from this analysis is that the upregulation of several genes which are target molecules of the downregulated miRNAs in Rag2 KO mice, can potentially trigger the degradation of l-tryptophan, thereby leading to the systemic impairment and alteration of biomolecules synthesis as well as changes in behavioral patterns (such as stress and fear responses, and social recognition memory) in Rag2 gene-depleted mice. These findings were either not observed or not explicitly described in other published Rag2 KO transcriptome analyses. In conclusion, we have provided an indication of miRNA-dependent regulations of clinical and pathological conditions in cardiac, renal, hepatic, nervous, and hematopoietic systems in Rag2 KO mice. These results may significantly contribute to the prediction of clinical disease caused by Rag2 deficiency.

Costimulation dependent expression of miR-214 increases the ability of T cells to proliferate by targeting Pten

PubMed Central

Jindra, Peter T.; Bagley, Jessamyn; Godwin, Jonathan G.; Iacomini, John

2010-01-01

T cell activation requires signaling through the T cell receptor (TCR) and costimulatory molecules such as CD28. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post transcriptionally and are also known to be involved in lymphocyte development and function. Here we set out to examine potential roles of miRNAs in T cell activation by using genome-wide expression profiling to identify miRNAs differentially regulated following T cell activation. One of the miRNAs up-regulated after T cell activation, miR-214, was predicted to be capable of targeting Pten based on bioinformatics and reports suggesting that it targets Pten in ovarian tumor cells. Up-regulation of miR-214 in T cells inversely correlated with PTEN levels. In vivo, transcripts containing the 3' untranslated region (3' UTR) of Pten including the miR-214 target sequence were negatively regulated after T cell activation, and forced expression of miR-214 in T cells led to increased proliferation after stimulation. Blocking CD28 signaling in vivo prevented miR-214 up-regulation in alloreactive T cells. Stimulation of T cells through the TCR alone was not sufficient to result in upregulation of miR-214. Thus, costimulation dependent up-regulation of miR-214 promotes T cell activation by targeting the negative regulator Pten. Thus, the requirement for T cell costimulation is in part related to its ability to regulate expression of miRNAs that control T cell activation. PMID:20548023

Current knowledge of microRNA-mediated regulation of drug metabolism in humans.

PubMed

Nakano, Masataka; Nakajima, Miki

2018-05-01

Understanding the factors causing inter- and intra-individual differences in drug metabolism potencies is required for the practice of personalized or precision medicine, as well as for the promotion of efficient drug development. The expression of drug-metabolizing enzymes is controlled by transcriptional regulation by nuclear receptors and transcriptional factors, epigenetic regulation, such as DNA methylation and histone acetylation, and post-translational modification. In addition to such regulation mechanisms, recent studies revealed that microRNAs (miRNAs), endogenous ~22-nucleotide non-coding RNAs that regulate gene expression through the translational repression and degradation of mRNAs, significantly contribute to post-transcriptional regulation of drug-metabolizing enzymes. Areas covered: This review summarizes the current knowledge regarding miRNAs-dependent regulation of drug-metabolizing enzymes and transcriptional factors and its physiological and clinical significance. We also describe recent advances in miRNA-dependent regulation research, showing that the presence of pseudogenes, single-nucleotide polymorphisms, and RNA editing affects miRNA targeting. Expert opinion: It is unwavering fact that miRNAs are critical factors causing inter- and intra-individual differences in the expression of drug-metabolizing enzymes. Consideration of miRNA-dependent regulation would be a helpful tool for optimizing personalized and precision medicine.

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.

A Screen in Mice Uncovers Repression of Lipoprotein Lipase by MicroRNA-29a as a Mechanism for Lipid Distribution Away From the Liver

PubMed Central

Mattis, Aras N.; Song, Guisheng; Hitchner, Kelly; Kim, Roy Y.; Lee, Andrew Y.; Sharma, Amar D.; Malato, Yann; McManus, Michael T.; Esau, Christine C.; Koller, Erich; Koliwad, Suneil; Lim, Lee P.; Maher, Jacquelyn J.; Raffai, Robert L.; Willenbring, Holger

2015-01-01

Identification of microRNAs (miRNAs) that regulate lipid metabolism is important to advance the understanding and treatment of some of the most common human diseases. In the liver, a few key miRNAs have been reported that regulate lipid metabolism, but since many genes contribute to hepatic lipid metabolism, we hypothesized that other such miRNAs exist. To identify genes repressed by miRNAs in mature hepatocytes in vivo, we injected adult mice carrying floxed Dicer1 alleles with an adenoassociated viral vector expressing Cre recombinase specifically in hepatocytes. By inactivating Dicer in adult quiescent hepatocytes we avoided the hepatocyte injury and regeneration observed in previous mouse models of global miRNA deficiency in hepatocytes. Next, we combined gene and miRNA expression profiling to identify candidate gene/miRNA interactions involved in hepatic lipid metabolism, and validated their function in vivo using antisense oligonucleotides. A candidate gene that emerged from our screen was lipoprotein lipase (Lpl), which encodes an enzyme that facilitates cellular uptake of lipids from the circulation. Unlike in energy-dependent cells like myocytes, Lpl is normally repressed in adult hepatocytes. We identified miR-29a as the miRNA responsible for repressing Lpl in hepatocytes, and found that decreasing hepatic miR-29a levels causes lipids to accumulate in mouse livers. Conclusion Our screen suggests several new miRNAs are regulators of hepatic lipid metabolism. We show that one of these, miR-29a, contributes to physiological lipid distribution away from the liver and protects hepatocytes from steatosis. Our results, together with miR-29a’s known anti-fibrotic effect, suggest miR-29a is a therapeutic target in fatty liver disease. PMID:25131933

MicroRNA Related Polymorphisms and Breast Cancer Risk

PubMed Central

Khan, Sofia; Greco, Dario; Michailidou, Kyriaki; Milne, Roger L.; Muranen, Taru A.; Heikkinen, Tuomas; Aaltonen, Kirsimari; Dennis, Joe; Bolla, Manjeet K.; Liu, Jianjun; Hall, Per; Irwanto, Astrid; Humphreys, Keith; Li, Jingmei; Czene, Kamila; Chang-Claude, Jenny; Hein, Rebecca; Rudolph, Anja; Seibold, Petra; Flesch-Janys, Dieter; Fletcher, Olivia; Peto, Julian; dos Santos Silva, Isabel; Johnson, Nichola; Gibson, Lorna; Aitken, Zoe; Hopper, John L.; Tsimiklis, Helen; Bui, Minh; Makalic, Enes; Schmidt, Daniel F.; Southey, Melissa C.; Apicella, Carmel; Stone, Jennifer; Waisfisz, Quinten; Meijers-Heijboer, Hanne; Adank, Muriel A.; van der Luijt, Rob B.; Meindl, Alfons; Schmutzler, Rita K.; Müller-Myhsok, Bertram; Lichtner, Peter; Turnbull, Clare; Rahman, Nazneen; Chanock, Stephen J.; Hunter, David J.; Cox, Angela; Cross, Simon S.; Reed, Malcolm W. R.; Schmidt, Marjanka K.; Broeks, Annegien; Veer, Laura J. V. a. n't.; Hogervorst, Frans B.; Fasching, Peter A.; Schrauder, Michael G.; Ekici, Arif B.; Beckmann, Matthias W.; Bojesen, Stig E.; Nordestgaard, Børge G.; Nielsen, Sune F.; Flyger, Henrik; Benitez, Javier; Zamora, Pilar M.; Perez, Jose I. A.; Haiman, Christopher A.; Henderson, Brian E.; Schumacher, Fredrick; Le Marchand, Loic; Pharoah, Paul D. P.; Dunning, Alison M.; Shah, Mitul; Luben, Robert; Brown, Judith; Couch, Fergus J.; Wang, Xianshu; Vachon, Celine; Olson, Janet E.; Lambrechts, Diether; Moisse, Matthieu; Paridaens, Robert; Christiaens, Marie-Rose; Guénel, Pascal; Truong, Thérèse; Laurent-Puig, Pierre; Mulot, Claire; Marme, Frederick; Burwinkel, Barbara; Schneeweiss, Andreas; Sohn, Christof; Sawyer, Elinor J.; Tomlinson, Ian; Kerin, Michael J.; Miller, Nicola; Andrulis, Irene L.; Knight, Julia A.; Tchatchou, Sandrine; Mulligan, Anna Marie; Dörk, Thilo; Bogdanova, Natalia V.; Antonenkova, Natalia N.; Anton-Culver, Hoda; Darabi, Hatef; Eriksson, Mikael; Garcia-Closas, Montserrat; Figueroa, Jonine; Lissowska, Jolanta; Brinton, Louise; Devilee, Peter; Tollenaar, Robert A. E. M.; Seynaeve, Caroline; van Asperen, Christi J.; Kristensen, Vessela N.; Slager, Susan; Toland, Amanda E.; Ambrosone, Christine B.; Yannoukakos, Drakoulis; Lindblom, Annika; Margolin, Sara; Radice, Paolo; Peterlongo, Paolo; Barile, Monica; Mariani, Paolo; Hooning, Maartje J.; Martens, John W. M.; Collée, J. Margriet; Jager, Agnes; Jakubowska, Anna; Lubinski, Jan; Jaworska-Bieniek, Katarzyna; Durda, Katarzyna; Giles, Graham G.; McLean, Catriona; Brauch, Hiltrud; Brüning, Thomas; Ko, Yon-Dschun; Brenner, Hermann; Dieffenbach, Aida Karina; Arndt, Volker; Stegmaier, Christa; Swerdlow, Anthony; Ashworth, Alan; Orr, Nick; Jones, Michael; Simard, Jacques; Goldberg, Mark S.; Labrèche, France; Dumont, Martine; Winqvist, Robert; Pylkäs, Katri; Jukkola-Vuorinen, Arja; Grip, Mervi; Kataja, Vesa; Kosma, Veli-Matti; Hartikainen, Jaana M.; Mannermaa, Arto; Hamann, Ute; Chenevix-Trench, Georgia; Blomqvist, Carl; Aittomäki, Kristiina; Easton, Douglas F.; Nevanlinna, Heli

2014-01-01

Genetic variations, such as single nucleotide polymorphisms (SNPs) in microRNAs (miRNA) or in the miRNA binding sites may affect the miRNA dependent gene expression regulation, which has been implicated in various cancers, including breast cancer, and may alter individual susceptibility to cancer. We investigated associations between miRNA related SNPs and breast cancer risk. First we evaluated 2,196 SNPs in a case-control study combining nine genome wide association studies (GWAS). Second, we further investigated 42 SNPs with suggestive evidence for association using 41,785 cases and 41,880 controls from 41 studies included in the Breast Cancer Association Consortium (BCAC). Combining the GWAS and BCAC data within a meta-analysis, we estimated main effects on breast cancer risk as well as risks for estrogen receptor (ER) and age defined subgroups. Five miRNA binding site SNPs associated significantly with breast cancer risk: rs1045494 (odds ratio (OR) 0.92; 95% confidence interval (CI): 0.88–0.96), rs1052532 (OR 0.97; 95% CI: 0.95–0.99), rs10719 (OR 0.97; 95% CI: 0.94–0.99), rs4687554 (OR 0.97; 95% CI: 0.95–0.99, and rs3134615 (OR 1.03; 95% CI: 1.01–1.05) located in the 3′ UTR of CASP8, HDDC3, DROSHA, MUSTN1, and MYCL1, respectively. DROSHA belongs to miRNA machinery genes and has a central role in initial miRNA processing. The remaining genes are involved in different molecular functions, including apoptosis and gene expression regulation. Further studies are warranted to elucidate whether the miRNA binding site SNPs are the causative variants for the observed risk effects. PMID:25390939

MicroRNA related polymorphisms and breast cancer risk.

PubMed

Khan, Sofia; Greco, Dario; Michailidou, Kyriaki; Milne, Roger L; Muranen, Taru A; Heikkinen, Tuomas; Aaltonen, Kirsimari; Dennis, Joe; Bolla, Manjeet K; Liu, Jianjun; Hall, Per; Irwanto, Astrid; Humphreys, Keith; Li, Jingmei; Czene, Kamila; Chang-Claude, Jenny; Hein, Rebecca; Rudolph, Anja; Seibold, Petra; Flesch-Janys, Dieter; Fletcher, Olivia; Peto, Julian; dos Santos Silva, Isabel; Johnson, Nichola; Gibson, Lorna; Aitken, Zoe; Hopper, John L; Tsimiklis, Helen; Bui, Minh; Makalic, Enes; Schmidt, Daniel F; Southey, Melissa C; Apicella, Carmel; Stone, Jennifer; Waisfisz, Quinten; Meijers-Heijboer, Hanne; Adank, Muriel A; van der Luijt, Rob B; Meindl, Alfons; Schmutzler, Rita K; Müller-Myhsok, Bertram; Lichtner, Peter; Turnbull, Clare; Rahman, Nazneen; Chanock, Stephen J; Hunter, David J; Cox, Angela; Cross, Simon S; Reed, Malcolm W R; Schmidt, Marjanka K; Broeks, Annegien; Van't Veer, Laura J; Hogervorst, Frans B; Fasching, Peter A; Schrauder, Michael G; Ekici, Arif B; Beckmann, Matthias W; Bojesen, Stig E; Nordestgaard, Børge G; Nielsen, Sune F; Flyger, Henrik; Benitez, Javier; Zamora, Pilar M; Perez, Jose I A; Haiman, Christopher A; Henderson, Brian E; Schumacher, Fredrick; Le Marchand, Loic; Pharoah, Paul D P; Dunning, Alison M; Shah, Mitul; Luben, Robert; Brown, Judith; Couch, Fergus J; Wang, Xianshu; Vachon, Celine; Olson, Janet E; Lambrechts, Diether; Moisse, Matthieu; Paridaens, Robert; Christiaens, Marie-Rose; Guénel, Pascal; Truong, Thérèse; Laurent-Puig, Pierre; Mulot, Claire; Marme, Frederick; Burwinkel, Barbara; Schneeweiss, Andreas; Sohn, Christof; Sawyer, Elinor J; Tomlinson, Ian; Kerin, Michael J; Miller, Nicola; Andrulis, Irene L; Knight, Julia A; Tchatchou, Sandrine; Mulligan, Anna Marie; Dörk, Thilo; Bogdanova, Natalia V; Antonenkova, Natalia N; Anton-Culver, Hoda; Darabi, Hatef; Eriksson, Mikael; Garcia-Closas, Montserrat; Figueroa, Jonine; Lissowska, Jolanta; Brinton, Louise; Devilee, Peter; Tollenaar, Robert A E M; Seynaeve, Caroline; van Asperen, Christi J; Kristensen, Vessela N; Slager, Susan; Toland, Amanda E; Ambrosone, Christine B; Yannoukakos, Drakoulis; Lindblom, Annika; Margolin, Sara; Radice, Paolo; Peterlongo, Paolo; Barile, Monica; Mariani, Paolo; Hooning, Maartje J; Martens, John W M; Collée, J Margriet; Jager, Agnes; Jakubowska, Anna; Lubinski, Jan; Jaworska-Bieniek, Katarzyna; Durda, Katarzyna; Giles, Graham G; McLean, Catriona; Brauch, Hiltrud; Brüning, Thomas; Ko, Yon-Dschun; Brenner, Hermann; Dieffenbach, Aida Karina; Arndt, Volker; Stegmaier, Christa; Swerdlow, Anthony; Ashworth, Alan; Orr, Nick; Jones, Michael; Simard, Jacques; Goldberg, Mark S; Labrèche, France; Dumont, Martine; Winqvist, Robert; Pylkäs, Katri; Jukkola-Vuorinen, Arja; Grip, Mervi; Kataja, Vesa; Kosma, Veli-Matti; Hartikainen, Jaana M; Mannermaa, Arto; Hamann, Ute; Chenevix-Trench, Georgia; Blomqvist, Carl; Aittomäki, Kristiina; Easton, Douglas F; Nevanlinna, Heli

2014-01-01

Genetic variations, such as single nucleotide polymorphisms (SNPs) in microRNAs (miRNA) or in the miRNA binding sites may affect the miRNA dependent gene expression regulation, which has been implicated in various cancers, including breast cancer, and may alter individual susceptibility to cancer. We investigated associations between miRNA related SNPs and breast cancer risk. First we evaluated 2,196 SNPs in a case-control study combining nine genome wide association studies (GWAS). Second, we further investigated 42 SNPs with suggestive evidence for association using 41,785 cases and 41,880 controls from 41 studies included in the Breast Cancer Association Consortium (BCAC). Combining the GWAS and BCAC data within a meta-analysis, we estimated main effects on breast cancer risk as well as risks for estrogen receptor (ER) and age defined subgroups. Five miRNA binding site SNPs associated significantly with breast cancer risk: rs1045494 (odds ratio (OR) 0.92; 95% confidence interval (CI): 0.88-0.96), rs1052532 (OR 0.97; 95% CI: 0.95-0.99), rs10719 (OR 0.97; 95% CI: 0.94-0.99), rs4687554 (OR 0.97; 95% CI: 0.95-0.99, and rs3134615 (OR 1.03; 95% CI: 1.01-1.05) located in the 3' UTR of CASP8, HDDC3, DROSHA, MUSTN1, and MYCL1, respectively. DROSHA belongs to miRNA machinery genes and has a central role in initial miRNA processing. The remaining genes are involved in different molecular functions, including apoptosis and gene expression regulation. Further studies are warranted to elucidate whether the miRNA binding site SNPs are the causative variants for the observed risk effects.

Polycyclic aromatic hydrocarbon (PAH)-mediated upregulation of hepatic microRNA-181 family promotes cancer cell migration by targeting MAPK phosphatase-5, regulating the activation of p38 MAPK

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

Song, Mi-Kyung; School of Life Sciences and Biotechnology, Korea University, Anam-Dong, Seoungbuk-Gu, Seoul 136-701; Park, Yong-Keun

2013-11-15

Growing evidence indicates that changes in microRNA (miRNA) expression in cancer induced by chemical carcinogens play an important role in cancer development and progression by regulating related genes. However, the mechanisms underlying miRNA involvement in hepatocarcinogenesis induced by polycyclic aromatic hydrocarbons (PAHs) remain unclear. Thus, the identification of aberrant miRNA expression during PAH-induced cancer cell migration will lead to a better understanding of the substantial role of miRNAs in cancer progression. In the present study, miRNA expression profiling showed significant upregulation of miR-181a, -181b, and -181d in human hepatocellular carcinoma cells (HepG2 line) exposed to benzo[a]anthracene (BA) and benzo[k]fluoranthene (BF).more » MAPK phosphatase-5 (MKP-5), a validated miR-181 target that deactivates MAPKs, was markedly suppressed while phosphorylation of p38 MAPK was increased after BA and BF exposure. The migration of HepG2 cells, observed using the scratch wound-healing assay, also increased in a dose-dependent manner. Depletion of miR-181 family members by miRNA inhibitors enhanced the expression of MKP-5 and suppressed the phosphorylation of p38 MAPK. Furthermore, the depletion of the miR-181 family inhibited cancer cell migration. Based on these results, we conclude that the miR-181 family plays a critical role in PAH-induced hepatocarcinogenesis by targeting MKP-5, resulting in the regulation of p38 MAPK activation. - Highlights: • We found significant upregulation of miR-181 family in HCC exposed to BA and BF. • We identified the MKP-5 as a putative target of miR-181 family. • MKP-5 was suppressed while p-P38 was increased after BA and BF exposure. • The migration of HepG2 cells increased in a dose-dependent manner.« less

Steps to achieve quantitative measurements of microRNA using two step droplet digital PCR.

PubMed

Stein, Erica V; Duewer, David L; Farkas, Natalia; Romsos, Erica L; Wang, Lili; Cole, Kenneth D

2017-01-01

Droplet digital PCR (ddPCR) is being advocated as a reference method to measure rare genomic targets. It has consistently been proven to be more sensitive and direct at discerning copy numbers of DNA than other quantitative methods. However, one of the largest obstacles to measuring microRNA (miRNA) using ddPCR is that reverse transcription efficiency depends upon the target, meaning small RNA nucleotide composition directly effects primer specificity in a manner that prevents traditional quantitation optimization strategies. Additionally, the use of reagents that are optimized for miRNA measurements using quantitative real-time PCR (qRT-PCR) appear to either cause false positive or false negative detection of certain targets when used with traditional ddPCR quantification methods. False readings are often related to using inadequate enzymes, primers and probes. Given that two-step miRNA quantification using ddPCR relies solely on reverse transcription and uses proprietary reagents previously optimized only for qRT-PCR, these barriers are substantial. Therefore, here we outline essential controls, optimization techniques, and an efficacy model to improve the quality of ddPCR miRNA measurements. We have applied two-step principles used for miRNA qRT-PCR measurements and leveraged the use of synthetic miRNA targets to evaluate ddPCR following cDNA synthesis with four different commercial kits. We have identified inefficiencies and limitations as well as proposed ways to circumvent identified obstacles. Lastly, we show that we can apply these criteria to a model system to confidently quantify miRNA copy number. Our measurement technique is a novel way to quantify specific miRNA copy number in a single sample, without using standard curves for individual experiments. Our methodology can be used for validation and control measurements, as well as a diagnostic technique that allows scientists, technicians, clinicians, and regulators to base miRNA measures on a single unit of measurement rather than a ratio of values.

Steps to achieve quantitative measurements of microRNA using two step droplet digital PCR

PubMed Central

Duewer, David L.; Farkas, Natalia; Romsos, Erica L.; Wang, Lili; Cole, Kenneth D.

2017-01-01

Droplet digital PCR (ddPCR) is being advocated as a reference method to measure rare genomic targets. It has consistently been proven to be more sensitive and direct at discerning copy numbers of DNA than other quantitative methods. However, one of the largest obstacles to measuring microRNA (miRNA) using ddPCR is that reverse transcription efficiency depends upon the target, meaning small RNA nucleotide composition directly effects primer specificity in a manner that prevents traditional quantitation optimization strategies. Additionally, the use of reagents that are optimized for miRNA measurements using quantitative real-time PCR (qRT-PCR) appear to either cause false positive or false negative detection of certain targets when used with traditional ddPCR quantification methods. False readings are often related to using inadequate enzymes, primers and probes. Given that two-step miRNA quantification using ddPCR relies solely on reverse transcription and uses proprietary reagents previously optimized only for qRT-PCR, these barriers are substantial. Therefore, here we outline essential controls, optimization techniques, and an efficacy model to improve the quality of ddPCR miRNA measurements. We have applied two-step principles used for miRNA qRT-PCR measurements and leveraged the use of synthetic miRNA targets to evaluate ddPCR following cDNA synthesis with four different commercial kits. We have identified inefficiencies and limitations as well as proposed ways to circumvent identified obstacles. Lastly, we show that we can apply these criteria to a model system to confidently quantify miRNA copy number. Our measurement technique is a novel way to quantify specific miRNA copy number in a single sample, without using standard curves for individual experiments. Our methodology can be used for validation and control measurements, as well as a diagnostic technique that allows scientists, technicians, clinicians, and regulators to base miRNA measures on a single unit of measurement rather than a ratio of values. PMID:29145448

mir-125a-5p-mediated Regulation of Lfng is Essential for the Avian Segmentation Clock

PubMed Central

Riley, Maurisa F.; Bochter, Matthew S.; Wahi, Kanu; Nuovo, Gerard J.; Cole, Susan E.

2013-01-01

Summary Somites are embryonic precursors of the axial skeleton and skeletal muscles, and establish the segmental vertebrate body plan. Somitogenesis is controlled in part by a segmentation clock that requires oscillatory expression of genes including Lunatic fringe (Lfng). Oscillatory genes must be tightly regulated both at the transcriptional and post-transcriptional levels for proper clock function. Here we demonstrate that microRNA-mediated regulation of Lfng is essential for proper segmentation during chick somitogenesis. We find that mir-125a-5p targets evolutionarily conserved sequences in the Lfng 3′UTR, and that preventing interactions between mir-125a-5p and Lfng transcripts in vivo causes abnormal segmentation and perturbs clock activity. This provides strong evidence that miRNAs function in the post-transcriptional regulation of oscillatory genes in the segmentation clock. Further, this demonstrates that the relatively subtle effects of miRNAs on target genes can have broad effects in developmental situations that have critical requirements for tight post-transcriptional regulation. PMID:23484856

A Pitx2-MicroRNA Pathway Modulates Cell Proliferation in Myoblasts and Skeletal-Muscle Satellite Cells and Promotes Their Commitment to a Myogenic Cell Fate

PubMed Central

Lozano-Velasco, Estefanía; Vallejo, Daniel; Esteban, Francisco J.; Doherty, Chris; Hernández-Torres, Francisco; Franco, Diego

2015-01-01

The acquisition of a proliferating-cell status from a quiescent state as well as the shift between proliferation and differentiation are key developmental steps in skeletal-muscle stem cells (satellite cells) to provide proper muscle regeneration. However, how satellite cell proliferation is regulated is not fully understood. Here, we report that the c-isoform of the transcription factor Pitx2 increases cell proliferation in myoblasts by downregulating microRNA 15b (miR-15b), miR-23b, miR-106b, and miR-503. This Pitx2c-microRNA (miRNA) pathway also regulates cell proliferation in early-activated satellite cells, enhancing Myf5+ satellite cells and thereby promoting their commitment to a myogenic cell fate. This study reveals unknown functions of several miRNAs in myoblast and satellite cell behavior and thus may have future applications in regenerative medicine. PMID:26055324

The let-7 microRNA target gene, Mlin41/Trim71 is required for mouse embryonic survival and neural tube closure

PubMed Central

Schulman, Betsy R. Maller; Liang, Xianping; Stahlhut, Carlos; DelConte, Casey; Stefani, Giovanni; Slack, Frank J.

2010-01-01

In the nematode Caenorhabditis elegans, the let-7 microRNA (miRNA) controls the timing of key developmental events and terminal differentiation in part by directly regulating lin-41. C. elegans lin-41 mutants display precocious cell cycle exit and terminal differentiation of epidermal skin cells. lin-41 orthologues are found in more complex organisms including both mice and humans, but their roles are not known. We generated Mlin41 mouse mutants to ascertain a functional role for Mlin41. Strong loss of function Mlin41 gene-trap mutants demonstrated a striking neural tube closure defect during development, and embryonic lethality. Like C. elegans lin-41, Mlin41 also appears to be regulated by the let-7 and mir-125 miRNAs. Since Mlin41 is required for neural tube closure and survival it points to human lin-41 (HLIN41/TRIM71) as a potential human development and disease gene. PMID:19098426

Reciprocal Expression of lin-41 and the microRNAs let-7 and mir-125 During Mouse Embryogenesis

PubMed Central

Schulman, Betsy R. Maller; Esquela-Kerscher, Aurora; Slack, Frank J.

2008-01-01

In C. elegans, heterochronic genes control the timing of cell fate determination during development. Two heterochronic genes, let-7 and lin-4, encode microRNAs (miRNAs) that down-regulate a third heterochronic gene lin-41 by binding to complementary sites in its 3’UTR. let-7 and lin-4 are conserved in mammals. Here we report the cloning and sequencing of mammalian lin-41 orthologs. We find that mouse and human lin-41 genes contain predicted conserved complementary sites for let-7 and the lin-4 ortholog, mir-125, in their 3’UTRs. Mouse lin-41 (Mlin-41) is temporally expressed in developing mouse embryos, most dramatically in the limb buds. Mlin-41 is down-regulated during mid-embryogenesis at the time when mouse let-7c and mir-125 RNA levels are up-regulated. Our results suggest that mammalian lin-41 is temporally regulated by miRNAs in order to direct key developmental events such as limb formation. PMID:16247770

Identification and characterization of microRNAs from in vitro-grown pear shoots infected with Apple stem grooving virus in response to high temperature using small RNA sequencing.

PubMed

Liu, Juan; Zhang, XueJiao; Zhang, FangPeng; Hong, Ni; Wang, GuoPing; Wang, Aiming; Wang, LiPing

2015-11-16

MicroRNAs (miRNAs) have functions in diverse biological processes such as growth, signal transduction, disease resistance, and stress responses in plants. Thermotherapy is an effective approach for elimination of viruses from fruit trees. However, the role of miRNAs in this process remains elusive. Previously, we showed that high temperature treatment reduces the titers of Apple stem grooving virus (ASGV) from the tips of in vitro-grown Pyrus pyrifolia plants. In this study, we identified high temperature-altered pear miRNAs using the next generation sequencing technology, and futher molecularly characterized miRNA-mediated regulaton of target gene expression in the meristem tip and base tissues of in vitro-grown, ASGV-infected pear shoots under different temperatures. Using in vitro-grown P. pyrifolia shoot meristem tips infected with ASGV, a total of 22,592,997 and 20,411,254 clean reads were obtained from Illumina high-throughput sequencing of small RNA libraries at 24 °C and 37 °C, respectively. We identified 149 conserved and 141 novel miRNAs. Seven conserved miRNAs and 77 novel miRNAs were differentially expressed at different temperatures. Target genes for differentially expressed known and novel miRNAs were predicted and functionally annotated. Gene Ontology (GO) analysis showed that high-ranking miRNA target genes were involved in metabolic processes, responses to stress, and signaling, indicating that these high temperature-responsive miRNAs have functions in diverse gene regulatory networks. Spatial expression patterns of the miRNAs and their target genes were found to be expressed in shoot tip and base tissues by qRT-PCR. In addition, high temperature reduced viral titers in the shoot meristem tip, while negatively regulated miRNA-mediated target genes related to resistance disease defense and hormone signal transduction pathway were up-regulated in the P. pyrifolia shoot tip in response to high temperature. These results suggested that miRNAs may have important functions in the high temperature-dependent decrease of ASGV titer in in vitro-grown pear shoots. This is the first report of miRNAs differentially expressed at 24 °C and 37 °C in the meristem tip of pear shoots infected with ASGV. The results of this study provide valuable information for further exploration of the function of high temperature-altered miRNAs in suppressing viral infections in pear and other fruit trees.

Tethering of human Ago proteins to mRNA mimics the miRNA-mediated repression of protein synthesis.

PubMed

Pillai, Ramesh S; Artus, Caroline G; Filipowicz, Witold

2004-10-01

MicroRNAs (miRNAs) are approximately 21-nt-long RNAs involved in regulating development, differentiation, and other processes in eukaryotes. In metazoa, nearly all miRNAs control gene expression by imperfectly base-pairing with the 3'-untranslated region (3'-UTR) of target mRNAs and repressing protein synthesis by an unknown mechanism. It is also unknown whether miRNA-mRNA duplexes containing mismatches and bulges provide specific features that are recognized by factors mediating the repression. miRNAs form part of ribonucleoprotein complexes, miRNPs, that contain Argonaute (Ago) and other proteins. Here we demonstrate that effects of miRNAs on translation can be mimicked in human HeLa cells by the miRNA-independent tethering of Ago proteins to the 3'-UTR of a reporter mRNA. Inhibition of protein synthesis occurred without a change in the reporter mRNA level and was dependent on the number, but not the position, of the hairpins tethering hAgo2 to the 3'-UTR. These findings indicate that a primary function of miRNAs is to guide their associated proteins to the mRNA. Copyright 2004 RNA Society

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.

Small RNA analysis in Petunia hybrida identifies unusual tissue-specific expression patterns of conserved miRNAs and of a 24mer RNA

PubMed Central

Tedder, Philip; Zubko, Elena; Westhead, David R.; Meyer, Peter

2009-01-01

Two pools of small RNAs were cloned from inflorescences of Petunia hybrida using a 5′-ligation dependent and a 5′-ligation independent approach. The two libraries were integrated into a public website that allows the screening of individual sequences against 359,769 unique clones. The library contains 15 clones with 100% identity and 53 clones with one mismatch to miRNAs described for other plant species. For two conserved miRNAs, miR159 and miR390, we find clear differences in tissue-specific distribution, compared with other species. This shows that evolutionary conservation of miRNA sequences does not necessarily include a conservation of the miRNA expression profile. Almost 60% of all clones in the database are 24-nucleotide clones. In accordance with the role of 24mers in marking repetitive regions, we find them distributed across retroviral and transposable element sequences but other 24mers map to promoter regions and to different transcript regions. For one target region we observe tissue-specific variation of matching 24mers, which demonstrates that, as for 21mers, 24mer concentrations are not necessarily identical in different tissues. Asymmetric distribution of a putative novel miRNA in the two libraries suggests that the cloning method can be selective for the representation of certain small RNAs in a collection. PMID:19369427

MicroRNAs in islet immunobiology and transplantation.

PubMed

Pileggi, Antonello; Klein, Dagmar; Fotino, Carmen; Bravo-Egaña, Valia; Rosero, Samuel; Doni, Marco; Podetta, Michele; Ricordi, Camillo; Molano, R Damaris; Pastori, Ricardo L

2013-12-01

The ultimate goal of diabetes therapy is the restoration of physiologic metabolic control. For type 1 diabetes, research efforts are focused on the prevention or early intervention to halt the autoimmune process and preserve β cell function. Replacement of pancreatic β cells via islet transplantation reestablishes physiologic β cell function in patients with diabetes. Emerging research shows that microRNAs (miRNAs), noncoding small RNA molecules produced by a newly discovered class of genes, negatively regulate gene expression. MiRNAs recognize and bind to partially complementary sequences of target messenger RNA (mRNA), regulating mRNA translation and affecting gene expression. Correlation between miRNA signatures and genome-wide RNA expression allows identification of multiple miRNA-mRNA pairs in biological processes. Because miRNAs target functionally related genes, they represent an exciting and indispensable approach for biomarkers and drug discovery. We are studying the role of miRNA in the context of islet immunobiology. Our research aims at understanding the mechanisms underlying pancreatic β cell loss and developing clinically relevant approaches for preservation and restoration of β cell function to treat insulin-dependent diabetes. Herein, we discuss some of our recent efforts related to the study of miRNA in islet inflammation and islet engraftment. Our working hypothesis is that modulation of the expression of specific microRNAs in the transplant microenvironment will be of assistance in enhancing islet engraftment and promoting long-term function.

Next-generation sequencing identifies deregulation of microRNAs involved in both innate and adaptive immune response in ALK+ ALCL.

PubMed

Steinhilber, Julia; Bonin, Michael; Walter, Michael; Fend, Falko; Bonzheim, Irina; Quintanilla-Martinez, Leticia

2015-01-01

Anaplastic large cell lymphoma (ALCL) is divided into two systemic diseases according to the expression of the anaplastic lymphoma kinase (ALK). We investigated the differential expression of miRNAs between ALK+ ALCL, ALK- ALCL cells and normal T-cells using next generation sequencing (NGS). In addition, a C/EBPβ-dependent miRNA profile was generated. The data were validated in primary ALCL cases. NGS identified 106 miRNAs significantly differentially expressed between ALK+ and ALK- ALCL and 228 between ALK+ ALCL and normal T-cells. We identified a signature of 56 miRNAs distinguishing ALK+ ALCL, ALK- ALCL and T-cells. The top candidates significant differentially expressed between ALK+ and ALK- ALCL included 5 upregulated miRNAs: miR-340, miR-203, miR-135b, miR-182, miR-183; and 7 downregulated: miR-196b, miR-155, miR-146a, miR-424, miR-503, miR-424*, miR-542-3p. The miR-17-92 cluster was also upregulated in ALK+ cells. Additionally, we identified a signature of 3 miRNAs significantly regulated by the transcription factor C/EBPβ, which is specifically overexpressed in ALK+ ALCL, including the miR-181 family. Of interest, miR-181a, which regulates T-cell differentiation and modulates TCR signalling strength, was significantly downregulated in ALK+ ALCL cases. In summary, our data reveal a miRNA signature linking ALK+ ALCL to a deregulated immune response and may reflect the abnormal TCR antigen expression known in ALK+ ALCL.

Understanding the effect of locked nucleic acid and 2'-O-methyl modification on the hybridization thermodynamics of a miRNA-mRNA pair in the presence and absence of AfPiwi protein.

PubMed

Kumar, Santosh; Mapa, Koyeli; Maiti, Souvik

2014-03-18

miRNAs are some of the key epigenetic regulators of gene expression. They act through hybridization with their target mRNA and modulate the level of respective proteins via different mechanisms. Various cancer conditions are known to be associated with up- and downregulation of the oncogenic and tumor suppressor miRNAs, respectively. The levels of aberrantly expressed oncogenic miRNAs can be downregulated in different ways. Similarly, restoration of tumor suppressor miRNAs to their normal levels can be achieved using miRNA mimics. However, the use of miRNA mimics is limited by their reduced biostability and function. We have studied the hybridization thermodynamics of the miRNA 26a (11-mer, including the seed sequence) guide strand with the mRNA (11-mer) target strand in the absence and presence of AfPiwi protein. We have also inserted locked nucleic acids (LNAs) and 2'-O-methyl-modified nucleotides into the guide strand, in a walk-through manner, to assess their effect on the binding efficiency between guide and target RNA. Insertion of LNA and 2'-O-methyl-modified nucleotides into the guide strand helped to strengthen the binding affinity irrespective of the position of insertion. However, in the presence of AfPiwi protein, these modifications reduced the binding affinity to different extents depending on the position of insertion. Insertion of a modification leads to an increase in the enthalpic contribution with an increased unfavorable entropic contribution, which negatively compensates for the higher favorable enthalpy.

Ketamine up-regulates a cluster of intronic miRNAs within the serotonin receptor 2C gene by inhibiting glycogen synthase kinase-3.

PubMed

Grieco, Steven F; Velmeshev, Dmitry; Magistri, Marco; Eldar-Finkelman, Hagit; Faghihi, Mohammad A; Jope, Richard S; Beurel, Eleonore

2017-09-01

We examined mechanisms that contribute to the rapid antidepressant effect of ketamine in mice that is dependent on glycogen synthase kinase-3 (GSK3) inhibition. We measured serotonergic (5HT)-2C-receptor (5HTR2C) cluster microRNA (miRNA) levels in mouse hippocampus after administering an antidepressant dose of ketamine (10 mg/kg) in wild-type and GSK3 knockin mice, after GSK3 inhibition with L803-mts, and in learned helpless mice. Ketamine up-regulated cluster miRNAs 448-3p, 764-5p, 1264-3p, 1298-5p and 1912-3p (2- to 11-fold). This up-regulation was abolished in GSK3 knockin mice that express mutant constitutively active GSK3. The GSK3 specific inhibitor L803-mts was antidepressant in the learned helplessness and novelty suppressed feeding depression-like behaviours and up-regulated the 5HTR2C miRNA cluster in mouse hippocampus. After administration of the learned helplessness paradigm mice were divided into cohorts that were resilient (non-depressed) or were susceptible (depressed) to learned helplessness. The resilient, but not depressed, mice displayed increased hippocampal levels of miRNAs 448-3p and 1264-3p. Administration of an antagonist to miRNA 448-3p diminished the antidepressant effect of ketamine in the learned helplessness paradigm, indicating that up-regulation of miRNA 448-3p provides an antidepressant action. These findings identify a new outcome of GSK3 inhibition by ketamine that may contribute to antidepressant effects.

p53 shapes genome-wide and cell type-specific changes in microRNA expression during the human DNA damage response.

PubMed

Hattori, Hiroyoshi; Janky, Rekin's; Nietfeld, Wilfried; Aerts, Stein; Madan Babu, M; Venkitaraman, Ashok R

2014-01-01

The human DNA damage response (DDR) triggers profound changes in gene expression, whose nature and regulation remain uncertain. Although certain micro-(mi)RNA species including miR34, miR-18, miR-16 and miR-143 have been implicated in the DDR, there is as yet no comprehensive description of genome-wide changes in the expression of miRNAs triggered by DNA breakage in human cells. We have used next-generation sequencing (NGS), combined with rigorous integrative computational analyses, to describe genome-wide changes in the expression of miRNAs during the human DDR. The changes affect 150 of 1523 miRNAs known in miRBase v18 from 4-24 h after the induction of DNA breakage, in cell-type dependent patterns. The regulatory regions of the most-highly regulated miRNA species are enriched in conserved binding sites for p53. Indeed, genome-wide changes in miRNA expression during the DDR are markedly altered in TP53-/- cells compared to otherwise isogenic controls. The expression levels of certain damage-induced, p53-regulated miRNAs in cancer samples correlate with patient survival. Our work reveals genome-wide and cell type-specific alterations in miRNA expression during the human DDR, which are regulated by the tumor suppressor protein p53. These findings provide a genomic resource to identify new molecules and mechanisms involved in the DDR, and to examine their role in tumor suppression and the clinical outcome of cancer patients.

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.

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

Modulation of Neuroblastoma Disease Pathogenesis By An Extensive Network of Epigenetically Regulated MicroRNAs

PubMed Central

Das, Sudipto; Bryan, Kenneth; Buckley, Patrick G; Piskareva, Olga; Bray, Isabella M; Foley, Niamh; Ryan, Jacqueline; Lynch, Jennifer; Creevey, Laura; Fay, Joanna; Prenter, Suzanne; Koster, Jan; van Sluis, Peter; Versteeg, Rogier; Eggert, Angelika; Schulte, Johannes H; Schramm, Alexander; Mesdagh, Pieter; Vandesompele, Jo; Speleman, Frank

2012-01-01

MicroRNAs contribute to the pathogenesis of many forms of cancer, including the pediatric cancer neuroblastoma, but the underlying mechanisms leading to altered miRNA expression are often unknown. Here, a novel integrated approach for analyzing DNA methylation coupled with miRNA and mRNA expression data sets identified 67 epigenetically regulated miRNA in neuroblastoma. A large proportion (42%) of these miRNAs were associated with poor patient survival when under-expressed in tumors. Moreover, we demonstrate that this panel of epigenetically silenced miRNAs targets a large set of genes that are over-expressed in tumors from patients with poor survival in a highly redundant manner. The genes targeted by the epigenetically regulated miRNAs are enriched for a number of biological processes, including regulation of cell differentiation. Functional studies involving ectopic over-expression of several of the epigenetically silenced miRNAs had a negative impact on neuroblastoma cell viability, providing further support to the concept that inactivation of these miRNAs is important for neuroblastoma disease pathogenesis. One locus, miR-340, induced either differentiation or apoptosis in a cell context dependent manner, indicating a tumor suppressive function for this miRNA. Intriguingly, it was determined that miR-340 is up-regulated by demethylation of an upstream genomic region that occurs during the process of neuroblastoma cell differentiation induced by all-trans retinoic acid (ATRA). Further biological studies of miR-340 revealed that it directly represses the SOX2 transcription factor by targeting of its 3’ UTR, explaining the mechanism by which SOX2 is down-regulated by ATRA. Although SOX2 contributes to the maintenance of stem cells in an undifferentiated state, we demonstrate that miR-340 mediated down-regulation of SOX2 is not required for ATRA induced differentiation to occur. In summary, our results exemplify the dynamic nature of the miRNA epigenome and identify a remarkable network of miRNA/mRNA interactions that significantly contribute to neuroblastoma disease pathogenesis. PMID:22797059

Use of biomarkers to establish potential role and function of circulating microRNAs in acute heart failure.

PubMed

Vegter, Eline L; Schmitter, Daniela; Hagemeijer, Yanick; Ovchinnikova, Ekaterina S; van der Harst, Pim; Teerlink, John R; O'Connor, Christopher M; Metra, Marco; Davison, Beth A; Bloomfield, Daniel; Cotter, Gad; Cleland, John G; Givertz, Michael M; Ponikowski, Piotr; van Veldhuisen, Dirk J; van der Meer, Peter; Berezikov, Eugene; Voors, Adriaan A; Khan, Mohsin A F

2016-12-01

Circulating microRNAs (miRNAs) emerge as potential heart failure biomarkers. We aimed to identify associations between acute heart failure (AHF)-specific circulating miRNAs and well-known heart failure biomarkers. Associations between 16 biomarkers predictive for 180day mortality and the levels of 12 AHF-specific miRNAs were determined in 100 hospitalized AHF patients, at baseline and 48hours. Patients were divided in 4 pre-defined groups, based on clinical parameters during hospitalization. Correlation analyses between miRNAs and biomarkers were performed and complemented by miRNA target prediction and pathway analysis. No significant correlations were found at hospital admission. However, after 48hours, 7 miRNAs were significantly negatively correlated to biomarkers indicative for a worse clinical outcome in the patient group with the most unfavorable in-hospital course (n=21); miR-16-5p was correlated to C-reactive protein (R=-0.66, p-value=0.0027), miR-106a-5p to creatinine (R=-0.68, p-value=0.002), miR-223-3p to growth differentiation factor 15 (R=-0.69, p-value=0.0015), miR-652-3p to soluble ST-2 (R=-0.77, p-value<0.001), miR-199a-3p to procalcitonin (R=-0.72, p-value<0.001) and galectin-3 (R=-0.73, p-value<0.001) and miR-18a-5p to procalcitonin (R=-0.68, p-value=0.002). MiRNA target prediction and pathway analysis identified several pathways related to cardiac diseases, which could be linked to some of the miRNA-biomarker correlations. The majority of correlations between circulating AHF-specific miRNAs were related to biomarkers predictive for a worse clinical outcome in a subgroup of worsening heart failure patients at 48hours of hospitalization. The selective findings suggest a time-dependent effect of circulating miRNAs and highlight the susceptibility to individual patient characteristics influencing potential relations between miRNAs and biomarkers. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Transposable element-associated microRNA hairpins produce 21-nt sRNAs integrated into typical microRNA pathways in rice

PubMed Central

Ou-Yang, Fangqian; Luo, Qing-Jun; Zhang, Yue; Richardson, Casey R.; Jiang, Yingwen; Rock, Christopher D.

2013-01-01

microRNAs (miRNAs) are a class of small RNAs (sRNAs) of ~21 nucleotides (nt) in length processed from foldback hairpins by DICER-LIKE1 (DCL1) or DCL4. They regulate the expression of target mRNAs by base pairing through RNA-Induced Silencing Complex (RISC). In the RISC, ARGONAUTE1 (AGO1) is the key protein that cleaves miRNA targets at position ten of a miRNA:target duplex. The authenticity of many annotated rice miRNA hairpins is under debate because of their homology to repeat sequences. Some of them, like miR1884b, have been removed from the current release of miRBase based on incomplete information. In this study, we investigated the association of transposable element (TE)-derived miRNAs with typical miRNA pathways (DCL1/4- and AGO1-dependent) using publicly available deep sequencing datasets. Seven miRNA hairpins with 13 unique sRNAs were specifically enriched in AGO1 immunoprecipitation samples and relatively reduced in DCL1/4 knockdown genotypes. Interestingly, these species are ~21-nt long, instead of 24-nt as annotated in miRBase and the literature. Their expression profiles meet current criteria for functional annotation of miRNAs. In addition, diagnostic cleavage tags were found in degradome datasets for predicted target mRNAs. Most of these miRNA hairpins share significant homology with miniature inverted-repeat transposable elements (MITEs), one type of abundant DNA transposons in rice. Finally, the root-specific production of a 24 nt miRNA-like sRNA was confirmed by RNA blot for a novel EST that maps to the 3'-UTR of a candidate pseudogene showing extensive sequence homology to miR1884b hairpin. Our data are consistent with the hypothesis that TEs can serve as a driving force for the evolution of some MIRNAs, where co-opting of DICER-LIKE1/4 processing and integration into AGO1 could exapt transcribed TE-associated hairpins into typical miRNA pathways. PMID:23420033

Post-transcriptional control of DGCR8 expression by the Microprocessor.

PubMed

Triboulet, Robinson; Chang, Hao-Ming; Lapierre, Robert J; Gregory, Richard I

2009-06-01

The Microprocessor, comprising the RNase III Drosha and the double-stranded RNA binding protein DGCR8, is essential for microRNA (miRNA) biogenesis. In the miRNA processing pathway certain hairpin structures within primary miRNA (pri-miRNA) transcripts are specifically cleaved by the Microprocessor to release approximately 60-70-nucleotide precursor miRNA (pre-miRNA) intermediates. Although both Drosha and DGCR8 are required for Microprocessor activity, the mechanisms regulating the expression of these proteins are unknown. Here we report that the Microprocessor negatively regulates DGCR8 expression. Using in vitro reconstitution and in vivo studies, we demonstrate that a hairpin, localized in the 5' untranslated region (5'UTR) of DGCR8 mRNA, is cleaved by the Microprocessor. Accordingly, knockdown of Drosha leads to an increase in DGCR8 mRNA and protein levels in cells. Furthermore, we found that the DGCR8 5'UTR confers Microprocessor-dependent repression of a luciferase reporter gene in vivo. Our results uncover a novel feedback loop that regulates DGCR8 levels.

Analysis of RDR1/RDR2/RDR6-independent small RNAs in Arabidopsis thaliana improves MIRNA annotations and reveals unexplained types of short interfering RNA loci.

PubMed

Polydore, Seth; Axtell, Michael J

2018-06-01

Plant small RNAs (sRNAs) modulate key physiological mechanisms through post-transcriptional and transcriptional silencing of gene expression. Small RNAs fall into two major categories: those are reliant on RNA-dependent RNA polymerases (RDRs) for biogenesis and those that are not. Known RDR1/2/6-dependent sRNAs include phased and repeat-associated short interfering RNAs, while known RDR1/2/6-independent sRNAs are primarily microRNAs (miRNA) and other hairpin-derived sRNAs. In this study we produced and analyzed sRNA-seq libraries from rdr1/rdr2/rdr6 triple mutant plants. We found 58 previously annotated miRNA loci that were reliant on RDR1, -2, or -6 function, casting doubt on their classification. We also found 38 RDR1/2/6-independent sRNA loci that are not MIRNAs or otherwise hairpin-derived, and did not fit into other known paradigms for sRNA biogenesis. These 38 sRNA-producing loci have as-yet-undescribed biogenesis mechanisms, and are frequently located in the vicinity of protein-coding genes. Altogether, our analysis suggests that these 38 loci represent one or more undescribed types of sRNA in Arabidopsis thaliana. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

Transcriptome - Scale characterization of salt responsive bean TCP transcription factors.

PubMed

İlhan, Emre; Büyük, İlker; İnal, Behcet

2018-02-05

TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) proteins are important regulators of growth and developmental processes including branching, floral organ morphogenesis and leaf growth as well as stress response. This study identified 27 TCP genes of Phaseolus vulgaris (common bean), which were divided into three clusters based on phylogenetic relationship. In addition, this study showed that some of TCP genes such as Pvul-TCP-4 and Pvul-TCP-15 located on chromosomes 3 and 7, Pvul-TCP-7 and Pvul-TCP-20 located on chromosome 7 and 9, were segmentally duplicated. On the other hand, a total of 20 Pvul-TCP genes have predicted to be targeted by microRNAs (miRNA). Most of the miRNA-target genes were Pvul-TCP-1, -11, -13 and -27, which were targeted by 13, 17, 22 and 13 plant miRNAs, respectively. miR319 was one of the highly represented regulatory miRNAs to target TCP transcripts. Promoter region analysis of TCP genes resulted that the GT-1 motif, which was related to salt stress, was found in 14 different Pvul-TCP genes. Expression profiling of 10 Pvul-TCP genes based on RNA-sequencing data further confirmed with quantitative real-time RT-PCR measurements identified that Pvul-TCP genes under salt stress are expressed in a cultivar- and tissue-specific manner. Copyright © 2017 Elsevier B.V. All rights reserved.

miR-132, an experience-dependent microRNA, is essential for visual cortex plasticity

PubMed Central

Mellios, Nikolaos; Sugihara, Hiroki; Castro, Jorge; Banerjee, Abhishek; Le, Chuong; Kumar, Arooshi; Crawford, Benjamin; Strathmann, Julia; Tropea, Daniela; Levine, Stuart S.; Edbauer, Dieter; Sur, Mriganka

2011-01-01

Using multiple quantitative analyses, we discovered microRNAs (miRNAs) abundantly expressed in visual cortex that respond to dark-rearing (DR) and/or monocular deprivation (MD). The most significantly altered miRNA, miR-132, was rapidly upregulated after eye-opening and delayed by DR. In vivo inhibition of miR-132 prevented ocular dominance plasticity in identified neurons following MD, and affected maturation of dendritic spines, demonstrating its critical role in the plasticity of visual cortex circuits. PMID:21892155

Small Molecules Targeting the miRNA-Binding Domain of Argonaute 2: From Computer-Aided Molecular Design to RNA Immunoprecipitation.

PubMed

Bellissimo, Teresa; Masciarelli, Silvia; Poser, Elena; Genovese, Ilaria; Del Rio, Alberto; Colotti, Gianni; Fazi, Francesco

2017-01-01

The development of small-molecule-based target therapy design for human disease and cancer is object of growing attention. Recently, specific microRNA (miRNA) mimicking compounds able to bind the miRNA-binding domain of Argonaute 2 protein (AGO2) to inhibit miRNA loading and its functional activity were described. Computer-aided molecular design techniques and RNA immunoprecipitation represent suitable approaches to identify and experimentally determine if a compound is able to impair the loading of miRNAs on AGO2 protein. Here, we describe these two methodologies that we recently used to select a specific compound able to interfere with the AGO2 functional activity and able to improve the retinoic acid-dependent myeloid differentiation of leukemic cells.

Aberrant methylation-mediated silencing of microRNAs contributes to HPV-induced anchorage independence

PubMed Central

Wilting, Saskia M.; Boon, Debby; Sørgård, Hanne; Lando, Malin; Snoek, Barbara C.; van Wieringen, Wessel N.; Meijer, Chris J.L.M.; Lyng, Heidi; Snijders, Peter J.F.; Steenbergen, Renske D.M.

2016-01-01

Cervical cancer and a subset of anogenital and head-and-neck carcinomas are caused by high-risk types of the human papillomavirus (hrHPV). During hrHPV-induced malignant transformation keratinocytes become able to grow anchorage independently, a tumorigenic trait at least partly associated with inactivation of tumor suppressor genes. We used hrHPV-containing keratinocytes to investigate the role of DNA methylation-mediated silencing of microRNAs (miRNAs) in the acquisition of anchorage independence. Anchorage dependent (n=11) and independent passages (n=19) of 4 hrHPV-immortalized keratinocyte cell lines were treated with 2′-deoxy-5-azacytidine (DAC). Genome-wide miRNA expression profiles before and after treatment were compared to identify miRNAs silenced by methylation. Bisulfite sequencing and methylation-specific PCR showed increased methylation of hsa-mir-129-2/-137/-935/-3663/-3665 and -4281 in anchorage independent HPV-transformed keratinocytes and cervical cancer cell lines. Mature miRNAs derived from hsa-mir-129-2/-137/-3663 and -3665 showed functional relevance as they decreased anchorage independence in cervical cancer cell lines. Cervical (pre)cancerous lesions demonstrated increased methylation of hsa-mir-129-2/-935/-3663/-3665 and -4281, underlining the clinical relevance of our findings. In conclusion, methylation-mediated silencing of tumor suppressive miRNAs contributes to acquisition of an anchorage independent phenotype. This study further substantiates the importance of miRNAs during early stages of carcinogenesis and underlines their potential as both disease markers and therapeutic targets. PMID:27270309

Aberrant methylation-mediated silencing of microRNAs contributes to HPV-induced anchorage independence.

PubMed

Wilting, Saskia M; Miok, Viktorian; Jaspers, Annelieke; Boon, Debby; Sørgård, Hanne; Lando, Malin; Snoek, Barbara C; van Wieringen, Wessel N; Meijer, Chris J L M; Lyng, Heidi; Snijders, Peter J F; Steenbergen, Renske D M

2016-07-12

Cervical cancer and a subset of anogenital and head-and-neck carcinomas are caused by high-risk types of the human papillomavirus (hrHPV). During hrHPV-induced malignant transformation keratinocytes become able to grow anchorage independently, a tumorigenic trait at least partly associated with inactivation of tumor suppressor genes. We used hrHPV-containing keratinocytes to investigate the role of DNA methylation-mediated silencing of microRNAs (miRNAs) in the acquisition of anchorage independence.Anchorage dependent (n=11) and independent passages (n=19) of 4 hrHPV-immortalized keratinocyte cell lines were treated with 2'-deoxy-5-azacytidine (DAC). Genome-wide miRNA expression profiles before and after treatment were compared to identify miRNAs silenced by methylation. Bisulfite sequencing and methylation-specific PCR showed increased methylation of hsa-mir-129-2/-137/-935/-3663/-3665 and -4281 in anchorage independent HPV-transformed keratinocytes and cervical cancer cell lines. Mature miRNAs derived from hsa-mir-129-2/-137/-3663 and -3665 showed functional relevance as they decreased anchorage independence in cervical cancer cell lines. Cervical (pre)cancerous lesions demonstrated increased methylation of hsa-mir-129-2/-935/-3663/-3665 and -4281, underlining the clinical relevance of our findings.In conclusion, methylation-mediated silencing of tumor suppressive miRNAs contributes to acquisition of an anchorage independent phenotype. This study further substantiates the importance of miRNAs during early stages of carcinogenesis and underlines their potential as both disease markers and therapeutic targets.

MiR-2 family regulates insect metamorphosis by controlling the juvenile hormone signaling pathway.

PubMed

Lozano, Jesus; Montañez, Raúl; Belles, Xavier

2015-03-24

In 2009 we reported that depletion of Dicer-1, the enzyme that catalyzes the final step of miRNA biosynthesis, prevents metamorphosis in Blattella germanica. However, the precise regulatory roles of miRNAs in the process have remained elusive. In the present work, we have observed that Dicer-1 depletion results in an increase of mRNA levels of Krüppel homolog 1 (Kr-h1), a juvenile hormone-dependent transcription factor that represses metamorphosis, and that depletion of Kr-h1 expression in Dicer-1 knockdown individuals rescues metamorphosis. We have also found that the 3'UTR of Kr-h1 mRNA contains a functional binding site for miR-2 family miRNAs (for miR-2, miR-13a, and miR-13b). These data suggest that metamorphosis impairment caused by Dicer-1 and miRNA depletion is due to a deregulation of Kr-h1 expression and that this deregulation is derived from a deficiency of miR-2 miRNAs. We corroborated this by treating the last nymphal instar of B. germanica with an miR-2 inhibitor, which impaired metamorphosis, and by treating Dicer-1-depleted individuals with an miR-2 mimic to allow nymphal-to-adult metamorphosis to proceed. Taken together, the data indicate that miR-2 miRNAs scavenge Kr-h1 transcripts when the transition from nymph to adult should be taking place, thus crucially contributing to the correct culmination of metamorphosis.

MicroRNA regulatory networks reflective of polyhexamethylene guanidine phosphate-induced fibrosis in A549 human alveolar adenocarcinoma cells.

PubMed

Shin, Da Young; Jeong, Mi Ho; Bang, In Jae; Kim, Ha Ryong; Chung, Kyu Hyuck

2018-05-01

Polyhexamethylene guanidine phosphate (PHMG-phosphate), an active component of humidifier disinfectant, is suspected to be a major cause of pulmonary fibrosis. Fibrosis, induced by recurrent epithelial damage, is significantly affected by epigenetic regulation, including microRNAs (miRNAs). The aim of this study was to investigate the fibrogenic mechanisms of PHMG-phosphate through the profiling of miRNAs and their target genes. A549 cells were treated with 0.75 μg/mL PHMG-phosphate for 24 and 48 h and miRNA microarray expression analysis was conducted. The putative mRNA targets of the miRNAs were identified and subjected to Gene Ontology analysis. After exposure to PHMG-phosphate for 24 and 48 h, 46 and 33 miRNAs, respectively, showed a significant change in expression over 1.5-fold compared with the control. The integrated analysis of miRNA and mRNA microarray results revealed the putative targets that were prominently enriched were associated with the epithelial-mesenchymal transition (EMT), cell cycle changes, and apoptosis. The dose-dependent induction of EMT by PHMG-phosphate exposure was confirmed by western blot. We identified 13 putative EMT-related targets that may play a role in PHMG-phosphate-induced fibrosis according to the Comparative Toxicogenomic Database. Our findings contribute to the comprehension of the fibrogenic mechanism of PHMG-phosphate and will aid further study on PHMG-phosphate-induced toxicity. Copyright © 2018 Elsevier B.V. All rights reserved.

Single nucleotide polymorphism-specific regulation of matrix metalloproteinase-9 by multiple miRNAs targeting the coding exon

PubMed Central

Duellman, Tyler; Warren, Christopher; Yang, Jay

2014-01-01

Microribonucleic acids (miRNAs) work with exquisite specificity and are able to distinguish a target from a non-target based on a single nucleotide mismatch in the core nucleotide domain. We questioned whether miRNA regulation of gene expression could occur in a single nucleotide polymorphism (SNP)-specific manner, manifesting as a post-transcriptional control of expression of genetic polymorphisms. In our recent study of the functional consequences of matrix metalloproteinase (MMP)-9 SNPs, we discovered that expression of a coding exon SNP in the pro-domain of the protein resulted in a profound decrease in the secreted protein. This missense SNP results in the N38S amino acid change and a loss of an N-glycosylation site. A systematic study demonstrated that the loss of secreted protein was due not to the loss of an N-glycosylation site, but rather an SNP-specific targeting by miR-671-3p and miR-657. Bioinformatics analysis identified 41 SNP-specific miRNA targeting MMP-9 SNPs, mostly in the coding exon and an extension of the analysis to chromosome 20, where the MMP-9 gene is located, suggesting that SNP-specific miRNAs targeting the coding exon are prevalent. This selective post-transcriptional regulation of a target messenger RNA harboring genetic polymorphisms by miRNAs offers an SNP-dependent post-transcriptional regulatory mechanism, allowing for polymorphic-specific differential gene regulation. PMID:24627221

The Effect of Oseltamivir on the Disease Progression of Lethal Influenza A Virus Infection: Plasma Cytokine and miRNA Responses in a Mouse Model

PubMed Central

Chockalingam, Ashok K.; Hamed, Salaheldin; Goodwin, David G.; Rosenzweig, Barry A.; Pang, Eric; Boyne II, Michael T.

2016-01-01

Lethal influenza A virus infection leads to acute lung injury and possibly lethal complications. There has been a continuous effort to identify the possible predictors of disease severity. Unlike earlier studies, where biomarkers were analyzed on certain time points or days after infection, in this study biomarkers were evaluated over the entire course of infection. Circulating proinflammatory cytokines and/or miRNAs that track with the onset and progression of lethal A/Puerto Rico/8/34 (PR8) influenza A virus infection and their response to oseltamivir treatment were investigated up to 10 days after infection. Changes in plasma cytokines (IL-1β, IL-10, IL-12p70, IL-6, KC, TNF-α, and IFN-γ) and several candidate miRNAs were profiled. Among the cytokines analyzed, IL-6 and KC/GRO cytokines appeared to correlate with peak viral titer. Over the selected 48 miRNAs profiled, certain miRNAs were up- or downregulated in a manner that was dependent on the oseltamivir treatment and disease severity. Our findings suggest that IL-6 and KC/GRO cytokines can be a potential disease severity biomarker and/or marker for the progression/remission of infection. Further studies to explore other cytokines, miRNAs, and lung injury proteins in serum with different subtypes of influenza A viruses with varying disease severity may provide new insight into other unique biomarkers. PMID:27110056

Autophagy-Regulating microRNAs and Cancer

PubMed Central

Gozuacik, Devrim; Akkoc, Yunus; Ozturk, Deniz Gulfem; Kocak, Muhammed

2017-01-01

Macroautophagy (autophagy herein) is a cellular stress response and a survival pathway that is responsible for the degradation of long-lived proteins, protein aggregates, as well as damaged organelles in order to maintain cellular homeostasis. Consequently, abnormalities of autophagy are associated with a number of diseases, including Alzheimers’s disease, Parkinson’s disease, and cancer. According to the current view, autophagy seems to serve as a tumor suppressor in the early phases of cancer formation, yet in later phases, autophagy may support and/or facilitate tumor growth, spread, and contribute to treatment resistance. Therefore, autophagy is considered as a stage-dependent dual player in cancer. microRNAs (miRNAs) are endogenous non-coding small RNAs that negatively regulate gene expression at a post-transcriptional level. miRNAs control several fundamental biological processes, and autophagy is no exception. Furthermore, accumulating data in the literature indicate that dysregulation of miRNA expression contribute to the mechanisms of cancer formation, invasion, metastasis, and affect responses to chemotherapy or radiotherapy. Therefore, considering the importance of autophagy for cancer biology, study of autophagy-regulating miRNA in cancer will allow a better understanding of malignancies and lead to the development of novel disease markers and therapeutic strategies. The potential to provide study of some of these cancer-related miRNAs were also implicated in autophagy regulation. In this review, we will focus on autophagy, miRNA, and cancer connection, and discuss its implications for cancer biology and cancer treatment. PMID:28459042

Transcripts and MicroRNAs Responding to Salt Stress in Musa acuminata Colla (AAA Group) cv. Berangan Roots

PubMed Central

Lee, Wan Sin; Gudimella, Ranganath; Wong, Gwo Rong; Tammi, Martti Tapani; Khalid, Norzulaani; Harikrishna, Jennifer Ann

2015-01-01

Physiological responses to stress are controlled by expression of a large number of genes, many of which are regulated by microRNAs. Since most banana cultivars are salt-sensitive, improved understanding of genetic regulation of salt induced stress responses in banana can support future crop management and improvement in the face of increasing soil salinity related to irrigation and climate change. In this study we focused on determining miRNA and their targets that respond to NaCl exposure and used transcriptome sequencing of RNA and small RNA from control and NaCl-treated banana roots to assemble a cultivar-specific reference transcriptome and identify orthologous and Musa-specific miRNA responding to salinity. We observed that, banana roots responded to salinity stress with changes in expression for a large number of genes (9.5% of 31,390 expressed unigenes) and reduction in levels of many miRNA, including several novel miRNA and banana-specific miRNA-target pairs. Banana roots expressed a unique set of orthologous and Musa-specific miRNAs of which 59 respond to salt stress in a dose-dependent manner. Gene expression patterns of miRNA compared with those of their predicted mRNA targets indicated that a majority of the differentially expressed miRNAs were down-regulated in response to increased salinity, allowing increased expression of targets involved in diverse biological processes including stress signaling, stress defence, transport, cellular homeostasis, metabolism and other stress-related functions. This study may contribute to the understanding of gene regulation and abiotic stress response of roots and the high-throughput sequencing data sets generated may serve as important resources related to salt tolerance traits for functional genomic studies and genetic improvement in banana. PMID:25993649

Transcripts and MicroRNAs Responding to Salt Stress in Musa acuminata Colla (AAA Group) cv. Berangan Roots.

PubMed

Lee, Wan Sin; Gudimella, Ranganath; Wong, Gwo Rong; Tammi, Martti Tapani; Khalid, Norzulaani; Harikrishna, Jennifer Ann

2015-01-01

Physiological responses to stress are controlled by expression of a large number of genes, many of which are regulated by microRNAs. Since most banana cultivars are salt-sensitive, improved understanding of genetic regulation of salt induced stress responses in banana can support future crop management and improvement in the face of increasing soil salinity related to irrigation and climate change. In this study we focused on determining miRNA and their targets that respond to NaCl exposure and used transcriptome sequencing of RNA and small RNA from control and NaCl-treated banana roots to assemble a cultivar-specific reference transcriptome and identify orthologous and Musa-specific miRNA responding to salinity. We observed that, banana roots responded to salinity stress with changes in expression for a large number of genes (9.5% of 31,390 expressed unigenes) and reduction in levels of many miRNA, including several novel miRNA and banana-specific miRNA-target pairs. Banana roots expressed a unique set of orthologous and Musa-specific miRNAs of which 59 respond to salt stress in a dose-dependent manner. Gene expression patterns of miRNA compared with those of their predicted mRNA targets indicated that a majority of the differentially expressed miRNAs were down-regulated in response to increased salinity, allowing increased expression of targets involved in diverse biological processes including stress signaling, stress defence, transport, cellular homeostasis, metabolism and other stress-related functions. This study may contribute to the understanding of gene regulation and abiotic stress response of roots and the high-throughput sequencing data sets generated may serve as important resources related to salt tolerance traits for functional genomic studies and genetic improvement in banana.

Micro RNA responses to chronic or acute exposures to low dose ionizing radiation

PubMed Central

Chaudhry, M. Ahmad; Omaruddin, Romaica A.; Kreger, Bridget; de Toledo, Sonia M.; Azzam, Edouard I.

2014-01-01

Human health risks of exposure to low dose ionizing radiation remain ambiguous and are the subject of intense debate. A wide variety of biological effects are induced after cellular exposure to ionizing radiation, but the underlying molecular mechanism(s) remain to be completely understood. We hypothesized that low dose c-radiation-induced effects are controlled by the modulation of micro RNA (miRNA) that participate in the control of gene expression at the posttranscriptional level and are involved in many cellular processes. We monitored the expression of several miRNA in human cells exposed to acute or chronic low doses of 10 cGy or a moderate dose of 400 cGy of 137Cs γ-rays. Dose, dose rate and time dependent differences in the relative expression of several miRNA were investigated. The expression patterns of many miRNA differed after exposure to either chronic or acute 10 cGy. The expression of miRNA let-7e, a negative regulator of RAS oncogene, and the c-MYC miRNA cluster were upregulated after 10 cGy chronic dose but were downregulated after 3 h of acute 10 cGy. The miR-21 was upregulated in chronic or acute low dose and moderate dose treated cells and its target genes hPDCD4, hPTEN, hSPRY2, and hTPM1 were found to be downregulated. These findings provide evidence that low dose and dose rate c-irradiation dictate the modulation of miRNA, which can result in a differential cellular response than occurs at high doses. This information will contribute to understanding the risks to human health after exposure to low dose radiation. PMID:22367372

Dysregulated RNA-Induced Silencing Complex (RISC) Assembly within CNS Corresponds with Abnormal miRNA Expression during Autoimmune Demyelination.

PubMed

Lewkowicz, Przemysław; Cwiklińska, Hanna; Mycko, Marcin P; Cichalewska, Maria; Domowicz, Małgorzata; Lewkowicz, Natalia; Jurewicz, Anna; Selmaj, Krzysztof W

2015-05-13

MicroRNAs (miRNAs) associate with Argonaute (Ago), GW182, and FXR1 proteins to form RNA-induced silencing complexes (RISCs). RISCs represent a critical checkpoint in the regulation and bioavailability of miRNAs. Recent studies have revealed dysregulation of miRNAs in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE); however, the function of RISCs in EAE and MS is largely unknown. Here, we examined the expression of Ago, GW182, and FXR1 in CNS tissue, oligodendrocytes (OLs), brain-infiltrating T lymphocytes, and CD3(+)splenocytes (SCs) of EAE mic, and found that global RISC protein levels were significantly dysregulated. Specifically, Ago2 and FXR1 levels were decreased in OLs and brain-infiltrating T cells in EAE mice. Accordingly, assembly of Ago2/GW182/FXR1 complexes in EAE brain tissues was disrupted, as confirmed by immunoprecipitation experiments. In parallel with alterations in RISC complex content in OLs, we found downregulation of miRNAs essential for differentiation and survival of OLs and myelin synthesis. In brain-infiltrating T lymphocytes, aberrant RISC formation contributed to miRNA-dependent proinflammatory helper T-cell polarization. In CD3(+) SCs, we found increased expression of both Ago2 and FXR1 in EAE compared with nonimmunized mice. Therefore, our results demonstrate a gradient in expression of miRNA between primary activated T cells in the periphery and polarized CNS-infiltrating T cells. These results suggest that, in polarized autoreactive effector T cells, miRNA synthesis is inhibited in response to dysregulated RISC assembly, allowing these cells to maintain a highly specific proinflammatory program. Therefore, our findings may provide a mechanism that leads to miRNA dysregulation in EAE/MS. Copyright © 2015 the authors 0270-6474/15/357521-17$15.00/0.

Isolation and Identification of Gene-Specific MicroRNAs.

PubMed

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

2018-01-01

Computer programming has identified hundreds of genomic hairpin sequences, many with functions yet to be determined. Because transfection of hairpin-like microRNA precursors (pre-miRNAs) into mammalian cells is not always sufficient to trigger RNA-induced gene silencing complex (RISC) assembly, a key step for inducing RNA interference (RNAi)-related gene silencing, we have developed an intronic miRNA expression system to overcome this problem by inserting a hairpin-like pre-miRNA structure into the intron region of a gene, and hence successfully increase the efficiency and effectiveness of miRNA-associated RNAi induction in vitro and in vivo. This intronic miRNA biogenesis mechanism has been found to depend on a coupled interaction of nascent messenger RNA transcription and intron excision within a specific nuclear region proximal to genomic perichromatin fibrils. The intronic miRNA so obtained is transcribed by type-II RNA polymerases, coexpressed within a primary gene transcript, and then excised out of the gene transcript by intracellular RNA splicing and processing machineries. After that, ribonuclease III (RNaseIII) endonucleases further process the spliced introns into mature miRNAs. Using this intronic miRNA expression system, we have shown for the first time that the intron-derived miRNAs are able to elicit strong RNAi effects in not only human and mouse cells in vitro but also in zebrafishes, chicken embryos, and adult mice in vivo. We have also developed a miRNA isolation protocol, based on the complementarity between the designed miRNA and its targeted gene sequence, to purify and identify the mature miRNAs generated. As a result, several intronic miRNA identities and structures have been confirmed. According to this proof-of-principle methodology, we now have full knowledge to design various intronic pre-miRNA inserts that are more efficient and effective for inducing specific gene silencing effects in vitro and in vivo.

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

IFN-β antiproliferative effect and miRNA regulation in Human Papilloma Virus E6- and E7-transformed keratinocytes.

PubMed

Chiantore, Maria Vincenza; Mangino, Giorgio; Iuliano, Marco; Zangrillo, Maria Simona; De Lillis, Ilaria; Vaccari, Gabriele; Accardi, Rosita; Tommasino, Massimo; Fiorucci, Gianna; Romeo, Giovanna

2017-01-01

Human Papilloma Viruses (HPVs) are the causative agents of cervical cancer although other types of cancers are associated with HPV infection. Type I Interferons can interfere with HPV E6- and/or E7-dependent transformation and can affect microRNA (miRNA) expression. Cancer cells show a specific pattern of miRNA expression and HPVs are able to modulate miRNAs expressed in infected cells. Keratinocytes transduced with E6 and E7 from mucosal HPV-16 or cutaneous HPV-38 (K16 and K38) were studied to analyze the involvement of HPV oncoproteins in the anti-proliferative activity of IFN-β. In view of our previous data showing senescence induction by the cytokine in K38 cells, we observe that IFN-β treatment leads to p53-indipendent apoptosis in K16 cells whereas induces senescence in K16 cells if E6 is silenced and p53 expression is restored. The levels of selected miRNAs, deregulated in K16 and K38 cells, can be modulated by IFN-β when E6 and E7 proteins of HPV-16, but not HPV-38, are expressed. Copyright © 2015 Elsevier Ltd. All rights reserved.

Integrated Strategy Improves the Prediction Accuracy of miRNA in Large Dataset

PubMed Central

Lipps, David; Devineni, Sree

2016-01-01

MiRNAs are short non-coding RNAs of about 22 nucleotides, which play critical roles in gene expression regulation. The biogenesis of miRNAs is largely determined by the sequence and structural features of their parental RNA molecules. Based on these features, multiple computational tools have been developed to predict if RNA transcripts contain miRNAs or not. Although being very successful, these predictors started to face multiple challenges in recent years. Many predictors were optimized using datasets of hundreds of miRNA samples. The sizes of these datasets are much smaller than the number of known miRNAs. Consequently, the prediction accuracy of these predictors in large dataset becomes unknown and needs to be re-tested. In addition, many predictors were optimized for either high sensitivity or high specificity. These optimization strategies may bring in serious limitations in applications. Moreover, to meet continuously raised expectations on these computational tools, improving the prediction accuracy becomes extremely important. In this study, a meta-predictor mirMeta was developed by integrating a set of non-linear transformations with meta-strategy. More specifically, the outputs of five individual predictors were first preprocessed using non-linear transformations, and then fed into an artificial neural network to make the meta-prediction. The prediction accuracy of meta-predictor was validated using both multi-fold cross-validation and independent dataset. The final accuracy of meta-predictor in newly-designed large dataset is improved by 7% to 93%. The meta-predictor is also proved to be less dependent on datasets, as well as has refined balance between sensitivity and specificity. This study has two folds of importance: First, it shows that the combination of non-linear transformations and artificial neural networks improves the prediction accuracy of individual predictors. Second, a new miRNA predictor with significantly improved prediction accuracy is developed for the community for identifying novel miRNAs and the complete set of miRNAs. Source code is available at: https://github.com/xueLab/mirMeta PMID:28002428

Comparative Genomics as a Foundation for Evo-Devo Studies in Birds.

PubMed

Grayson, Phil; Sin, Simon Y W; Sackton, Timothy B; Edwards, Scott V

2017-01-01

Developmental genomics is a rapidly growing field, and high-quality genomes are a useful foundation for comparative developmental studies. A high-quality genome forms an essential reference onto which the data from numerous assays and experiments, including ChIP-seq, ATAC-seq, and RNA-seq, can be mapped. A genome also streamlines and simplifies the development of primers used to amplify putative regulatory regions for enhancer screens, cDNA probes for in situ hybridization, microRNAs (miRNAs) or short hairpin RNAs (shRNA) for RNA interference (RNAi) knockdowns, mRNAs for misexpression studies, and even guide RNAs (gRNAs) for CRISPR knockouts. Finally, much can be gleaned from comparative genomics alone, including the identification of highly conserved putative regulatory regions. This chapter provides an overview of laboratory and bioinformatics protocols for DNA extraction, library preparation, library quantification, and genome assembly, from fresh or frozen tissue to a draft avian genome. Generating a high-quality draft genome can provide a developmental research group with excellent resources for their study organism, opening the doors to many additional assays and experiments.

Identification of side- and shear-dependent microRNAs regulating porcine aortic valve pathogenesis

NASA Astrophysics Data System (ADS)

Rathan, Swetha; Ankeny, Casey J.; Arjunon, Sivakkumar; Ferdous, Zannatul; Kumar, Sandeep; Fernandez Esmerats, Joan; Heath, Jack M.; Nerem, Robert M.; Yoganathan, Ajit P.; Jo, Hanjoong

2016-05-01

Aortic valve (AV) calcification is an inflammation driven process that occurs preferentially in the fibrosa. To explore the underlying mechanisms, we investigated if key microRNAs (miRNA) in the AV are differentially expressed due to disturbed blood flow (oscillatory shear (OS)) experienced by the fibrosa compared to the ventricularis. To identify the miRNAs involved, endothelial-enriched RNA was isolated from either side of healthy porcine AVs for microarray analysis. Validation using qPCR confirmed significantly higher expression of 7 miRNAs (miR-100, -130a, -181a/b, -199a-3p, -199a-5p, and -214) in the fibrosa versus the ventricularis. Upon bioinformatics analysis, miR-214 was selected for further investigation using porcine AV leaflets in an ex vivo shear system. Fibrosa and ventricularis sides were exposed to either oscillatory or unidirectional pulsatile shear for 2 days and 3 & 7 days in regular and osteogenic media, respectively. Higher expression of miR-214, increased thickness of the fibrosa, and calcification was observed when the fibrosa was exposed to OS compared to the ventricularis. Silencing of miR-214 by anti-miR-214 in whole AV leaflets with the fibrosa exposed to OS significantly increased the protein expression of TGFβ1 and moderately increased collagen content but did not affect AV calcification. Thus, miR-214 is identified as a side- and shear-dependent miRNA that regulates key mechanosensitive gene in AV such as TGFβ1.

MicroRNA Profiling Reveals Marker of Motor Neuron Disease in ALS Models.

PubMed

Hoye, Mariah L; Koval, Erica D; Wegener, Amy J; Hyman, Theodore S; Yang, Chengran; O'Brien, David R; Miller, Rebecca L; Cole, Tracy; Schoch, Kathleen M; Shen, Tao; Kunikata, Tomonori; Richard, Jean-Philippe; Gutmann, David H; Maragakis, Nicholas J; Kordasiewicz, Holly B; Dougherty, Joseph D; Miller, Timothy M

2017-05-31

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder marked by the loss of motor neurons (MNs) in the brain and spinal cord, leading to fatally debilitating weakness. Because this disease predominantly affects MNs, we aimed to characterize the distinct expression profile of that cell type to elucidate underlying disease mechanisms and to identify novel targets that inform on MN health during ALS disease time course. microRNAs (miRNAs) are short, noncoding RNAs that can shape the expression profile of a cell and thus often exhibit cell-type-enriched expression. To determine MN-enriched miRNA expression, we used Cre recombinase-dependent miRNA tagging and affinity purification in mice. By defining the in vivo miRNA expression of MNs, all neurons, astrocytes, and microglia, we then focused on MN-enriched miRNAs via a comparative analysis and found that they may functionally distinguish MNs postnatally from other spinal neurons. Characterizing the levels of the MN-enriched miRNAs in CSF harvested from ALS models of MN disease demonstrated that one miRNA (miR-218) tracked with MN loss and was responsive to an ALS therapy in rodent models. Therefore, we have used cellular expression profiling tools to define the distinct miRNA expression of MNs, which is likely to enrich future studies of MN disease. This approach enabled the development of a novel, drug-responsive marker of MN disease in ALS rodents. SIGNIFICANCE STATEMENT Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons (MNs) in the brain and spinal cord are selectively lost. To develop tools to aid in our understanding of the distinct expression profiles of MNs and, ultimately, to monitor MN disease progression, we identified small regulatory microRNAs (miRNAs) that were highly enriched or exclusive in MNs. The signal for one of these MN-enriched miRNAs is detectable in spinal tap biofluid from an ALS rat model, where its levels change as disease progresses, suggesting that it may be a clinically useful marker of disease status. Furthermore, rats treated with ALS therapy have restored expression of this MN RNA marker, making it an MN-specific and drug-responsive marker for ALS rodents. Copyright © 2017 the authors 0270-6474/17/375574-13$15.00/0.

MicroRNA Intercellular Transfer and Bioelectrical Regulation of Model Multicellular Ensembles by the Gap Junction Connectivity.

PubMed

Cervera, Javier; Meseguer, Salvador; Mafe, Salvador

2017-08-17

We have studied theoretically the microRNA (miRNA) intercellular transfer through voltage-gated gap junctions in terms of a biophysically grounded system of coupled differential equations. Instead of modeling a specific system, we use a general approach describing the interplay between the genetic mechanisms and the single-cell electric potentials. The dynamics of the multicellular ensemble are simulated under different conditions including spatially inhomogeneous transcription rates and local intercellular transfer of miRNAs. These processes result in spatiotemporal changes of miRNA, mRNA, and ion channel protein concentrations that eventually modify the bioelectrical states of small multicellular domains because of the ensemble average nature of the electrical potential. The simulations allow a qualitative understanding of the context-dependent nature of the effects observed when specific signaling molecules are transferred through gap junctions. The results suggest that an efficient miRNA intercellular transfer could permit the spatiotemporal control of small cellular domains by the conversion of single-cell genetic and bioelectric states into multicellular states regulated by the gap junction interconnectivity.

Overexpression of Trophoblast Stem Cell-Enriched MicroRNAs Promotes Trophoblast Fate in Embryonic Stem Cells.

PubMed

Nosi, Ursula; Lanner, Fredrik; Huang, Tsu; Cox, Brian

2017-05-09

The first cell fate choice of the preimplantation embryo generates the extraembryonic trophoblast and embryonic epiblast lineages. Embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) can be utilized to investigate molecular mechanisms of this first cell fate decision. It has been established that ESCs can be induced to acquire trophoblast lineage characteristics upon manipulation of lineage-determining transcription factors. Here, we have interrogated the potential of microRNAs (miRNAs) to drive trans-differentiation of ESCs into the trophoblast lineage. Analysis of gene expression data identified a network of TSC-enriched miRNAs that were predicted to target mRNAs enriched in ESCs. Ectopic expression of these miRNAs in ESCs resulted in a stable trophoblast phenotype, supported by gene expression changes and in vivo contribution potential. This process is highly miRNA-specific and dependent on Hdac2 inhibition. Our experimental evidence suggests that these miRNAs promote a mural trophectoderm (TE)-like cell fate with physiological properties that differentiate them from the polar TE. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Structural determinants of miRNAs for RISC loading and slicer-independent unwinding.

PubMed

Kawamata, Tomoko; Seitz, Hervé; Tomari, Yukihide

2009-09-01

MicroRNAs (miRNAs) regulate expression of their target mRNAs through the RNA-induced silencing complex (RISC), which contains an Argonaute (Ago) family protein as a core component. In Drosophila melanogaster, miRNAs are generally sorted into Ago1-containing RISC (Ago1-RISC). We established a native gel system that can biochemically dissect the Ago1-RISC assembly pathway. We found that miRNA-miRNA* duplexes are loaded into Ago1 as double-stranded RNAs in an ATP-dependent fashion. In contrast, unexpectedly, unwinding of miRNA-miRNA* duplexes is a passive process that does not require ATP or slicer activity of Ago1. Central mismatches direct miRNA-miRNA* duplexes into pre-Ago1-RISC, whereas mismatches in the seed or guide strand positions 12-15 promote conversion of pre-Ago1-RISC into mature Ago1-RISC. Our findings show that unwinding of miRNAs is a precise mirror-image process of target recognition, and both processes reflect the unique geometry of RNAs in Ago proteins.

Microprocessor depends on hemin to recognize the apical loop of primary microRNA

PubMed Central

Park, Joha; Dang, Thi Lieu; Choi, Yeon-Gil; Kim, V Narry

2018-01-01

Abstract Microprocessor, which consists of a ribonuclease III DROSHA and its cofactor DGCR8, initiates microRNA (miRNA) maturation by cleaving primary miRNA transcripts (pri-miRNAs). We recently demonstrated that the DGCR8 dimer recognizes the apical elements of pri-miRNAs, including the UGU motif, to accurately locate and orient Microprocessor on pri-miRNAs. However, the mechanism underlying the selective RNA binding remains unknown. In this study, we find that hemin, a ferric ion-containing porphyrin, enhances the specific interaction between the apical UGU motif and the DGCR8 dimer, allowing Microprocessor to achieve high efficiency and fidelity of pri-miRNA processing in vitro. Furthermore, by generating a DGCR8 mutant cell line and carrying out rescue experiments, we discover that hemin preferentially stimulates the expression of miRNAs possessing the UGU motif, thereby conferring differential regulation of miRNA maturation. Our findings reveal the molecular action mechanism of hemin in pri-miRNA processing and establish a novel function of hemin in inducing specific RNA-protein interaction. PMID:29750274

Microprocessor depends on hemin to recognize the apical loop of primary microRNA.

PubMed

Nguyen, Tuan Anh; Park, Joha; Dang, Thi Lieu; Choi, Yeon-Gil; Kim, V Narry

2018-06-20

Microprocessor, which consists of a ribonuclease III DROSHA and its cofactor DGCR8, initiates microRNA (miRNA) maturation by cleaving primary miRNA transcripts (pri-miRNAs). We recently demonstrated that the DGCR8 dimer recognizes the apical elements of pri-miRNAs, including the UGU motif, to accurately locate and orient Microprocessor on pri-miRNAs. However, the mechanism underlying the selective RNA binding remains unknown. In this study, we find that hemin, a ferric ion-containing porphyrin, enhances the specific interaction between the apical UGU motif and the DGCR8 dimer, allowing Microprocessor to achieve high efficiency and fidelity of pri-miRNA processing in vitro. Furthermore, by generating a DGCR8 mutant cell line and carrying out rescue experiments, we discover that hemin preferentially stimulates the expression of miRNAs possessing the UGU motif, thereby conferring differential regulation of miRNA maturation. Our findings reveal the molecular action mechanism of hemin in pri-miRNA processing and establish a novel function of hemin in inducing specific RNA-protein interaction.

Plasma miRNA levels correlate with sensitivity to bone mineral density in postmenopausal osteoporosis patients.

PubMed

Li, Hongqiu; Wang, Zhe; Fu, Qin; Zhang, Jing

2014-11-01

In our study, we detect the levels of three micro-RNAs (miRNAs; miR-21, miR-133a and miR-146a) in the plasma of 120 Chinese postmenopausal women who were divided into three groups (normal, osteopenia and osteoporosis) according to the T-scores. Downregulation of miR-21, as well as upregulation of miR-133a, was validated in the plasma of osteoporosis and osteopenia patients versus the normal group. The difference in expression regarding the miR-146a level in plasma among the three groups was not significant (p > 0.01). The circulating miRNA expression levels and bone mineral density (BMD) were examined during a multiple correlation analysis as a dependent variable after adjusting for age, weight and height. We have demonstrated that specific miRNAs species are significantly changed in the plasma of osteoporosis and osteopenia patients and correlated with the BMD. Our study suggested a potential use of miR-21 and miR-133a as sensitive and plasma biomarkers for postmenopausal osteoporosis.

Chronic constriction injury-induced microRNA-146a-5p alleviates neuropathic pain through suppression of IRAK1/TRAF6 signaling pathway.

PubMed

Wang, Zhiyao; Liu, Fan; Wei, Min; Qiu, Yue; Ma, Chao; Shen, Le; Huang, Yuguang

2018-06-09

microRNA-146a-5p (miRNA-146a-5p) is a key molecule in the negative regulation pathway of TLRs and IL-1 receptor (TIR) signaling. Our recent study demonstrated that MyD88-dependent signaling pathway of TIR in the dorsal root ganglion (DRG) and spinal dorsal horn (SDH) plays a role in peripheral nerve injury-induced neuropathic pain. However, it was not clear whether and how miRNA-146a-5p regulates the TIR pathway of DRG and SDH in the development of neuropathic pain. The sciatic nerve chronic constriction injury (CCI) model of rat was used to induce chronic neuropathic pain. The levels and cellular distribution of miRNA-146a-5p were detected with quantitative real-time PCR (qPCR) and fluorescent in situ hybridization (FISH). The RNA level, protein level, and cellular distribution of IRAK1 and TRAF6 that is targeted by miRNA-146a-5p were detected with qPCR, western blot, and immunofluorescent. The pain-related behavioral effect of miRNA-146a-5p was accessed after intrathecal administration. Mechanical stimuli and radiant heat were used to evaluate mechanical allodynia and thermal hyperalgesia. We found that the level of miRNA-146a-5p significantly increased in L4-L6 DRGs and SDH after CCI surgery; meanwhile, the protein level of IRAK1 and TRAF6 in DRGs was significantly increased after CCI. Intrathecal injection of miR146a-5p agomir or miRNA-146a-5p antagomir regulates miRNA-146a-5p level of L4-L6 DRGs and SDH. We found that intrathecal injection of miR146a-5p agomir can alleviate mechanical and thermal hyperalgesia in CCI rats and reverse the upregulation of IRAK1 and TRAF6 of L4-L6 DRGs and SDH induced by CCI. We furthermore found that intrathecal injection of miRNA-146a-5p antagomir can exacerbate the mechanical and thermal pain-related behavior of CCI rats and meanwhile increase IRAK1 and TRAF6 of L4-L6 DRGs and SDH expression even further. miRNA-146a-5p of DRG and SDH can modulate the development of CCI-induced neuropathic pain through inhibition of IRAK1 and TRAF6 in the TIR signaling pathway. Hence, miRNA-146a-5p may serve as a potential therapeutic target for neuropathic pain.

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.

An investigation into anti-proliferative effects of microRNAs encoded by the miR-106a-363 cluster on human carcinoma cells and keratinocytes using microarray profiling of miRNA transcriptomes

PubMed Central

Khuu, Cuong; Jevnaker, Anne-Marthe; Bryne, Magne; Osmundsen, Harald

2014-01-01

Transfection of human oral squamous carcinoma cells (clone E10) with mimics for unexpressed miR-20b or miR-363-5p, encoded by the miR-106a-363 cluster (miR-20b, miR-106a, miR-363-3p, or miR-363-5p), caused 40–50% decrease in proliferation. Transfection with mimics for miR-18a or miR-92a, encoded by the miR-17-92 cluster (all members being expressed in E10 cells), had no effect on proliferation. In contrast, mimic for the sibling miRNA-19a yielded about 20% inhibition of proliferation. To investigate miRNA involvement profiling of miRNA transcriptomes were carried out using deoxyoligonucleotide microarrays. In transfectants for miR-19a, or miR-20b or miR-363-5p most differentially expressed miRNAs exhibited decreased expression, including some miRNAs encoded in paralogous miR-17-92—or miR-106b-25 cluster. Only in cells transfected with miR-19a mimic significantly increased expression of miR-20b observed—about 50-fold as judged by qRT-PCR. Further studies using qRT-PCR showed that transfection of E10 cells with mimic for miRNAs encoded by miR-17-92 - or miR-106a-363 - or the miR-106b-25 cluster confirmed selective effect on expression on sibling miRNAs. We conclude that high levels of miRNAs encoded by the miR-106a-363 cluster may contribute to inhibition of proliferation by decreasing expression of several sibling miRNAs encoded by miR-17-92 or by the miR-106b-25 cluster. The inhibition of proliferation observed in miR-19a-mimic transfectants is likely caused by the miR-19a-dependent increase in the levels of miR-20b and miR-106a. Bioinformatic analysis of differentially expressed miRNAs from miR-106a, miR-20b and miR-363-5p transfectants, but not miR-92a transfectants, yielded significant associations to “Cellular Growth and Proliferation” and “Cell Cycle.” Western blotting results showed that levels of affected proteins to differ between transfectants, suggesting that different anti-proliferative mechanisms may operate in these transfectants. PMID:25202322

The Draft Genome and Transcriptome of Panagrellus redivivus Are Shaped by the Harsh Demands of a Free-Living Lifestyle

PubMed Central

Srinivasan, Jagan; Dillman, Adler R.; Macchietto, Marissa G.; Heikkinen, Liisa; Lakso, Merja; Fracchia, Kelley M.; Antoshechkin, Igor; Mortazavi, Ali; Wong, Garry; Sternberg, Paul W.

2013-01-01

Nematodes compose an abundant and diverse invertebrate phylum with members inhabiting nearly every ecological niche. Panagrellus redivivus (the “microworm”) is a free-living nematode frequently used to understand the evolution of developmental and behavioral processes given its phylogenetic distance to Caenorhabditis elegans. Here we report the de novo sequencing of the genome, transcriptome, and small RNAs of P. redivivus. Using a combination of automated gene finders and RNA-seq data, we predict 24,249 genes and 32,676 transcripts. Small RNA analysis revealed 248 microRNA (miRNA) hairpins, of which 63 had orthologs in other species. Fourteen miRNA clusters containing 42 miRNA precursors were found. The RNA interference, dauer development, and programmed cell death pathways are largely conserved. Analysis of protein family domain abundance revealed that P. redivivus has experienced a striking expansion of BTB domain-containing proteins and an unprecedented expansion of the cullin scaffold family of proteins involved in multi-subunit ubiquitin ligases, suggesting proteolytic plasticity and/or tighter regulation of protein turnover. The eukaryotic release factor protein family has also been dramatically expanded and suggests an ongoing evolutionary arms race with viruses and transposons. The P. redivivus genome provides a resource to advance our understanding of nematode evolution and biology and to further elucidate the genomic architecture leading to free-living lineages, taking advantage of the many fascinating features of this worm revealed by comparative studies. PMID:23410827

Coevolution Pattern and Functional Conservation or Divergence of miR167s and their targets across Diverse Plant Species

PubMed Central

Barik, Suvakanta; Kumar, Ashutosh; Sarkar Das, Shabari; Yadav, Sandeep; Gautam, Vibhav; Singh, Archita; Singh, Sharmila; Sarkar, Ananda K.

2015-01-01

microRNAs (miRNAs), a class of endogenously produced small non-coding RNAs of 20–21 nt length, processed from precursor miRNAs, regulate many developmental processes by negatively regulating the target genes in both animals and plants. The coevolutionary pattern of a miRNA family and their targets underscores its functional conservation or diversification. The miR167 regulates various aspects of plant development in Arabidopsis by targeting ARF6 and ARF8. The evolutionary conservation or divergence of miR167s and their target genes are poorly understood till now. Here we show the evolutionary relationship among 153 MIR167 genes obtained from 33 diverse plant species. We found that out of the 153 of miR167 sequences retrieved from the “miRBase”, 27 have been annotated to be processed from the 3′ end, and have diverged distinctively from the other miR167s produced from 5′ end. Our analysis reveals that gma-miR167h/i and mdm-miR167a are processed from 3′ end and have evolved separately, diverged most resulting in novel targets other than their known ones, and thus led to functional diversification, especially in apple and soybean. We also show that mostly conserved miR167 sequences and their target AUXIN RESPONSE FACTORS (ARFs) have gone through parallel evolution leading to functional diversification among diverse plant species. PMID:26459056

Coevolution Pattern and Functional Conservation or Divergence of miR167s and their targets across Diverse Plant Species.

PubMed

Barik, Suvakanta; Kumar, Ashutosh; Sarkar Das, Shabari; Yadav, Sandeep; Gautam, Vibhav; Singh, Archita; Singh, Sharmila; Sarkar, Ananda K

2015-10-13

microRNAs (miRNAs), a class of endogenously produced small non-coding RNAs of 20-21 nt length, processed from precursor miRNAs, regulate many developmental processes by negatively regulating the target genes in both animals and plants. The coevolutionary pattern of a miRNA family and their targets underscores its functional conservation or diversification. The miR167 regulates various aspects of plant development in Arabidopsis by targeting ARF6 and ARF8. The evolutionary conservation or divergence of miR167s and their target genes are poorly understood till now. Here we show the evolutionary relationship among 153 MIR167 genes obtained from 33 diverse plant species. We found that out of the 153 of miR167 sequences retrieved from the "miRBase", 27 have been annotated to be processed from the 3' end, and have diverged distinctively from the other miR167s produced from 5' end. Our analysis reveals that gma-miR167h/i and mdm-miR167a are processed from 3' end and have evolved separately, diverged most resulting in novel targets other than their known ones, and thus led to functional diversification, especially in apple and soybean. We also show that mostly conserved miR167 sequences and their target AUXIN RESPONSE FACTORS (ARFs) have gone through parallel evolution leading to functional diversification among diverse plant species.

Circular RNAs: A novel type of biomarker and genetic tools in cancer

PubMed Central

Han, Yi-Neng; Xia, Sheng-Qiang; Zhang, Yuan-Yuan; Zheng, Jun-Hua; Li, Wei

2017-01-01

Circular RNAs (circRNAs) are a novel type of universal and diverse endogenous noncoding RNAs (ncRNAs) and they form a covalently closed continuous loop without 5′ or 3′ tails unlike linear RNAs. Most circRNAs are presented with characteristics of abundance, stability, conservatism, and often exhibiting tissue/developmental-stage-specific expression. CircRNAs are generated either from exons or introns by back splicing or lariat introns. CircRNAs play important roles as miRNA sponges, gene transcription and expression regulators, RNA-binding protein (RBP) sponges and protein/peptide translators. Emerging evidence revealed the function of circRNAs in cancer and may potentially serve as a required novel biomarker and therapeutic target for cancer treatment. In this review, we discuss about the origins, characteristics and functions of circRNA and how they work as miRNA sponges, gene transcription and expression regulators, RBP sponges in cancer as well as current research methods of circRNAs, providing evidence for the significance of circRNAs in cancer diagnosis and clinical treatment. PMID:28969093

A stele-enriched gene regulatory network in the Arabidopsis root

PubMed Central

Brady, Siobhan M; Zhang, Lifang; Megraw, Molly; Martinez, Natalia J; Jiang, Eric; Yi, Charles S; Liu, Weilin; Zeng, Anna; Taylor-Teeples, Mallorie; Kim, Dahae; Ahnert, Sebastian; Ohler, Uwe; Ware, Doreen; Walhout, Albertha J M; Benfey, Philip N

2011-01-01

Tightly controlled gene expression is a hallmark of multicellular development and is accomplished by transcription factors (TFs) and microRNAs (miRNAs). Although many studies have focused on identifying downstream targets of these molecules, less is known about the factors that regulate their differential expression. We used data from high spatial resolution gene expression experiments and yeast one-hybrid (Y1H) and two-hybrid (Y2H) assays to delineate a subset of interactions occurring within a gene regulatory network (GRN) that determines tissue-specific TF and miRNA expression in plants. We find that upstream TFs are expressed in more diverse cell types than their targets and that promoters that are bound by a relatively large number of TFs correspond to key developmental regulators. The regulatory consequence of many TFs for their target was experimentally determined using genetic analysis. Remarkably, molecular phenotypes were identified for 65% of the TFs, but morphological phenotypes were associated with only 16%. This indicates that the GRN is robust, and that gene expression changes may be canalized or buffered. PMID:21245844

Exosomes Derived from HIV-1-infected Cells Contain Trans-activation Response Element RNA*

PubMed Central

Narayanan, Aarthi; Iordanskiy, Sergey; Das, Ravi; Van Duyne, Rachel; Santos, Steven; Jaworski, Elizabeth; Guendel, Irene; Sampey, Gavin; Dalby, Elizabeth; Iglesias-Ussel, Maria; Popratiloff, Anastas; Hakami, Ramin; Kehn-Hall, Kylene; Young, Mary; Subra, Caroline; Gilbert, Caroline; Bailey, Charles; Romerio, Fabio; Kashanchi, Fatah

2013-01-01

Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 104–106 copies/ml TAR RNA in exosomes derived from infected culture supernatants and 103 copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS. PMID:23661700

Olfactory discrimination training up-regulates and reorganizes expression of microRNAs in adult mouse hippocampus.

PubMed

Smalheiser, Neil R; Lugli, Giovanni; Lenon, Angela L; Davis, John M; Torvik, Vetle I; Larson, John

2010-02-26

Adult male mice (strain C57Bl/6J) were trained to execute nose-poke responses for water reinforcement; then they were randomly assigned to either of two groups: olfactory discrimination training (exposed to two odours with reward contingent upon correctly responding to one odour) or pseudo-training (exposed to two odours with reward not contingent upon response). These were run in yoked fashion and killed when the discrimination-trained mouse reached a learning criterion of 70% correct responses in 20 trials, occurring after three sessions (a total of approximately 40 min of training). The hippocampus was dissected bilaterally from each mouse (N = 7 in each group) and profiling of 585 miRNAs (microRNAs) was carried out using multiplex RT-PCR (reverse transcription-PCR) plates. A significant global up-regulation of miRNA expression was observed in the discrimination training versus pseudo-training comparison; when tested individually, 29 miRNAs achieved significance at P = 0.05. miR-10a showed a 2.7-fold increase with training, and is predicted to target several learning-related mRNAs including BDNF (brain-derived neurotrophic factor), CAMK2b (calcium/calmodulin-dependent protein kinase IIβ), CREB1 (cAMP-response-element-binding protein 1) and ELAVL2 [ELAV (embryonic lethal, abnormal vision, Drosophila)-like; Hu B]. Analysis of miRNA pairwise correlations revealed the existence of several miRNA co-expression modules that were specific to the training group. These in vivo results indicate that significant, dynamic and co-ordinated changes in miRNA expression accompany early stages of learning.

Transcriptome analysis reveals regional and temporal differences in mucosal immune system development in the small intestine of neonatal calves.

PubMed

Liang, Guanxiang; Malmuthuge, Nilusha; Bao, Hua; Stothard, Paul; Griebel, Philip J; Guan, Le Luo

2016-08-11

Postnatal development of the mammalian mucosal immune system is crucial for responding to the rapid colonization by commensal bacteria and possible exposure to pathogens. This study analyzed expression patterns for mRNAs and their relationship with microRNAs (miRNAs) in the bovine small intestine during the critical neonatal period (0 to 42 days). This analysis revealed molecular mechanisms regulating the postnatal development of the intestinal mucosal immune system. Small intestine samples (jejunum and ileum) were collected from newborn male, Holstein calves immediately post-partum (n = 3) and at 7 (n = 5), 21 (n = 5), and 42 (n = 5) days of age and the transcriptomes were profiled using RNA-Seq. When analyzing all time points collectively, greater expression of genes encoding the complement functional pathway, as well as lower expression of genes encoding Toll-like receptors and NOD-like receptors were observed in the jejunum when compared to the ileum. In addition, significant changes in the expression of immune-related genes were detected within the first week post-partum in both jejunum and ileum. For example, increased expression of genes encoding tight junction proteins (claudin 1, claudin 4 and occludin), an antimicrobial peptide (Regenerating Islet-Derived 3-γ), NOD-like receptors (NACHT, LRR and PYD domain-containing protein 3), regulatory T cell marker (forkhead box P3), and both anti-inflammatory (interleukin 10) and pro-inflammatory (interleukin 8) cytokines was observed throughout the small intestine of 7-day-old calves when compared to newborn calves. Moreover, the expression of mucosal immune-related genes were either positively or negatively correlated with total bacterial population depending on both intestinal region and age. The integrated analysis of miRNAs and mRNAs supported the conclusion that miRNAs may regulate temporal changes in the expression of genes encoding tight junction proteins (miR-335), cytokines (miR-335) and bacterial recognition (miR-100) during the first week of small intestine development. The rapid development of transcriptional differences between jejunum and ileum reveal that these two intestinal regions make distinct contributions to the intestinal mucosal immune system during the early neonatal period. In addition, transcriptome analysis indicates that the first week after birth is a very dynamic developmental period for the intestinal mucosal immune system and these changes may be regulated by both miRNAs and microbial colonization. Findings from this study indicate that a detailed analysis of both the abundance and diversity of the colonizing microbiome may be necessary to understand factors regulating the rapid development of the mucosal immune system during the first week of life.

Exposure to febrile-range hyperthermia potentiates Wnt signalling and epithelial-mesenchymal transition gene expression in lung epithelium.

PubMed

Potla, Ratnakar; Tulapurkar, Mohan E; Luzina, Irina G; Atamas, Sergei P; Singh, Ishwar S; Hasday, Jeffrey D

2018-02-01

As environmental and body temperatures vary, lung epithelial cells experience temperatures significantly different from normal core temperature. Our previous studies in human lung epithelium showed that: (i) heat shock accelerates wound healing and activates profibrotic gene expression through heat shock factor-1 (HSF1); (ii) HSF1 is activated at febrile temperatures (38-41 °C) and (iii) hypothermia (32 °C) activates and hyperthermia (39.5 °C) reduces expression of a subset of miRNAs that target protein kinase-Cα (PKCα) and enhance proliferation. We analysed the effect of hypo- and hyperthermia exposure on Wnt signalling by exposing human small airway epithelial cells (SAECs) and HEK293T cells to 32, 37 or 39.5 °C for 24 h, then analysing Wnt-3a-induced epithelial-mesenchymal transition (EMT) gene expression by qRT-PCR and TOPFlash reporter plasmid activity. Effects of miRNA mimics and inhibitors and the HSF1 inhibitor, KNK437, were evaluated. Exposure to 39.5 °C for 24 h increased subsequent Wnt-3a-induced EMT gene expression in SAECs and Wnt-3a-induced TOPFlash activity in HEK293T cells. Increased Wnt responsiveness was associated with HSF1 activation and blocked by KNK437. Overexpressing temperature-responsive miRNA mimics reduced Wnt responsiveness in 39.5 °C-exposed HEK293T cells, but inhibitors of the same miRNAs failed to restore Wnt responsiveness in 32 °C-exposed HEK293T cells. Wnt responsiveness, including expression of genes associated with EMT, increases after exposure to febrile-range temperature through an HSF1-dependent mechanism that is independent of previously identified temperature-dependent miRNAs. This process may be relevant to febrile fibrosing lung diseases, including the fibroproliferative phase of acute respiratory distress syndrome (ARDS) and exacerbations of idiopathic pulmonary fibrosis (IPF).

Differential microRNA expression is associated with androgen receptor expression in breast cancer.

PubMed

Shi, Yaqin; Yang, Fang; Sun, Zijia; Zhang, Wenwen; Gu, Jun; Guan, Xiaoxiang

2017-01-01

The androgen receptor (AR) is frequently expressed in breast cancer; however, its prognostic value remains unclear. AR expression in breast cancer has been associated with improved outcomes in estrogen receptor (ER)‑positive breast cancer compared with ER‑negative disease. Eliminating AR function in breast cancer is critically important for breast cancer progression. However, the mechanism underlying AR regulation remains poorly understood. The study of microRNAs (miRNAs) has provided important insights into the pathogenesis of hormone‑dependent cancer. To determine whether miRNAs function in the AR regulation of breast cancer, the present study performed miRNA expression profiling in AR‑positive and ‑negative breast cancer cell lines. A total of 153 miRNAs were differentially expressed in AR‑positive compared with AR‑negative breast cancer cells; 52 were upregulated and 101 were downregulated. A number of these have been extensively associated with breast cancer cell functions, including proliferation, invasion and drug‑resistance. Furthermore, through pathway enrichment analysis, signaling pathways associated with the prediction targets of the miRNAs were characterized, including the vascular endothelial growth factor and mammalian target of rapamycin signaling pathways. In conclusion, the results of the present study indicated that the expression of miRNAs may be involved in the mechanism underlying AR regulation of breast cancer, and may improve understanding of the role of AR in breast cancer.

The human circulating miRNome reflects multiple organ disease risks in association with short-term exposure to traffic-related air pollution.

PubMed

Krauskopf, Julian; Caiment, Florian; van Veldhoven, Karin; Chadeau-Hyam, Marc; Sinharay, Rudy; Chung, Kian Fan; Cullinan, Paul; Collins, Peter; Barratt, Benjamin; Kelly, Frank J; Vermeulen, Roel; Vineis, Paolo; de Kok, Theo M; Kleinjans, Jos C

2018-04-01

Traffic-related air pollution is a complex mixture of particulate matter (PM) and gaseous pollutants, such as nitrogen dioxide (NO2). PM exposure contributes to the pathogenesis of many diseases including several types of cancer, as well as pulmonary, cardiovascular and neurodegenerative diseases. Also exposure to NO2 has been related to increased cardiovascular mortality. In search of an early diagnostic biomarker for improved air pollution-associated health risk assessment, recent human studies have shown that certain circulating miRNAs are altered upon exposure to traffic-related air pollutants. Here, we present for the first time a global analysis of the circulating miRNA genome in an experimental cross-over study of a human population exposed to traffic-related air pollution. By utilizing next-generation sequencing technology and detailed real-time exposure measurements we identified 54 circulating miRNAs to be dose- and pollutant species-dependently associated with PM10, PM2.5, black carbon, ultrafine particles and NO2 already after 2 h of exposure. Bioinformatics analysis suggests that these circulating miRNAs actually reflect the adverse consequences of traffic pollution-induced toxicity in target tissues including the lung, heart, kidney and brain. This study shows the strong potential of circulating miRNAs as novel biomarkers for environmental health risk assessment. Copyright © 2018 Elsevier Ltd. All rights reserved.

3,3′-Diindolylmethane Ameliorates Staphylococcal Enterotoxin B–Induced Acute Lung Injury through Alterations in the Expression of MicroRNA that Target Apoptosis and Cell-Cycle Arrest in Activated T Cells

PubMed Central

Elliott, David M.; Nagarkatti, Mitzi

2016-01-01

3,3′-Diindolylmethane (DIM), a natural indole found in cruciferous vegetables, has significant anti-cancer and anti-inflammatory properties. In this current study, we investigated the effects of DIM on acute lung injury (ALI) induced by exposure to staphylococcal enterotoxin B (SEB). We found that pretreatment of mice with DIM led to attenuation of SEB-induced inflammation in the lungs, vascular leak, and IFN-γ secretion. Additionally, DIM could induce cell-cycle arrest and cell death in SEB-activated T cells in a concentration-dependent manner. Interestingly, microRNA (miRNA) microarray analysis uncovered an altered miRNA profile in lung-infiltrating mononuclear cells after DIM treatment of SEB-exposed mice. Moreover, computational analysis of miRNA gene targets and regulation networks indicated that DIM alters miRNA in the cell death and cell-cycle progression pathways. Specifically, DIM treatment significantly downregulated several miRNA and a correlative increase associated gene targets. Furthermore, overexpression and inhibition studies demonstrated that DIM-induced cell death, at least in part, used miR-222. Collectively, these studies demonstrate for the first time that DIM treatment attenuates SEB-induced ALI and may do so through the induction of microRNAs that promote apoptosis and cell-cycle arrest in SEB-activated T cells. PMID:26818958

Insulin-like growth factor-1 receptor is regulated by microRNA-133 during skeletal myogenesis.

PubMed

Huang, Mian-Bo; Xu, Hui; Xie, Shu-Juan; Zhou, Hui; Qu, Liang-Hu

2011-01-01

The insulin-like growth factor (IGF) signaling pathway has long been established as playing critical roles in skeletal muscle development. However, the underlying regulatory mechanism is poorly understood. Recently, a large family of small RNAs, named microRNAs (miRNAs), has been identified as key regulators for many developmental processes. Because miRNAs participate in the regulation of various signaling pathways, we hypothesized that miRNAs may be involved in the regulation of IGF signaling in skeletal myogenesis. In the present study, we determined that the cell-surface receptor IGF-1R is directly regulated by a muscle-specific miRNA, microRNA-133 (miR-133). A conserved and functional binding site for miR-133 was identified in the 3'untranslated region (3'UTR) of IGF-1R. During differentiation of C2C12 myoblasts, IGF-1R protein, but not messenger RNA (mRNA) expression, was gradually reduced, concurrent with the upregulation of miR-133. Overexpression of miR-133 in C2C12 cells significantly suppressed IGF-1R expression at the posttranscriptional level. We also demonstrated that both overexpression of miR-133 and knockdown of IGF-1R downregulated the phosphorylation of Akt, the central mediator of the PI3K/Akt signaling pathway. Furthermore, upregulation of miR-133 during C2C12 differentiation was significantly accelerated by the addition of IGF-1. Mechanistically, we found that the expression of myogenin, a myogenic transcription factor reported to transactivate miR-133, was increased by IGF-1 stimulation. Our results elucidate a negative feedback circuit in which IGF-1-stimulated miR-133 in turn represses IGF-1R expression to modulate the IGF-1R signaling pathway during skeletal myogenesis. These findings also suggest that miR-133 may be a potential therapeutic target in muscle diseases.

Insulin-Like Growth Factor-1 Receptor Is Regulated by microRNA-133 during Skeletal Myogenesis

PubMed Central

Huang, Mian-Bo; Xu, Hui; Xie, Shu-Juan; Zhou, Hui; Qu, Liang-Hu

2011-01-01

Background The insulin-like growth factor (IGF) signaling pathway has long been established as playing critical roles in skeletal muscle development. However, the underlying regulatory mechanism is poorly understood. Recently, a large family of small RNAs, named microRNAs (miRNAs), has been identified as key regulators for many developmental processes. Because miRNAs participate in the regulation of various signaling pathways, we hypothesized that miRNAs may be involved in the regulation of IGF signaling in skeletal myogenesis. Methodology/Principal Findings In the present study, we determined that the cell-surface receptor IGF-1R is directly regulated by a muscle-specific miRNA, microRNA-133 (miR-133). A conserved and functional binding site for miR-133 was identified in the 3′untranslated region (3′UTR) of IGF-1R. During differentiation of C2C12 myoblasts, IGF-1R protein, but not messenger RNA (mRNA) expression, was gradually reduced, concurrent with the upregulation of miR-133. Overexpression of miR-133 in C2C12 cells significantly suppressed IGF-1R expression at the posttranscriptional level. We also demonstrated that both overexpression of miR-133 and knockdown of IGF-1R downregulated the phosphorylation of Akt, the central mediator of the PI3K/Akt signaling pathway. Furthermore, upregulation of miR-133 during C2C12 differentiation was significantly accelerated by the addition of IGF-1. Mechanistically, we found that the expression of myogenin, a myogenic transcription factor reported to transactivate miR-133, was increased by IGF-1 stimulation. Conclusion/Significance Our results elucidate a negative feedback circuit in which IGF-1-stimulated miR-133 in turn represses IGF-1R expression to modulate the IGF-1R signaling pathway during skeletal myogenesis. These findings also suggest that miR-133 may be a potential therapeutic target in muscle diseases. PMID:22195016

Uncovering Small RNA-Mediated Responses to Cold Stress in a Wheat Thermosensitive Genic Male-Sterile Line by Deep Sequencing1[W][OA

PubMed Central

Tang, Zhonghui; Zhang, Liping; Xu, Chenguang; Yuan, Shaohua; Zhang, Fengting; Zheng, Yonglian; Zhao, Changping

2012-01-01

The male sterility of thermosensitive genic male sterile (TGMS) lines of wheat (Triticum aestivum) is strictly controlled by temperature. The early phase of anther development is especially susceptible to cold stress. MicroRNAs (miRNAs) play an important role in plant development and in responses to environmental stress. In this study, deep sequencing of small RNA (smRNA) libraries obtained from spike tissues of the TGMS line under cold and control conditions identified a total of 78 unique miRNA sequences from 30 families and trans-acting small interfering RNAs (tasiRNAs) derived from two TAS3 genes. To identify smRNA targets in the wheat TGMS line, we applied the degradome sequencing method, which globally and directly identifies the remnants of smRNA-directed target cleavage. We identified 26 targets of 16 miRNA families and three targets of tasiRNAs. Comparing smRNA sequencing data sets and TaqMan quantitative polymerase chain reaction results, we identified six miRNAs and one tasiRNA (tasiRNA-ARF [for Auxin-Responsive Factor]) as cold stress-responsive smRNAs in spike tissues of the TGMS line. We also determined the expression profiles of target genes that encode transcription factors in response to cold stress. Interestingly, the expression of cold stress-responsive smRNAs integrated in the auxin-signaling pathway and their target genes was largely noncorrelated. We investigated the tissue-specific expression of smRNAs using a tissue microarray approach. Our data indicated that miR167 and tasiRNA-ARF play roles in regulating the auxin-signaling pathway and possibly in the developmental response to cold stress. These data provide evidence that smRNA regulatory pathways are linked with male sterility in the TGMS line during cold stress. PMID:22508932

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.

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

Identifying optimal reference genes for the normalization of microRNA expression in cucumber under viral stress

PubMed Central

Liang, Chaoqiong; Hao, Jianjun; Meng, Yan; Luo, Laixin; Li, Jianqiang

2018-01-01

Cucumber green mottle mosaic virus (CGMMV) is an economically important pathogen and causes significant reduction of both yield and quality of cucumber (Cucumis sativus). Currently, there were no satisfied strategies for controlling the disease. A better understanding of microRNA (miRNA) expression related to the regulation of plant-virus interactions and virus resistance would be of great assistance when developing control strategies for CGMMV. However, accurate expression analysis is highly dependent on robust and reliable reference gene used as an internal control for normalization of miRNA expression. Most commonly used reference genes involved in CGMMV-infected cucumber are not universally expressed depending on tissue types and stages of plant development. It is therefore crucial to identify suitable reference genes in investigating the role of miRNA expression. In this study, seven reference genes, including Actin, Tubulin, EF-1α, 18S rRNA, Ubiquitin, GAPDH and Cyclophilin, were evaluated for the most accurate results in analyses using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Gene expression was assayed on cucumber leaves, stems and roots that were collected at different days post inoculation with CGMMV. The expression data were analyzed using algorithms including delta-Ct, geNorm, NormFinder, and BestKeeper as well as the comparative tool RefFinder. The reference genes were subsequently validated using miR159. The results showed that EF-1α and GAPDH were the most reliable reference genes for normalizing miRNA expression in leaf, root and stem samples, while Ubiquitin and EF-1α were the most suitable combination overall. PMID:29543906

Density-Dependent Regulation of Glioma Cell Proliferation and Invasion Mediated by miR-9.

PubMed

Katakowski, Mark; Charteris, Nicholas; Chopp, Michael; Khain, Evgeniy

2016-12-01

The phenotypic axis of invasion and proliferation in malignant glioma cells is a well-documented phenomenon. Invasive glioma cells exhibit a decreased proliferation rate and a resistance to apoptosis, and invasive tumor cells dispersed in brain subsequently revert to proliferation and contribute to secondary tumor formation. One miRNA can affect dozens of mRNAs, and some miRNAs are potent oncogenes. Multiple miRNAs are implicated in glioma malignancy, and several of which have been identified to regulate tumor cell motility and division. Using rat 9 L gliosarcoma and human U87 glioblastoma cell lines, we investigated miRNAs associated with the switch between glioma cell invasion and proliferation. Using micro-dissection of 9 L glioma tumor xenografts in rat brain, we identified disparate expression of miR-9 between cells within the periphery of the primary tumor, and those comprising tumor islets within the invasive zone. Modifying miR-9 expression in in vitro assays, we report that miR-9 controls the axis of glioma cell invasion/proliferation, and that its contribution to invasion or proliferation is biphasic and dependent upon local tumor cell density. In addition, immunohistochemistry revealed elevated hypoxia inducible factor 1 alpha (HIF-1α) in the invasive zone as compared to the primary tumor periphery. We also found that hypoxia promotes miR-9 expression in glioma cells. Based upon these findings, we propose a hypothesis for the contribution of miR-9 to the dynamics glioma invasion and satellite tumor formation in brain adjacent to tumor.

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

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.

Parameter optimization for constructing competing endogenous RNA regulatory network in glioblastoma multiforme and other cancers.

PubMed

Chiu, Yu-Chiao; Hsiao, Tzu-Hung; Chen, Yidong; Chuang, Eric Y

2015-01-01

In addition to direct targeting and repressing mRNAs, recent studies reported that microRNAs (miRNAs) can bridge up an alternative layer of post-transcriptional gene regulatory networks. The competing endogenous RNA (ceRNA) regulation depicts the scenario where pairs of genes (ceRNAs) sharing, fully or partially, common binding miRNAs (miRNA program) can establish coexpression through competition for a limited pool of the miRNA program. While the dynamics of ceRNA regulation among cellular conditions have been verified based on in silico and in vitro experiments, comprehensive investigation into the strength of ceRNA regulation in human datasets remains largely unexplored. Furthermore, pan-cancer analysis of ceRNA regulation, to our knowledge, has not been systematically investigated. In the present study we explored optimal conditions for ceRNA regulation, investigated functions governed by ceRNA regulation, and evaluated pan-cancer effects. We started by investigating how essential factors, such as the size of miRNA programs, the number of miRNA program binding sites, and expression levels of miRNA programs and ceRNAs affect the ceRNA regulation capacity in tumors derived from glioblastoma multiforme patients captured by The Cancer Genome Atlas (TCGA). We demonstrated that increased numbers of common targeting miRNAs as well as the abundance of binding sites enhance ceRNA regulation and strengthen coexpression of ceRNA pairs. Also, our investigation revealed that the strength of ceRNA regulation is dependent on expression levels of both miRNA programs and ceRNAs. Through functional annotation analysis, our results indicated that ceRNA regulation is highly associated with essential cellular functions and diseases including cancer. Furthermore, the highly intertwined ceRNA regulatory relationship enables constitutive and effective intra-function regulation of genes in diverse types of cancer. Using gene and microRNA expression datasets from TCGA, we successfully quantified the optimal conditions for ceRNA regulation, which hinge on four essential parameters of ceRNAs. Our analysis suggests optimized ceRNA regulation is related to disease pathways and essential cellular functions. Furthermore, although the strength of ceRNA regulation is dynamic among cancers, its governing functions are stably maintained. The findings of this report contribute to better understanding of ceRNA dynamics and its crucial roles in cancers.

Milk: an epigenetic amplifier of FTO-mediated transcription? Implications for Western diseases.

PubMed

Melnik, Bodo C

2015-12-21

Single-nucleotide polymorphisms within intron 1 of the FTO (fat mass and obesity-associated) gene are associated with enhanced FTO expression, increased body weight, obesity and type 2 diabetes mellitus (T2DM). The N (6) -methyladenosine (m(6)A) demethylase FTO plays a pivotal regulatory role for postnatal growth and energy expenditure. The purpose of this review is to provide translational evidence that links milk signaling with FTO-activated transcription of the milk recipient. FTO-dependent demethylation of m(6)A regulates mRNA splicing required for adipogenesis, increases the stability of mRNAs, and affects microRNA (miRNA) expression and miRNA biosynthesis. FTO senses branched-chain amino acids (BCAAs) and activates the nutrient sensitive kinase mechanistic target of rapamycin complex 1 (mTORC1), which plays a key role in translation. Milk provides abundant BCAAs and glutamine, critical components increasing FTO expression. CpG hypomethylation in the first intron of FTO has recently been associated with T2DM. CpG methylation is generally associated with gene silencing. In contrast, CpG demethylation generally increases transcription. DNA de novo methylation of CpG sites is facilitated by DNA methyltransferases (DNMT) 3A and 3B, whereas DNA maintenance methylation is controlled by DNMT1. MiRNA-29s target all DNMTs and thus reduce DNA CpG methylation. Cow´s milk provides substantial amounts of exosomal miRNA-29s that reach the systemic circulation and target mRNAs of the milk recipient. Via DNMT suppression, milk exosomal miRNA-29s may reduce the magnitude of FTO methylation, thereby epigenetically increasing FTO expression in the milk consumer. High lactation performance with increased milk yield has recently been associated with excessive miRNA-29 expression of dairy cow mammary epithelial cells (DCMECs). Notably, the galactopoietic hormone prolactin upregulates the transcription factor STAT3, which induces miRNA-29 expression. In a retrovirus-like manner milk exosomes may transfer DCMEC-derived miRNA-29s and bovine FTO mRNA to the milk consumer amplifying FTO expression. There is compelling evidence that obesity, T2DM, prostate and breast cancer, and neurodegenerative diseases are all associated with increased FTO expression. Maximization of lactation performance by veterinary medicine with enhanced miRNA-29s and FTO expression associated with increased exosomal miRNA-29 and FTO mRNA transfer to the milk consumer may represent key epigenetic mechanisms promoting FTO/mTORC1-mediated diseases of civilization.

MicroRNA-122 regulates polyploidization in the murine liver.

PubMed

Hsu, Shu-Hao; Delgado, Evan R; Otero, P Anthony; Teng, Kun-Yu; Kutay, Huban; Meehan, Kolin M; Moroney, Justin B; Monga, Jappmann K; Hand, Nicholas J; Friedman, Joshua R; Ghoshal, Kalpana; Duncan, Andrew W

2016-08-01

A defining feature of the mammalian liver is polyploidy, a numerical change in the entire complement of chromosomes. The first step of polyploidization involves cell division with failed cytokinesis. Although polyploidy is common, affecting ∼90% of hepatocytes in mice and 50% in humans, the specialized role played by polyploid cells in liver homeostasis and disease remains poorly understood. The goal of this study was to identify novel signals that regulate polyploidization, and we focused on microRNAs (miRNAs). First, to test whether miRNAs could regulate hepatic polyploidy, we examined livers from Dicer1 liver-specific knockout mice, which are devoid of mature miRNAs. Loss of miRNAs resulted in a 3-fold reduction in binucleate hepatocytes, indicating that miRNAs regulate polyploidization. Second, we surveyed age-dependent expression of miRNAs in wild-type mice and identified a subset of miRNAs, including miR-122, that is differentially expressed at 2-3 weeks, a period when extensive polyploidization occurs. Next, we examined Mir122 knockout mice and observed profound, lifelong depletion of polyploid hepatocytes, proving that miR-122 is required for complete hepatic polyploidization. Moreover, the polyploidy defect in Mir122 knockout mice was ameliorated by adenovirus-mediated overexpression of miR-122, underscoring the critical role miR-122 plays in polyploidization. Finally, we identified direct targets of miR-122 (Cux1, Rhoa, Iqgap1, Mapre1, Nedd4l, and Slc25a34) that regulate cytokinesis. Inhibition of each target induced cytokinesis failure and promoted hepatic binucleation. Among the different signals that have been associated with hepatic polyploidy, miR-122 is the first liver-specific signal identified; our data demonstrate that miR-122 is both necessary and sufficient in liver polyploidization, and these studies will serve as the foundation for future work investigating miR-122 in liver maturation, homeostasis, and disease. (Hepatology 2016;64:599-615). © 2016 by the American Association for the Study of Liver Diseases.

MicroRNA-122 Regulates Polyploidization in the Murine Liver

PubMed Central

Hsu, Shu-hao; Delgado, Evan R.; Otero, P. Anthony; Teng, Kun-yu; Kutay, Huban; Meehan, Kolin M.; Moroney, Justin B.; Monga, Jappmann K.; Hand, Nicholas J.; Friedman, Joshua R.; Ghoshal, Kalpana; Duncan, Andrew W.

2016-01-01

A defining feature of the mammalian liver is polyploidy, a numerical change in the entire complement of chromosomes. The first step of polyploidization involves cell division with failed cytokinesis. Although polyploidy is common, affecting ~90% of hepatocytes in mice and 50% in humans, the specialized role played by polyploid cells in liver homeostasis and disease remains poorly understood. The goal of this study was to identify novel signals that regulate polyploidization, and we focused on microRNAs (miRNAs). First, to test whether miRNAs could regulate hepatic polyploidy we examined livers from Dicer1 liver-specific knockout mice, which are devoid of mature miRNAs. Loss of miRNAs resulted in a 3-fold reduction in binucleate hepatocytes, indicating that miRNAs regulate polyploidization. Secondly, we surveyed age-dependent expression of miRNAs in wild-type mice and identified a subset of miRNAs, including miR-122, that is differentially expressed at 2–3 weeks, a period when extensive polyploidization occurs. Next, we examined Mir122 knockout mice and observed profound, life-long depletion of polyploid hepatocytes, proving that miR-122 is required for complete hepatic polyploidization. Moreover, the polyploidy defect in Mir122 knockout mice was ameliorated by adenovirus-mediated over-expression of miR-122, underscoring the critical role miR-122 plays in polyploidization. Finally, we identified direct targets of miR-122 (Cux1, Rhoa, Iqgap1, Mapre1, Nedd4l and Slc25a34) that regulate cytokinesis. Inhibition of each target induced cytokinesis failure and promoted hepatic binucleation. Conclusion Our data demonstrate that miR-122 is both necessary and sufficient in liver polyploidization. Among the different signals that have been associated with hepatic polyploidy, miR-122 is the first liver-specific signal identified. These studies will serve as the foundation for future work investigating miR-122 in liver maturation, homeostasis and disease. PMID:27016325

miR-30-HNF4γ and miR-194-NR2F2 regulatory networks contribute to the up-regulation of metaplasia markers in the stomach

PubMed Central

Sousa, Josane F.; Nam, Ki Taek; Petersen, Christine P.; Lee, Hyuk-Joon; Yang, Han-Kwang; Kim, Woo Ho; Goldenring, James R.

2016-01-01

Objective Intestinal metaplasia and spasmolytic polypeptide-expressing metaplasia (SPEM) are considered neoplastic precursors of gastric adenocarcinoma and are both marked by gene expression alterations in comparison to normal stomach. Since miRNAs are important regulators of gene expression, we sought to investigate the role of miRNAs on the development of stomach metaplasias. Design We performed miRNA profiling using a qRT-PCR approach on laser capture microdissected human intestinal metaplasia and SPEM. Data integration of the miRNA profile with a previous mRNA profile from the same samples was performed to detect potential miRNA-mRNA regulatory circuits. Transfection of gastric cancer cell lines with selected miRNA mimics and inhibitors was used to evaluate their effects on the expression of putative targets and additional metaplasia markers. Results We identified several genes as potential targets of miRNAs altered during metaplasia progression. We showed evidence that HNF4γ (upregulated in intestinal metaplasia) is targeted by miR-30 and that miR-194 targets a known co-regulator of HNF4 activity, NR2F2 (downregulated in intestinal metaplasia). Intestinal metaplasia markers such as VIL1, TFF2 and TFF3 were down-regulated after overexpression of miR-30a in a HNF4γ-dependent manner. In addition, overexpression of HNF4γ was sufficient to induce the expression of VIL1 and this effect was potentiated by down-regulation of NR2F2. Conclusion The interplay of the two transcription factors HNF4γ and NR2F2 and their coordinate regulation by miR-30 and miR-194, respectively, represent a miRNA to transcription factor network responsible for the expression of intestinal transcripts in stomach cell lineages during the development of intestinal metaplasia. PMID:25800782

Magnetic Beads-Based Sensor with Tailored Sensitivity for Rapid and Single-Step Amperometric Determination of miRNAs.

PubMed

Vargas, Eva; Torrente-Rodríguez, Rebeca M; Ruiz-Valdepeñas Montiel, Víctor; Povedano, Eloy; Pedrero, María; Montoya, Juan J; Campuzano, Susana; Pingarrón, José M

2017-11-09

This work describes a sensitive amperometric magneto-biosensor for single-step and rapid determination of microRNAs (miRNAs). The developed strategy involves the use of direct hybridization of the target miRNA (miRNA-21) with a specific biotinylated DNA probe immobilized on streptavidin-modified magnetic beads (MBs), and labeling of the resulting heteroduplexes with a specific DNA-RNA antibody and the bacterial protein A (ProtA) conjugated with an horseradish peroxidase (HRP) homopolymer (Poly-HRP40) as an enzymatic label for signal amplification. Amperometric detection is performed upon magnetic capture of the modified MBs onto the working electrode surface of disposable screen-printed carbon electrodes (SPCEs) using the H₂O₂/hydroquinone (HQ) system. The magnitude of the cathodic signal obtained at -0.20 V (vs. the Ag pseudo-reference electrode) demonstrated linear dependence with the concentration of the synthetic target miRNA over the 1.0 to 100 pM range. The method provided a detection limit (LOD) of 10 attomoles (in a 25 μL sample) without any target miRNA amplification in just 30 min (once the DNA capture probe-MBs were prepared). This approach shows improved sensitivity compared with that of biosensors constructed with the same anti-DNA-RNA Ab as capture instead of a detector antibody and further labeling with a Strep-HRP conjugate instead of the Poly-HRP40 homopolymer. The developed strategy involves a single step working protocol, as well as the possibility to tailor the sensitivity by enlarging the length of the DNA/miRNA heteroduplexes using additional probes and/or performing the labelling with ProtA conjugated with homopolymers prepared with different numbers of HRP molecules. The practical usefulness was demonstrated by determination of the endogenous levels of the mature target miRNA in 250 ng raw total RNA (RNA t ) extracted from human mammary epithelial normal (MCF-10A) and cancer (MCF-7) cells and tumor tissues.

miR-125b promotes MLL-AF9–driven murine acute myeloid leukemia involving a VEGFA-mediated non–cell-intrinsic mechanism

PubMed Central

Liu, Jun; Guo, Bo; Chen, Zhuo; Wang, Nayi; Iacovino, Michelina; Cheng, Jijun; Roden, Christine; Pan, Wen; Khan, Sajid; Chen, Suning; Kyba, Michael; Fan, Rong; Guo, Shangqin

2017-01-01

The hematopoietic stem cell–enriched miR-125 family microRNAs (miRNAs) are critical regulators of hematopoiesis. Overexpression of miR-125a or miR-125b is frequent in human acute myeloid leukemia (AML), and the overexpression of these miRNAs in mice leads to expansion of hematopoietic stem cells accompanied by perturbed hematopoiesis with mostly myeloproliferative phenotypes. However, whether and how miR-125 family miRNAs cooperate with known AML oncogenes in vivo, and how the resultant leukemia is dependent on miR-125 overexpression, are not well understood. We modeled the frequent co-occurrence of miR-125b overexpression and MLL translocations by examining functional cooperation between miR-125b and MLL-AF9. By generating a knock-in mouse model in which miR-125b overexpression is controlled by doxycycline induction, we demonstrated that miR-125b significantly enhances MLL-AF9–driven AML in vivo, and the resultant leukemia is partially dependent on continued overexpression of miR-125b. Surprisingly, miR-125b promotes AML cell expansion and suppresses apoptosis involving a non–cell-intrinsic mechanism. MiR-125b expression enhances VEGFA expression and production from leukemia cells, in part by suppressing TET2. Recombinant VEGFA recapitulates the leukemia-promoting effects of miR-125b, whereas knockdown of VEGFA or inhibition of VEGF receptor 2 abolishes the effects of miR-125b. In addition, significant correlation between miR-125b and VEGFA expression is observed in human AMLs. Our data reveal cooperative and dependent relationships between miR-125b and the MLL oncogene in AML leukemogenesis, and demonstrate a miR-125b-TET2-VEGFA pathway in mediating non–cell-intrinsic leukemia-promoting effects by an oncogenic miRNA. PMID:28053194

MiR-21 plays an Important Role in Radiation Induced Carcinogenesis in BALB/c Mice by Directly Targeting the Tumor Suppressor Gene Big-h3

PubMed Central

Liu, Cong; Li, Bailong; Cheng, Ying; Lin, Jing; Hao, Jun; Zhang, Shuyu; Mitchel, R.E.J.; Sun, Ding; Ni, Jin; Zhao, Luqian; Gao, Fu; Cai, Jianming

2011-01-01

Dysregulation of certain microRNAs (miRNAs) in cancer can promote tumorigenesis, metastasis and invasion. However, the functions and targets of only a few mammalian miRNAs are known. In particular, the miRNAs that participates in radiation induced carcinogenesis and the miRNAs that target the tumor suppressor gene Big-h3 remain undefined. Here in this study, using a radiation induced thymic lymphoma model in BALB/c mice, we found that the tumor suppressor gene Big-h3 is down-regulated and miR-21 is up-regulated in radiation induced thymic lymphoma tissue samples. We also found inverse correlations between Big-h3 protein and miR-21 expression level among different tissue samples. Furthermore, our data indicated that miR-21 could directly target Big-h3 in a 3′UTR dependent manner. Finally, we found that miR-21 could be induced by TGFβ, and miR-21 has both positive and negative effects in regulating TGFβ signaling. We conclude that miR-21 participates in radiation induced carcinogenesis and it regulates TGFβ signaling. PMID:21494432

Comprehensive Analysis of miRNome Alterations in Response to Sorafenib Treatment in Colorectal Cancer Cells

PubMed Central

Pehserl, Anna-Maria; Ress, Anna Lena; Stanzer, Stefanie; Resel, Margit; Karbiener, Michael; Stadelmeyer, Elke; Stiegelbauer, Verena; Gerger, Armin; Mayr, Christian; Scheideler, Marcel; Hutterer, Georg C.; Bauernhofer, Thomas; Kiesslich, Tobias; Pichler, Martin

2016-01-01

MicroRNAs (miRNAs) are master regulators of drug resistance and have been previously proposed as potential biomarkers for the prediction of therapeutic response in colorectal cancer (CRC). Sorafenib, a multi-kinase inhibitor which has been approved for the treatment of liver, renal and thyroid cancer, is currently being studied as a monotherapy in selected molecular subtypes or in combination with other drugs in metastatic CRC. In this study, we explored sorafenib-induced cellular effects in Kirsten rat sarcoma viral oncogene homolog olog (KRAS) wild-type and KRAS-mutated CRC cell lines (Caco-2 and HRT-18), and finally profiled expression changes of specific miRNAs within the miRNome (>1000 human miRNAs) after exposure to sorafenib. Overall, sorafenib induced a time- and dose-dependent growth-inhibitory effect through S-phase cell cycle arrest in KRAS wild-type and KRAS-mutated CRC cells. In HRT-18 cells, two human miRNAs (hsa-miR-597 and hsa-miR-720) and two small RNAs (SNORD 13 and hsa-miR-3182) were identified as specifically sorafenib-induced. In Caco-2 cells, nine human miRNAs (hsa-miR-3142, hsa-miR-20a, hsa-miR-4301, hsa-miR-1290, hsa-miR-4286, hsa-miR-3182, hsa-miR-3142, hsa-miR-1246 and hsa-miR-720) were identified to be differentially regulated post sorafenib treatment. In conclusion, we confirmed sorafenib as a potential anti-neoplastic treatment strategy for CRC cells by demonstrating a growth-inhibitory and cell cycle–arresting effect of this drug. Changes in the miRNome indicate that some specific miRNAs might be relevant as indicators for sorafenib response, drug resistance and potential targets for combinatorial miRNA-based drug strategies. PMID:27916938

Modulators of the microRNA biogenesis pathway via arrayed lentiviral enabled RNAi screening for drug and biomarker discovery

PubMed Central

Shum, David; Bhinder, Bhavneet; Djaballah, Hakim

2013-01-01

MicroRNAs (miRNAs) are small endogenous and conserved non-coding RNA molecules that regulate gene expression. Although the first miRNA was discovered well over sixteen years ago, little is known about their biogenesis and it is only recently that we have begun to understand their scope and diversity. For this purpose, we performed an RNAi screen aimed at identifying genes involved in their biogenesis pathway with a potential use as biomarkers. Using a previously developed miRNA 21 (miR-21) EGFP-based biosensor cell based assay monitoring green fluorescence enhancements, we performed an arrayed short hairpin RNA (shRNA) screen against a lentiviral particle ready TRC1 library covering 16,039 genes in 384-well plate format, and interrogating the genome one gene at a time building a panoramic view of endogenous miRNA activity. Using the BDA method for RNAi data analysis, we nominate 497 gene candidates the knockdown of which increased the EGFP fluorescence and yielding an initial hit rate of 3.09%; of which only 22, with reported validated clones, are deemed high-confidence gene candidates. An unexpected and surprising result was that only DROSHA was identified as a hit out of the seven core essential miRNA biogenesis genes; suggesting that perhaps intracellular shRNA processing into the correct duplex may be cell dependent and with differential outcome. Biological classification revealed several major control junctions among them genes involved in transport and vesicular trafficking. In summary, we report on 22 high confidence gene candidate regulators of miRNA biogenesis with potential use in drug and biomarker discovery. PMID:23977983

Mechanism of gemcitabine-induced suppression of human cholangiocellular carcinoma cell growth.

PubMed

Toyota, Yuka; Iwama, Hisakazu; Kato, Kiyohito; Tani, Joji; Katsura, Akiko; Miyata, Miwa; Fujiwara, Shintaro; Fujita, Koji; Sakamoto, Teppei; Fujimori, Takayuki; Okura, Ryoichi; Kobayashi, Kiyoyuki; Tadokoro, Tomoko; Mimura, Shima; Nomura, Takako; Miyoshi, Hisaaki; Morishita, Asahiro; Kamada, Hideki; Yoneyama, Hirohito; Okano, Keiichi; Suzuki, Yasuyuki; Masaki, Tsutomu

2015-10-01

Although gemcitabine (2',2'-difluorocytidine monohydrochloride) is a common anticancer agent of cholangiocellular carcinoma (CCC), its growth inhibitory effects and gemcitabine resistance in CCC cells are poorly understood. Our aims were to uncover the mechanism underlying the antitumor effect of gemcitabine and to analyze the mechanism regulating in vitro CCC cell gemcitabine resistance. In addition, we sought to identify miRNAs associated with the antitumor effects of gemcitabine in CCCs. Using a cell proliferation assay and flow cytometry, we examined the ability of gemcitabine to inhibit cell proliferation in three types of human CCC cell lines (HuCCT-1, Huh28, TKKK). We also employed western blotting to investigate the effects of gemcitabine on cell cycle-related molecules in CCC cells. In addition, we used array chips to assess gemcitabine-mediated changes in angiogenic molecules and activated tyrosine kinase receptors in CCC cells. We used miRNA array chips to comprehensively analyze gemcitabine-induced miRNAs and examined clusters of differentially expressed miRNAs in cells with and without gemcitabine treatment. Gemcitabine inhibited cell proliferation in a dose- and time-dependent manner in HuCCT-1 cells, whereas cell proliferation was unchanged in Huh28 and TKKK cells. Gemcitabine inhibited cell cycle progression in HuCCT-1 cells from G0/G1 to S phase, resulting in G1 cell cycle arrest due to the reduction of cyclin D1 expression. In addition, gemcitabine upregulated the angiogenic molecules IL-6, IL-8, ENA-78 and MCP-1. In TKKK cells, by contrast, gemcitabine did not arrest the cell cycle or modify angiogenic molecules. Furthermore, in gemcitabine-sensitive HuCCT-1 cells, gemcitabine markedly altered miRNA expression. The miRNAs and angiogenic molecules altered by gemcitabine contribute to the inhibition of tumor growth in vitro.

DELAY OF GERMINATION1 (DOG1) regulates both seed dormancy and flowering time through microRNA pathways

PubMed Central

Huo, Heqiang; Wei, Shouhui; Bradford, Kent J.

2016-01-01

Seed germination and flowering, two critical developmental transitions in plant life cycles, are coordinately regulated by genetic and environmental factors to match plant establishment and reproduction to seasonal cues. The DELAY OF GERMINATION1 (DOG1) gene is involved in regulating seed dormancy in response to temperature and has also been associated genetically with pleiotropic flowering phenotypes across diverse Arabidopsis thaliana accessions and locations. Here we show that DOG1 can regulate seed dormancy and flowering times in lettuce (Lactuca sativa, Ls) and Arabidopsis through an influence on levels of microRNAs (miRNAs) miR156 and miR172. In lettuce, suppression of LsDOG1 expression enabled seed germination at high temperature and promoted early flowering in association with reduced miR156 and increased miR172 levels. In Arabidopsis, higher miR156 levels resulting from overexpression of the MIR156 gene enhanced seed dormancy and delayed flowering. These phenotypic effects, as well as conversion of MIR156 transcripts to miR156, were compromised in DOG1 loss-of-function mutant plants, especially in seeds. Overexpression of MIR172 reduced seed dormancy and promoted early flowering in Arabidopsis, and the effect on flowering required functional DOG1. Transcript levels of several genes associated with miRNA processing were consistently lower in dry seeds of Arabidopsis and lettuce when DOG1 was mutated or its expression was reduced; in contrast, transcript levels of these genes were elevated in a DOG1 gain-of-function mutant. Our results reveal a previously unknown linkage between two critical developmental phase transitions in the plant life cycle through a DOG1–miR156–miR172 interaction. PMID:27035986

Simultaneous inhibition of multiple oncogenic miRNAs by a multi-potent microRNA sponge.

PubMed

Jung, Jaeyun; Yeom, Chanjoo; Choi, Yeon-Sook; Kim, Sinae; Lee, EunJi; Park, Min Ji; Kang, Sang Wook; Kim, Sung Bae; Chang, Suhwan

2015-08-21

The roles of oncogenic miRNAs are widely recognized in many cancers. Inhibition of single miRNA using antagomiR can efficiently knock-down a specific miRNA. However, the effect is transient and often results in subtle phenotype, as there are other miRNAs contribute to tumorigenesis. Here we report a multi-potent miRNA sponge inhibiting multiple miRNAs simultaneously. As a model system, we targeted miR-21, miR-155 and miR-221/222, known as oncogenic miRNAs in multiple tumors including breast and pancreatic cancers. To achieve efficient knockdown, we generated perfect and bulged-matched miRNA binding sites (MBS) and introduced multiple copies of MBS, ranging from one to five, in the multi-potent miRNA sponge. Luciferase reporter assay showed the multi-potent miRNA sponge efficiently inhibited 4 miRNAs in breast and pancreatic cancer cells. Furthermore, a stable and inducible version of the multi-potent miRNA sponge cell line showed the miRNA sponge efficiently reduces the level of 4 target miRNAs and increase target protein level of these oncogenic miRNAs. Finally, we showed the miRNA sponge sensitize cells to cancer drug and attenuate cell migratory activity. Altogether, our study demonstrates the multi-potent miRNA sponge is a useful tool to examine the functional impact of simultaneous inhibition of multiple miRNAs and proposes a therapeutic potential.

Comparative Analysis of Argonaute-dependent Small RNA Pathways in Drosophila

PubMed Central

Zhou, Rui; Hotta, Ikuko; Denli, Ahmet M.; Hong, Pengyu; Perrimon, Norbert; Hannon, Gregory J.

2008-01-01

Summary The specificity of RNAi pathways is determined by several classes of small RNAs, which include siRNAs, piRNAs, endo-siRNAs, and microRNAs (miRNAs). These small RNAs are invariably incorporated into large Argonaute (Ago)-containing effector complexes known as RNA-induced silencing complexes (RISCs), which they guide to silencing targets. Both genetic and biochemical strategies have yielded conserved molecular components of small RNA biogenesis and effector machineries. However, given the complexity of these pathways, there are likely to be additional components and regulators that remain to be uncovered. We have undertaken a comparative and comprehensive RNAi screen to identify genes that impact three major Ago-dependent small RNA pathways that operate in Drosophila S2 cells. We identify subsets of candidates that act positively or negatively in siRNA, endo-siRNA and miRNA pathways. Our studies indicate that many components are shared among all three Argonaute-dependent silencing pathways, though each is also impacted by discrete sets of genes. PMID:19026789

TAM: a method for enrichment and depletion analysis of a microRNA category in a list of microRNAs.

PubMed

Lu, Ming; Shi, Bing; Wang, Juan; Cao, Qun; Cui, Qinghua

2010-08-09

MicroRNAs (miRNAs) are a class of important gene regulators. The number of identified miRNAs has been increasing dramatically in recent years. An emerging major challenge is the interpretation of the genome-scale miRNA datasets, including those derived from microarray and deep-sequencing. It is interesting and important to know the common rules or patterns behind a list of miRNAs, (i.e. the deregulated miRNAs resulted from an experiment of miRNA microarray or deep-sequencing). For the above purpose, this study presents a method and develops a tool (TAM) for annotations of meaningful human miRNAs categories. We first integrated miRNAs into various meaningful categories according to prior knowledge, such as miRNA family, miRNA cluster, miRNA function, miRNA associated diseases, and tissue specificity. Using TAM, given lists of miRNAs can be rapidly annotated and summarized according to the integrated miRNA categorical data. Moreover, given a list of miRNAs, TAM can be used to predict novel related miRNAs. Finally, we confirmed the usefulness and reliability of TAM by applying it to deregulated miRNAs in acute myocardial infarction (AMI) from two independent experiments. TAM can efficiently identify meaningful categories for given miRNAs. In addition, TAM can be used to identify novel miRNA biomarkers. TAM tool, source codes, and miRNA category data are freely available at http://cmbi.bjmu.edu.cn/tam.

Methylation and microRNA-mediated epigenetic regulation of SOCS3

PubMed Central

Boosani, Chandra S.; Agrawal, Devendra K.

2017-01-01

Epigenetic gene silencing of several genes causes different pathological conditions in humans, and DNA methylation has been identified as one of the key mechanisms that underlie this evolutionarily conserved phenomenon associated with developmental and pathological gene regulation. Recent advances in the miRNA technology with high throughput analysis of gene regulation further increased our understanding on the role of miRNAs regulating multiple gene expression. There is increasing evidence supporting that the miRNAs not only regulate gene expression but they also are involved in the hypermethylation of promoter sequences, which cumulatively contributes to the epigenetic gene silencing. Here, we critically evaluated the recent progress on the transcriptional regulation of an important suppressor protein that inhibits cytokine-mediated signaling, SOCS3, whose expression is directly regulated both by promoter methylation and also by microRNAs, affecting its vital cell regulating functions. SOCS3 was identified as a potent inhibitor of Jak/STAT signaling pathway which is frequently upregulated in several pathologies, including cardiovascular disease, cancer, diabetes, viral infections, and the expression of SOCS3 was inhibited or greatly reduced due to hypermethylation of the CpG islands in its promoter region or suppression of its expression by different microRNAs. Additionally, we discuss key intracellular signaling pathways regulated by SOCS3 involving cellular events, including cell proliferation, cell growth, cell migration and apoptosis. Identification of the pathway intermediates as specific targets would not only aid in the development of novel therapeutic drugs, but, would also assist in developing new treatment strategies that could successfully be employed in combination therapy to target multiple signaling pathways. PMID:25682267

MiRNA-Target Interaction Reveals Cell-Specific Post-Transcriptional Regulation in Mammalian Cell Lines

PubMed Central

Kulkarni, Varun; Naqvi, Afsar Raza; Uttamani, Juhi Raju; Nares, Salvador

2016-01-01

MicroRNAs are 18–22 nucleotides long, non-coding RNAs that bind transcripts with complementary sequences leading to either mRNA degradation or translational suppression. However, the inherent differences in preferred mode of miRNA regulation among cells of different origin have not been examined. In our previous transcriptome profiling studies, we observed that post-transcriptional regulation can differ substantially depending on the cell in context. Here we examined mechanistic differences in the regulation of a let-7a targeted (wild type) or resistant (mutant) engineered renilla transcript across various mammalian cell lines of diverse origin. Dual luciferase assays show that compared to mutant (mut), the reporter gene containing wild type (wt) let-7a binding sites was efficiently suppressed upon transfection in various cell lines. Importantly, the strength of miRNA regulation varied across the cell lines. Total RNA analysis demonstrates that wt renilla mRNA was expressed to similar or higher levels compared to mut suggesting that translation repression is a predominant mode of miRNA regulation. Nonetheless, transcript degradation was observed in some cell lines. Ago-2 immunoprecipitation show that miRNA repressed renilla mRNA are associated with functional mi-RISC (miRNA-RNA induced silencing complex). Given the immense potential of miRNA as a therapeutic option, these findings highlight the necessity to thoroughly examine the mode of mRNA regulation in order to achieve the beneficial effects in targeting cells. PMID:26761000

Characterization of full-length sequenced cDNA inserts (FLIcs) from Atlantic salmon (Salmo salar)

PubMed Central

Andreassen, Rune; Lunner, Sigbjørn; Høyheim, Bjørn

2009-01-01

Background Sequencing of the Atlantic salmon genome is now being planned by an international research consortium. Full-length sequenced inserts from cDNAs (FLIcs) are an important tool for correct annotation and clustering of the genomic sequence in any species. The large amount of highly similar duplicate sequences caused by the relatively recent genome duplication in the salmonid ancestor represents a particular challenge for the genome project. FLIcs will therefore be an extremely useful resource for the Atlantic salmon sequencing project. In addition to be helpful in order to distinguish between duplicate genome regions and in determining correct gene structures, FLIcs are an important resource for functional genomic studies and for investigation of regulatory elements controlling gene expression. In contrast to the large number of ESTs available, including the ESTs from 23 developmental and tissue specific cDNA libraries contributed by the Salmon Genome Project (SGP), the number of sequences where the full-length of the cDNA insert has been determined has been small. Results High quality full-length insert sequences from 560 pre-smolt white muscle tissue specific cDNAs were generated, accession numbers [GenBank: BT043497 - BT044056]. Five hundred and ten (91%) of the transcripts were annotated using Gene Ontology (GO) terms and 440 of the FLIcs are likely to contain a complete coding sequence (cCDS). The sequence information was used to identify putative paralogs, characterize salmon Kozak motifs, polyadenylation signal variation and to identify motifs likely to be involved in the regulation of particular genes. Finally, conserved 7-mers in the 3'UTRs were identified, of which some were identical to miRNA target sequences. Conclusion This paper describes the first Atlantic salmon FLIcs from a tissue and developmental stage specific cDNA library. We have demonstrated that many FLIcs contained a complete coding sequence (cCDS). This suggests that the remaining cDNA libraries generated by SGP represent a valuable cCDS FLIc source. The conservation of 7-mers in 3'UTRs indicates that these motifs are functionally important. Identity between some of these 7-mers and miRNA target sequences suggests that they are miRNA targets in Salmo salar transcripts as well. PMID:19878547

Comprehensive thermodynamic analysis of 3′ double-nucleotide overhangs neighboring Watson–Crick terminal base pairs

PubMed Central

O'Toole, Amanda S.; Miller, Stacy; Haines, Nathan; Zink, M. Coleen; Serra, Martin J.

2006-01-01

Thermodynamic parameters are reported for duplex formation of 48 self-complementary RNA duplexes containing Watson–Crick terminal base pairs (GC, AU and UA) with all 16 possible 3′ double-nucleotide overhangs; mimicking the structures of short interfering RNAs (siRNA) and microRNAs (miRNA). Based on nearest-neighbor analysis, the addition of a second dangling nucleotide to a single 3′ dangling nucleotide increases stability of duplex formation up to 0.8 kcal/mol in a sequence dependent manner. Results from this study in conjunction with data from a previous study [A. S. O'Toole, S. Miller and M. J. Serra (2005) RNA, 11, 512.] allows for the development of a refined nearest-neighbor model to predict the influence of 3′ double-nucleotide overhangs on the stability of duplex formation. The model improves the prediction of free energy and melting temperature when tested against five oligomers with various core duplex sequences. Phylogenetic analysis of naturally occurring miRNAs was performed to support our results. Selection of the effector miR strand of the mature miRNA duplex appears to be dependent upon the identity of the 3′ double-nucleotide overhang. Thermodynamic parameters for 3′ single terminal overhangs adjacent to a UA pair are also presented. PMID:16820533

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.

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.

The PICK1 Ca2+ sensor modulates N-methyl-d-aspartate (NMDA) receptor-dependent microRNA-mediated translational repression in neurons.

PubMed

Rajgor, Dipen; Fiuza, Maria; Parkinson, Gabrielle T; Hanley, Jonathan G

2017-06-09

MicroRNAs (miRNAs) are important regulators of localized mRNA translation in neuronal dendrites. The presence of RNA-induced silencing complex proteins in these compartments and the dynamic miRNA expression changes that occur in response to neuronal stimulation highlight their importance in synaptic plasticity. Previously, we demonstrated a novel interaction between the major RNA-induced silencing complex component Argounaute-2 (Ago2) and the BAR (bin/amphiphysin/rvs) domain protein PICK1. PICK1 recruits Ago2 to recycling endosomes in dendrites, where it inhibits miRNA-mediated translational repression. Chemical induction of long-term depression via NMDA receptor activation causes the dissociation of Ago2 from PICK1 and a consequent increase in dendritic miRNA-mediated gene silencing. The mechanism that underlies the regulation of PICK1-Ago2 binding is unknown. In this study, we demonstrate that the PICK1-Ago2 interaction is directly sensitive to Ca 2+ ions so that high [Ca 2+ ] free reduces PICK1 binding to Ago2. Mutating a stretch of C-terminal Ca 2+ -binding residues in PICK1 results in a complete block of NMDA-induced PICK1-Ago2 disassociation in cortical neurons. Furthermore, the same mutant also blocks NMDA-stimulated miRNA-mediated gene silencing. This study defines a novel mechanism whereby elevated [Ca 2+ ] induced by NMDA receptor activation modulates Ago2 and miRNA activity via PICK1. Our work suggests a Ca 2+ -dependent process to regulate miRNA activity in neurons in response to the induction of long-term depression. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Combinatory RNA-Sequencing Analyses Reveal a Dual Mode of Gene Regulation by ADAR1 in Gastric Cancer.

PubMed

Cho, Charles J; Jung, Jaeeun; Jiang, Lushang; Lee, Eun Ji; Kim, Dae-Soo; Kim, Byung Sik; Kim, Hee Sung; Jung, Hwoon-Yong; Song, Ho-June; Hwang, Sung Wook; Park, Yangsoon; Jung, Min Kyo; Pack, Chan Gi; Myung, Seung-Jae; Chang, Suhwan

2018-04-25

Adenosine deaminase acting on RNA 1 (ADAR1) is known to mediate deamination of adenosine-to-inosine through binding to double-stranded RNA, the phenomenon known as RNA editing. Currently, the function of ADAR1 in gastric cancer is unclear. This study was aimed at investigating RNA editing-dependent and editing-independent functions of ADAR1 in gastric cancer, especially focusing on its influence on editing of 3' untranslated regions (UTRs) and subsequent changes in expression of messenger RNAs (mRNAs) as well as microRNAs (miRNAs). RNA-sequencing and small RNA-sequencing were performed on AGS and MKN-45 cells with a stable ADAR1 knockdown. Changed frequencies of editing and mRNA and miRNA expression were then identified by bioinformatic analyses. Targets of RNA editing were further validated in patients' samples. In the Alu region of both gastric cell lines, editing was most commonly of the A-to-I type in 3'-UTR or intron. mRNA and protein levels of PHACTR4 increased in ADAR1 knockdown cells, because of the loss of seed sequences in 3'-UTR of PHACTR4 mRNA that are required for miRNA-196a-3p binding. Immunohistochemical analyses of tumor and paired normal samples from 16 gastric cancer patients showed that ADAR1 expression was higher in tumors than in normal tissues and inversely correlated with PHACTR4 staining. On the other hand, decreased miRNA-148a-3p expression in ADAR1 knockdown cells led to increased mRNA and protein expression of NFYA, demonstrating ADAR1's editing-independent function. ADAR1 regulates post-transcriptional gene expression in gastric cancer through both RNA editing-dependent and editing-independent mechanisms.

Targeting oncomiRNAs and mimicking tumor suppressor miRNAs: New trends in the development of miRNA therapeutic strategies in oncology (Review)

PubMed Central

GAMBARI, ROBERTO; BROGNARA, ELEONORA; SPANDIDOS, DEMETRIOS A.; FABBRI, ENRICA

2016-01-01

MicroRNA (miRNA or miR) therapeutics in cancer are based on targeting or mimicking miRNAs involved in cancer onset, progression, angiogenesis, epithelial-mesenchymal transition and metastasis. Several studies conclusively have demonstrated that miRNAs are deeply involved in tumor onset and progression, either behaving as tumor-promoting miRNAs (oncomiRNAs and metastamiRNAs) or as tumor suppressor miRNAs. This review focuses on the most promising examples potentially leading to the development of anticancer, miRNA-based therapeutic protocols. The inhibition of miRNA activity can be readily achieved by the use of miRNA inhibitors and oligomers, including RNA, DNA and DNA analogues (miRNA antisense therapy), small molecule inhibitors, miRNA sponges or through miRNA masking. On the contrary, the enhancement of miRNA function (miRNA replacement therapy) can be achieved by the use of modified miRNA mimetics, such as plasmid or lentiviral vectors carrying miRNA sequences. Combination strategies have been recently developed based on the observation that i) the combined administration of different antagomiR molecules induces greater antitumor effects and ii) some anti-miR molecules can sensitize drug-resistant tumor cell lines to therapeutic drugs. In this review, we discuss two additional issues: i) the combination of miRNA replacement therapy with drug administration and ii) the combination of antagomiR and miRNA replacement therapy. One of the solid results emerging from different independent studies is that miRNA replacement therapy can enhance the antitumor effects of the antitumor drugs. The second important conclusion of the reviewed studies is that the combination of anti-miRNA and miRNA replacement strategies may lead to excellent results, in terms of antitumor effects. PMID:27175518

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

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.

Characterization of the mammalian miRNA turnover landscape

PubMed Central

Guo, Yanwen; Liu, Jun; Elfenbein, Sarah J.; Ma, Yinghong; Zhong, Mei; Qiu, Caihong; Ding, Ye; Lu, Jun

2015-01-01

Steady state cellular microRNA (miRNA) levels represent the balance between miRNA biogenesis and turnover. The kinetics and sequence determinants of mammalian miRNA turnover during and after miRNA maturation are not fully understood. Through a large-scale study on mammalian miRNA turnover, we report the co-existence of multiple cellular miRNA pools with distinct turnover kinetics and biogenesis properties and reveal previously unrecognized sequence features for fast turnover miRNAs. We measured miRNA turnover rates in eight mammalian cell types with a combination of expression profiling and deep sequencing. While most miRNAs are stable, a subset of miRNAs, mostly miRNA*s, turnovers quickly, many of which display a two-step turnover kinetics. Moreover, different sequence isoforms of the same miRNA can possess vastly different turnover rates. Fast turnover miRNA isoforms are enriched for 5′ nucleotide bias against Argonaute-(AGO)-loading, but also additional 3′ and central sequence features. Modeling based on two fast turnover miRNA*s miR-222-5p and miR-125b-1-3p, we unexpectedly found that while both miRNA*s are associated with AGO, they strongly differ in HSP90 association and sensitivity to HSP90 inhibition. Our data characterize the landscape of genome-wide miRNA turnover in cultured mammalian cells and reveal differential HSP90 requirements for different miRNA*s. Our findings also implicate rules for designing stable small RNAs, such as siRNAs. PMID:25653157

Use of Mature miRNA Strand Selection in miRNAs Families in Cervical Cancer Development

PubMed Central

Granados-López, Angelica Judith; Ruiz-Carrillo, José Luis; Servín-González, Luis Steven; Martínez-Rodríguez, José Luis; Reyes-Estrada, Claudia Araceli; Gutiérrez-Hernández, Rosalinda; López, Jesús Adrián

2017-01-01

Aberrant miRNA expression is well recognized as a cancer hallmark, nevertheless miRNA function and expression does not always correlate in patients tissues and cell lines studies. In addition to this issue, miRNA strand usage conduces to increased cell signaling pathways modulation diversifying cellular processes regulation. In cervical cancer, 20 miRNA families are involved in carcinogenesis induction and development to this moment. These families have 5p and 3p strands with different nucleotide (nt) chain sizes. In general, mature 5p strands are larger: two miRNAs of 24 nt, 24 miRNAs of 23 nt, 35 miRNAs of 22 nt and three miRNAs of 21 nt. On the other hand, the 3p strands lengths observed are: seven miRNAs of 23 nt, 50 miRNAs of 22 nt, six miRNAs of 21 nt and four miRNAs of 20 nt. Based on the analysis of the 20 miRNA families associated with cervical cancer, 67 3p strands and 65 5p strands are selected suggesting selectivity and specificity mechanisms regulating cell processes like proliferation, apoptosis, migration, invasion, metabolism and Warburg effect. The insight reviewed here could be used in the miRNA based therapy, diagnosis and prognosis approaches. PMID:28216603

Use of Mature miRNA Strand Selection in miRNAs Families in Cervical Cancer Development.

PubMed

Granados-López, Angelica Judith; Ruiz-Carrillo, José Luis; Servín-González, Luis Steven; Martínez-Rodríguez, José Luis; Reyes-Estrada, Claudia Araceli; Gutiérrez-Hernández, Rosalinda; López, Jesús Adrián

2017-02-14

Aberrant miRNA expression is well recognized as a cancer hallmark, nevertheless miRNA function and expression does not always correlate in patients tissues and cell lines studies. In addition to this issue, miRNA strand usage conduces to increased cell signaling pathways modulation diversifying cellular processes regulation. In cervical cancer, 20 miRNA families are involved in carcinogenesis induction and development to this moment. These families have 5p and 3p strands with different nucleotide (nt) chain sizes. In general, mature 5p strands are larger: two miRNAs of 24 nt, 24 miRNAs of 23 nt, 35 miRNAs of 22 nt and three miRNAs of 21 nt. On the other hand, the 3p strands lengths observed are: seven miRNAs of 23 nt, 50 miRNAs of 22 nt, six miRNAs of 21 nt and four miRNAs of 20 nt. Based on the analysis of the 20 miRNA families associated with cervical cancer, 67 3p strands and 65 5p strands are selected suggesting selectivity and specificity mechanisms regulating cell processes like proliferation, apoptosis, migration, invasion, metabolism and Warburg effect. The insight reviewed here could be used in the miRNA based therapy, diagnosis and prognosis approaches.

Expanding the horizons of microRNA bioinformatics.

PubMed

Huntley, Rachael P; Kramarz, Barbara; Sawford, Tony; Umrao, Zara; Kalea, Anastasia Z; Acquaah, Vanessa; Martin, Maria-Jesus; Mayr, Manuel; Lovering, Ruth C

2018-06-05

MicroRNA regulation of key biological and developmental pathways is a rapidly expanding area of research, accompanied by vast amounts of experimental data. This data, however, is not widely available in bioinformatic resources, making it difficult for researchers to find and analyse microRNA-related experimental data and define further research projects. We are addressing this problem by providing two new bioinformatics datasets that contain experimentally verified functional information for mammalian microRNAs involved in cardiovascular-relevant, and other, processes. To date, our resource provides over 3,900 Gene Ontology annotations associated with almost 500 miRNAs from human, mouse and rat and over 2,200 experimentally validated miRNA:target interactions. We illustrate how this resource can be used to create miRNA-focused interaction networks with a biological context using the known biological role of miRNAs and the mRNAs they regulate, enabling discovery of associations between gene products, biological pathways and, ultimately, diseases. This data will be crucial in advancing the field of microRNA bioinformatics and will establish consistent datasets for reproducible functional analysis of microRNAs across all biological research areas. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

The NS3 protein of Rice hoja blanca tenuivirus suppresses RNA silencing in plant and insect hosts by efficiently binding both siRNAs and miRNAs

PubMed Central

Hemmes, Hans; Lakatos, Lóránt; Goldbach, Rob; Burgyán, József; Prins, Marcel

2007-01-01

RNA silencing plays a key role in antiviral defense as well as in developmental processes in plants and insects. Negative strand RNA viruses such as the plant virus Rice hoja blanca tenuivirus (RHBV) replicate in plants and in their insect transmission vector. Like most plant-infecting viruses, RHBV encodes an RNA silencing suppressor, the NS3 protein, and here it is demonstrated that this protein is capable of suppressing RNA silencing in both plants and insect cells. Biochemical analyses showed that NS3 efficiently binds siRNA as well as miRNA molecules. Binding of NS3 is greatly influenced by the size of small RNA molecules, as 21 nucleotide (nt) siRNA molecules are bound > 100 times more efficiently than 26 nt species. Competition assays suggest that the activity of NS3 is based on binding to siRNAs prior to strand separation during the assembly of the RNA-induced silencing complex. In addition, NS3 has a high affinity for miRNA/miRNA* duplexes, indicating that its activity might also interfere with miRNA-regulated gene expression in both insects and plants. PMID:17513697

PAPERCLIP identifies microRNA targets and a role of CstF64/64tau in promoting non-canonical poly(A) site usage

PubMed Central

Hwang, Hun-Way; Park, Christopher Y.; Goodarzi, Hani; Fak, John J.; Mele, Aldo; Moore, Michael J.; Saito, Yuhki; Darnell, Robert B.

2016-01-01

Accurate and precise annotation of the 3′ untranslated regions (3′ UTRs) is critical in understanding how mRNAs are regulated by microRNAs (miRNAs) and RNA-binding proteins (RBPs). Here we describe a method, PAPERCLIP (Poly(A) binding Protein-mediated mRNA 3′ End Retrieval by CrossLinking ImmunoPrecipitation), which shows high specificity for the mRNA 3′ ends and compares favorably to existing 3′ end mapping methods. PAPERCLIP uncovers a previously unrecognized role of CstF64/64tau in promoting the usage of a selected group of non-canonical poly(A) sites, the majority of them containing a downstream GUKKU motif. Furthermore, in mouse brain, PAPERCLIP discovers extended 3′ UTR sequences harboring functional miRNA binding sites and reveals developmentally regulated APA shifts including one in Atp2b2 that is evolutionarily conserved in human and results in a gain of a functional binding site of miR-137. PAPERCLIP provides a powerful tool to decipher post-transcriptional regulation of mRNAs through APA in vivo. PMID:27050522

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

Characterization of herpes simplex virus 2 primary microRNA Transcript regulation.

PubMed

Tang, Shuang; Bosch-Marce, Marta; Patel, Amita; Margolis, Todd P; Krause, Philip R

2015-05-01

In order to understand factors that may influence latency-associated transcription and latency-associated transcript (LAT) phenotypes, we studied the expression of the herpes simplex virus 2 (HSV-2) LAT-associated microRNAs (miRNAs). We mapped the transcription initiation sites of all three primary miRNA transcripts and identified the ICP4-binding sequences at the transcription initiation sites of both HSV-2 LAT (pri-miRNA for miR-I and miR-II, which target ICP34.5, and miR-III, which targets ICP0) and L/ST (a pri-miRNA for miR-I and miR-II) but not at that of the primary miR-H6 (for which the target is unknown). We confirmed activity of the putative HSV-2 L/ST promoter and found that ICP4 trans-activates the L/ST promoter when the ICP4-binding site at its transcription initiation site is mutated, suggesting that ICP4 may play a dual role in regulating transcription of L/ST and, consequently, of miR-I and miR-II. LAT exon 1 (containing LAT enhancer sequences), together with the LAT promoter region, comprises a bidirectional promoter required for the expression of both LAT-encoded miRNAs and miR-H6 in latently infected mouse ganglia. The ability of ICP4 to suppress ICP34.5-targeting miRNAs and to activate lytic viral genes suggests that ICP4 could play a key role in the switch between latency and reactivation. The HSV-2 LAT and viral miRNAs expressed in the LAT region are the most abundant viral transcripts during HSV latency. The balance between the expression of LAT and LAT-associated miRNAs and the expression of lytic viral transcripts from the opposite strand appears to influence whether individual HSV-infected neurons will be latently or productively infected. The outcome of neuronal infection may thus depend on regulation of gene expression of the corresponding primary miRNAs. In the present study, we characterize promoter sequences responsible for miRNA expression, including identification of the primary miRNA 5' ends and evaluation of ICP4 response. These findings provide further insight into the virus' strategy to tightly control expression of lytic cycle genes (especially the neurovirulence factor, ICP34.5) and suggest a mechanism (via ICP4) for the transition from latency to reactivated productive infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Characterization of Herpes Simplex Virus 2 Primary MicroRNA Transcript Regulation

PubMed Central

Bosch-Marce, Marta; Patel, Amita; Margolis, Todd P.

2015-01-01

ABSTRACT In order to understand factors that may influence latency-associated transcription and latency-associated transcript (LAT) phenotypes, we studied the expression of the herpes simplex virus 2 (HSV-2) LAT-associated microRNAs (miRNAs). We mapped the transcription initiation sites of all three primary miRNA transcripts and identified the ICP4-binding sequences at the transcription initiation sites of both HSV-2 LAT (pri-miRNA for miR-I and miR-II, which target ICP34.5, and miR-III, which targets ICP0) and L/ST (a pri-miRNA for miR-I and miR-II) but not at that of the primary miR-H6 (for which the target is unknown). We confirmed activity of the putative HSV-2 L/ST promoter and found that ICP4 trans-activates the L/ST promoter when the ICP4-binding site at its transcription initiation site is mutated, suggesting that ICP4 may play a dual role in regulating transcription of L/ST and, consequently, of miR-I and miR-II. LAT exon 1 (containing LAT enhancer sequences), together with the LAT promoter region, comprises a bidirectional promoter required for the expression of both LAT-encoded miRNAs and miR-H6 in latently infected mouse ganglia. The ability of ICP4 to suppress ICP34.5-targeting miRNAs and to activate lytic viral genes suggests that ICP4 could play a key role in the switch between latency and reactivation. IMPORTANCE The HSV-2 LAT and viral miRNAs expressed in the LAT region are the most abundant viral transcripts during HSV latency. The balance between the expression of LAT and LAT-associated miRNAs and the expression of lytic viral transcripts from the opposite strand appears to influence whether individual HSV-infected neurons will be latently or productively infected. The outcome of neuronal infection may thus depend on regulation of gene expression of the corresponding primary miRNAs. In the present study, we characterize promoter sequences responsible for miRNA expression, including identification of the primary miRNA 5′ ends and evaluation of ICP4 response. These findings provide further insight into the virus' strategy to tightly control expression of lytic cycle genes (especially the neurovirulence factor, ICP34.5) and suggest a mechanism (via ICP4) for the transition from latency to reactivated productive infection. PMID:25673716

microRNA biogenesis and turnover in plants.

PubMed

Rogers, K; Chen, X

2012-01-01

microRNAs (miRNAs) are short RNAs that regulate gene expression in eukaryotes. The biogenesis and turnover of miRNAs determine their spatiotemporal accumulation within tissues. miRNA biogenesis is a multistep process that entails transcription, processing, nuclear export, and formation of the miRNA-ARGONAUTE complex. Factors that perform each of these steps have been identified. Generation of mature miRNAs from primary transcripts, i.e., miRNA processing, is a key step in miRNA biogenesis. Our understanding of miRNA processing has expanded beyond the enzyme that performs the reactions, as more and more additional factors that impact the efficiency and accuracy of miRNA processing are uncovered. In contrast to miRNA biogenesis, miRNA turnover is an important but poorly understood process that contributes to the steady-state levels of miRNAs. Enzymes responsible for miRNA degradation have only recently been identified. This review describes the processes of miRNA maturation and degradation in plants.

Mice lacking microRNAs in Pax8-expressing cells develop hypothyroidism and end-stage renal failure.

PubMed

Bartram, Malte P; Amendola, Elena; Benzing, Thomas; Schermer, Bernhard; de Vita, Gabriella; Müller, Roman-Ulrich

2016-04-18

Non-coding RNAs have gained increasing attention during the last decade. The first large group of non-coding RNAs to be characterized systematically starting at the beginning of the 21st century were small oligonucleotides--the so-called microRNAs (miRNAs). By now we have learnt that microRNAs are indispensable for most biological processes including organogenesis and maintenance of organ structure and function. The role of microRNAs has been studied extensively in the development of a number of organs, so far most studies focussed on e.g. the heart or the brain whilst the role of microRNAs in the development and maintenance of complex epithelial organs is less well understood. Furthermore most analyses regarding microRNA function in epithelial organs employed conditional knockout mouse models of the RNAse III Dicer to abrogate microRNA biogenesis. However, there is increasing evidence for Dicer to have multiple functions independent from microRNA maturation. Therefore Dicer independent models are needed to gain further insight into the complex biology of miRNA dependent processes. Here we analyze the contribution of microRNA-dependent transcriptional control in Pax8-expressing epithelial cells. Pax8 is a transcription factor that is crucial to the development of epithelial organs. The miRNA machinery was disrupted by crossing conditional DiGeorge syndrome critical region 8 (Dgcr8) fl/fl mice to Pax8Cre mice. The Dgcr8/Drosha complex processes pri-miRNAs in the nucleus before they are exported as pre-miRNAs for further maturation by Dicer in the cytoplasm. Dgcr8 fl/fl; Pax8Cre+ knockout mice died prematurely, developed massive hypothyroidism and end stage renal disease due to a loss of miRNAs in Pax8 expressing tissue. Pax8Cre-mediated conditional loss of DiGeorge syndrome critical region 8 (Dgcr8), an essential component of the nuclear machinery that is required for microRNA biogenesis, resulted in severe hypothyroidism, massively reduced body weight and ultimately led to renal failure and death of the animals. These data provide further insight into the importance of miRNAs in organ homeostasis using a Dicer independent model.

Parecoxib inhibits glioblastoma cell proliferation, migration and invasion by upregulating miRNA-29c.

PubMed

Li, Lin-Yong; Xiao, Jie; Liu, Qiang; Xia, Kun

2017-03-15

Glioblastoma (GBM) is one of the most lethal brain cancers worldwide, and there is an urgent need for development of novel therapeutic approaches. Parecoxib is a well-known cyclooxygenase-2 (COX-2) inhibitor, and had already been developed for postoperative analgesia with high efficacy and low adverse reaction. A recent study has suggested that parecoxib potently enhances immunotherapeutic efficacy of GBM, but its effects on GBM growth, migration and invasion have not previously been studied. In the present study, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and BrdU (5-bromo-2-deoxyuridine) incorporation assays were used to evaluate the cell proliferation of GBM cells. Wound-healing and transwell assays were preformed to analyze GBM cell migration and invasion, respectively. The results suggested that parecoxib inhibits cell proliferation, migration and invasion of GBM cells in a dose-dependent manner. RT-qPCR (real-time quantitative PCR) analysis demonstrated that miRNA-29c can be significantly induced by parecoxib. Furthermore, our data suggests that a miRNA-29c inhibitor can significantly attenuate parecoxib's effect on proliferation, migration and invasion of GBM. In conclusion, the present study suggests that parecoxib inhibits GBM cell proliferation, migration and invasion by upregulating miRNA-29c. © 2017. Published by The Company of Biologists Ltd.

Parecoxib inhibits glioblastoma cell proliferation, migration and invasion by upregulating miRNA-29c

PubMed Central

Li, Lin-Yong; Xiao, Jie; Liu, Qiang

2017-01-01

ABSTRACT Glioblastoma (GBM) is one of the most lethal brain cancers worldwide, and there is an urgent need for development of novel therapeutic approaches. Parecoxib is a well-known cyclooxygenase-2 (COX-2) inhibitor, and had already been developed for postoperative analgesia with high efficacy and low adverse reaction. A recent study has suggested that parecoxib potently enhances immunotherapeutic efficacy of GBM, but its effects on GBM growth, migration and invasion have not previously been studied. In the present study, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and BrdU (5-bromo-2-deoxyuridine) incorporation assays were used to evaluate the cell proliferation of GBM cells. Wound-healing and transwell assays were preformed to analyze GBM cell migration and invasion, respectively. The results suggested that parecoxib inhibits cell proliferation, migration and invasion of GBM cells in a dose-dependent manner. RT-qPCR (real-time quantitative PCR) analysis demonstrated that miRNA-29c can be significantly induced by parecoxib. Furthermore, our data suggests that a miRNA-29c inhibitor can significantly attenuate parecoxib's effect on proliferation, migration and invasion of GBM. In conclusion, the present study suggests that parecoxib inhibits GBM cell proliferation, migration and invasion by upregulating miRNA-29c. PMID:27895048

Bistable switch in let-7 miRNA biogenesis pathway involving Lin28.

PubMed

Shi, Fei; Yu, Wenbao; Wang, Xia

2014-10-21

miRNAs are small noncoding RNAs capable of regulating gene expression at the post-transcriptional level. A growing body of evidence demonstrated that let-7 family of miRNAs, as one of the highly conserved miRNAs, plays an important role in cell differentiation and development, as well as tumor suppressor function depending on their levels of expression. To explore the physiological significance of let-7 in regulating cell fate decisions, we present a coarse grained model of let-7 biogenesis network, in which let-7 and its regulator Lin28 inhibit mutually. The dynamics of this minimal network architecture indicates that, as the concentration of Lin28 increases, the system undergoes a transition from monostability to a bistability and then to a one-way switch with increasing strength of positive feedback of let-7, while in the absence of Lin28 inhibition, the system loses bistability. Moreover, the ratio of degradation rates of let-7 and Lin28 is critical for the switching sensitivity and resistance to stimulus fluctuations. These findings may highlight why let-7 is required for normal gene expression in the context of embryonic development and oncogenesis, which will facilitate the development of approaches to exploit this regulatory pathway by manipulating Lin28/let-7 axis for novel treatments of human diseases.

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.

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.

Detection of the Argonaute Protein Ago2 and microRNAs in the RNA Induced Silencing Complex (RISC) Using a Monoclonal Antibody

PubMed Central

Ikeda, Keigo; Satoh, Minoru; Pauley, Kaleb M.; Fritzler, Marvin J.; Reeves, Westley H.; Chan, Edward K.L.

2007-01-01

MicroRNAs (miRNAs) are short RNA molecules responsible for post-transcriptional gene silencing by the degradation or translational inhibition of their target messenger RNAs (mRNAs). This process of gene silencing, known as RNA interference (RNAi), is mediated by highly conserved Argonaute (Ago) proteins which are the key components of the RNA induced silencing complex (RISC). In humans, Ago2 is responsible for the endonuclease cleavage of targeted mRNA and it interacts with the mRNA-binding protein GW182, which is a marker for cytoplasmic foci referred to as GW bodies (GWBs). We demonstrated that the anti-Ago2 monoclonal antibody 4F9 recognized GWBs in a cell cycle dependent manner and was capable of capturing miRNAs associated with Ago2. Since Ago2 protein is the effector protein of RNAi, anti-Ago2 monoclonal antibody may be useful in capturing functional miRNAs. PMID:17054975

Detection of the argonaute protein Ago2 and microRNAs in the RNA induced silencing complex (RISC) using a monoclonal antibody.

PubMed

Ikeda, Keigo; Satoh, Minoru; Pauley, Kaleb M; Fritzler, Marvin J; Reeves, Westley H; Chan, Edward K L

2006-12-20

MicroRNAs (miRNAs) are short RNA molecules responsible for post-transcriptional gene silencing by the degradation or translational inhibition of their target messenger RNAs (mRNAs). This process of gene silencing, known as RNA interference (RNAi), is mediated by highly conserved Argonaute (Ago) proteins which are the key components of the RNA induced silencing complex (RISC). In humans, Ago2 is responsible for the endonuclease cleavage of targeted mRNA and it interacts with the mRNA-binding protein GW182, which is a marker for cytoplasmic foci referred to as GW bodies (GWBs). We demonstrated that the anti-Ago2 monoclonal antibody 4F9 recognized GWBs in a cell cycle dependent manner and was capable of capturing miRNAs associated with Ago2. Since Ago2 protein is the effector protein of RNAi, anti-Ago2 monoclonal antibody may be useful in capturing functional miRNAs.

A biochemical approach to identifying microRNA targets

PubMed Central

Karginov, Fedor V.; Conaco, Cecilia; Xuan, Zhenyu; Schmidt, Bryan H.; Parker, Joel S.; Mandel, Gail; Hannon, Gregory J.

2007-01-01

Identifying the downstream targets of microRNAs (miRNAs) is essential to understanding cellular regulatory networks. We devised a direct biochemical method for miRNA target discovery that combined RNA-induced silencing complex (RISC) purification with microarray analysis of bound mRNAs. Because targets of miR-124a have been analyzed, we chose it as our model. We honed our approach both by examining the determinants of stable binding between RISC and synthetic target RNAs in vitro and by determining the dependency of both repression and RISC coimmunoprecipitation on miR-124a seed sites in two of its well characterized targets in vivo. Examining the complete spectrum of miR-124 targets in 293 cells yielded both a set that were down-regulated at the mRNA level, as previously observed, and a set whose mRNA levels were unaffected by miR-124a. Reporter assays validated both classes, extending the spectrum of mRNA targets that can be experimentally linked to the miRNA pathway. PMID:18042700

Hepatitis B virus pathogenesis: Fresh insights into hepatitis B virus RNA.

PubMed

Sekiba, Kazuma; Otsuka, Motoyuki; Ohno, Motoko; Yamagami, Mari; Kishikawa, Takahiro; Suzuki, Tatsunori; Ishibashi, Rei; Seimiya, Takahiro; Tanaka, Eri; Koike, Kazuhiko

2018-06-07

Hepatitis B virus (HBV) is still a worldwide health concern. While divergent factors are involved in its pathogenesis, it is now clear that HBV RNAs, principally templates for viral proteins and viral DNAs, have diverse biological functions involved in HBV pathogenesis. These functions include viral replication, hepatic fibrosis and hepatocarcinogenesis. Depending on the sequence similarities, HBV RNAs may act as sponges for host miRNAs and may deregulate miRNA functions, possibly leading to pathological consequences. Some parts of the HBV RNA molecule may function as viral-derived miRNA, which regulates viral replication. HBV DNA can integrate into the host genomic DNA and produce novel viral-host fusion RNA, which may have pathological functions. To date, elimination of HBV-derived covalently closed circular DNA has not been achieved. However, RNA transcription silencing may be an alternative practical approach to treat HBV-induced pathogenesis. A full understanding of HBV RNA transcription and the biological functions of HBV RNA may open a new avenue for the development of novel HBV therapeutics.

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

Analysis of microRNA profile of Anopheles sinensis by deep sequencing and bioinformatic approaches.

PubMed

Feng, Xinyu; Zhou, Xiaojian; Zhou, Shuisen; Wang, Jingwen; Hu, Wei

2018-03-12

microRNAs (miRNAs) are small non-coding RNAs widely identified in many mosquitoes. They are reported to play important roles in development, differentiation and innate immunity. However, miRNAs in Anopheles sinensis, one of the Chinese malaria mosquitoes, remain largely unknown. We investigated the global miRNA expression profile of An. sinensis using Illumina Hiseq 2000 sequencing. Meanwhile, we applied a bioinformatic approach to identify potential miRNAs in An. sinensis. The identified miRNA profiles were compared and analyzed by two approaches. The selected miRNAs from the sequencing result and the bioinformatic approach were confirmed with qRT-PCR. Moreover, target prediction, GO annotation and pathway analysis were carried out to understand the role of miRNAs in An. sinensis. We identified 49 conserved miRNAs and 12 novel miRNAs by next-generation high-throughput sequencing technology. In contrast, 43 miRNAs were predicted by the bioinformatic approach, of which two were assigned as novel. Comparative analysis of miRNA profiles by two approaches showed that 21 miRNAs were shared between them. Twelve novel miRNAs did not match any known miRNAs of any organism, indicating that they are possibly species-specific. Forty miRNAs were found in many mosquito species, indicating that these miRNAs are evolutionally conserved and may have critical roles in the process of life. Both the selected known and novel miRNAs (asi-miR-281, asi-miR-184, asi-miR-14, asi-miR-nov5, asi-miR-nov4, asi-miR-9383, and asi-miR-2a) could be detected by quantitative real-time PCR (qRT-PCR) in the sequenced sample, and the expression patterns of these miRNAs measured by qRT-PCR were in concordance with the original miRNA sequencing data. The predicted targets for the known and the novel miRNAs covered many important biological roles and pathways indicating the diversity of miRNA functions. We also found 21 conserved miRNAs and eight counterparts of target immune pathway genes in An. sinensis based on the analysis of An. gambiae. Our results provide the first lead to the elucidation of the miRNA profile in An. sinensis. Unveiling the roles of mosquito miRNAs will undoubtedly lead to a better understanding of mosquito biology and mosquito-pathogen interactions. This work lays the foundation for the further functional study of An. sinensis miRNAs and will facilitate their application in vector control.

A transgenic resource for conditional competitive inhibition of conserved Drosophila microRNAs

PubMed Central

Fulga, Tudor A.; McNeill, Elizabeth M.; Binari, Richard; Yelick, Julia; Blanche, Alexandra; Booker, Matthew; Steinkraus, Bruno R.; Schnall-Levin, Michael; Zhao, Yong; DeLuca, Todd; Bejarano, Fernando; Han, Zhe; Lai, Eric C.; Wall, Dennis P.; Perrimon, Norbert; Van Vactor, David

2015-01-01

Although the impact of microRNAs (miRNAs) in development and disease is well established, understanding the function of individual miRNAs remains challenging. Development of competitive inhibitor molecules such as miRNA sponges has allowed the community to address individual miRNA function in vivo. However, the application of these loss-of-function strategies has been limited. Here we offer a comprehensive library of 141 conditional miRNA sponges targeting well-conserved miRNAs in Drosophila. Ubiquitous miRNA sponge delivery and consequent systemic miRNA inhibition uncovers a relatively small number of miRNA families underlying viability and gross morphogenesis, with false discovery rates in the 4–8% range. In contrast, tissue-specific silencing of muscle-enriched miRNAs reveals a surprisingly large number of novel miRNA contributions to the maintenance of adult indirect flight muscle structure and function. A strong correlation between miRNA abundance and physiological relevance is not observed, underscoring the importance of unbiased screens when assessing the contributions of miRNAs to complex biological processes. PMID:26081261

Substantial Loss of Conserved and Gain of Novel MicroRNA Families in Flatworms

PubMed Central

Fromm, Bastian; Worren, Merete Molton; Hahn, Christoph; Hovig, Eivind; Bachmann, Lutz

2013-01-01

Recent studies on microRNA (miRNA) evolution focused mainly on the comparison of miRNA complements between animal clades. However, evolution of miRNAs within such groups is poorly explored despite the availability of comparable data that in some cases lack only a few key taxa. For flatworms (Platyhelminthes), miRNA complements are available for some free-living flatworms and all major parasitic lineages, except for the Monogenea. We present the miRNA complement of the monogenean flatworm Gyrodactylus salaris that facilitates a comprehensive analysis of miRNA evolution in Platyhelminthes. Using the newly designed bioinformatics pipeline miRCandRef, the miRNA complement was disentangled from next-generation sequencing of small RNAs and genomic DNA without a priori genome assembly. It consists of 39 miRNA hairpin loci of conserved miRNA families, and 22 novel miRNAs. A comparison with the miRNA complements of Schmidtea mediterranea (Turbellaria), Schistosoma japonicum (Trematoda), and Echinococcus granulosus (Cestoda) reveals a substantial loss of conserved bilaterian, protostomian, and lophotrochozoan miRNAs. Eight of the 46 expected conserved miRNAs were lost in all flatworms, 16 in Neodermata and 24 conserved miRNAs could not be detected in the cestode and the trematode. Such a gradual loss of miRNAs has not been reported before for other animal phyla. Currently, little is known about miRNAs in Platyhelminthes, and for the majority of the lost miRNAs there is no prediction of function. As suggested earlier they might be related to morphological simplifications. The presence and absence of 153 conserved miRNAs was compared for platyhelminths and 32 other metazoan taxa. Phylogenetic analyses support the monophyly of Platyhelminthes (Turbellaria + Neodermata [Monogenea {Trematoda + Cestoda}]). PMID:24025793

Genome-wide characterization of microRNA in foxtail millet (Setaria italica)

PubMed Central

2013-01-01

Background MicroRNAs (miRNAs) are a class of short non-coding, endogenous RNAs that play key roles in many biological processes in both animals and plants. Although many miRNAs have been identified in a large number of organisms, the miRNAs in foxtail millet (Setaria italica) have, until now, been poorly understood. Results In this study, two replicate small RNA libraries from foxtail millet shoots were sequenced, and 40 million reads representing over 10 million unique sequences were generated. We identified 43 known miRNAs, 172 novel miRNAs and 2 mirtron precursor candidates in foxtail millet. Some miRNA*s of the known and novel miRNAs were detected as well. Further, eight novel miRNAs were validated by stem-loop RT-PCR. Potential targets of the foxtail millet miRNAs were predicted based on our strict criteria. Of the predicted target genes, 79% (351) had functional annotations in InterPro and GO analyses, indicating the targets of the miRNAs were involved in a wide range of regulatory functions and some specific biological processes. A total of 69 pairs of syntenic miRNA precursors that were conserved between foxtail millet and sorghum were found. Additionally, stem-loop RT-PCR was conducted to confirm the tissue-specific expression of some miRNAs in the four tissues identified by deep-sequencing. Conclusions We predicted, for the first time, 215 miRNAs and 447 miRNA targets in foxtail millet at a genome-wide level. The precursors, expression levels, miRNA* sequences, target functions, conservation, and evolution of miRNAs we identified were investigated. Some of the novel foxtail millet miRNAs and miRNA targets were validated experimentally. PMID:24330712

Genome-wide characterization of microRNA in foxtail millet (Setaria italica).

PubMed

Yi, Fei; Xie, Shaojun; Liu, Yuwei; Qi, Xin; Yu, Jingjuan

2013-12-13

MicroRNAs (miRNAs) are a class of short non-coding, endogenous RNAs that play key roles in many biological processes in both animals and plants. Although many miRNAs have been identified in a large number of organisms, the miRNAs in foxtail millet (Setaria italica) have, until now, been poorly understood. In this study, two replicate small RNA libraries from foxtail millet shoots were sequenced, and 40 million reads representing over 10 million unique sequences were generated. We identified 43 known miRNAs, 172 novel miRNAs and 2 mirtron precursor candidates in foxtail millet. Some miRNA*s of the known and novel miRNAs were detected as well. Further, eight novel miRNAs were validated by stem-loop RT-PCR. Potential targets of the foxtail millet miRNAs were predicted based on our strict criteria. Of the predicted target genes, 79% (351) had functional annotations in InterPro and GO analyses, indicating the targets of the miRNAs were involved in a wide range of regulatory functions and some specific biological processes. A total of 69 pairs of syntenic miRNA precursors that were conserved between foxtail millet and sorghum were found. Additionally, stem-loop RT-PCR was conducted to confirm the tissue-specific expression of some miRNAs in the four tissues identified by deep-sequencing. We predicted, for the first time, 215 miRNAs and 447 miRNA targets in foxtail millet at a genome-wide level. The precursors, expression levels, miRNA* sequences, target functions, conservation, and evolution of miRNAs we identified were investigated. Some of the novel foxtail millet miRNAs and miRNA targets were validated experimentally.

Distinct AGO1 and AGO2 associated miRNA profiles in human cells and blood plasma

PubMed Central

Turchinovich, Andrey; Burwinkel, Barbara

2012-01-01

Studies of miRNA association with Argonaute (AGO) proteins in mammalian cells have indicated lack of bias toward particular AGO. However, to our knowledge, the use of quantitative methods for studying miRNA association with different AGOs has not been reported so far. In this work we compared the total miRNA content in AGO1 and AGO2 immunoprecipitates obtained from MCF7 adenocarcinoma cells using TaqMan Low Density miRNA Arrays and successfully verified selected miRNAs with qPCR. For most of the miRNA species AGO1 and AGO2 profiles were well correlated, however, some miRNAs demonstrated consistent biases toward one of the Argonautes. Furthermore, miRNAs which were predominantly AGO2-associated derived mostly from sense strands of the corresponding pre-miRNAs while the majority of AGO1 biased miRNAs originated from antisense strands of the pre-miRNAs. Additionally, we show that circulating miRNA in human blood plasma can be immunoprecipitated with both AGO1 and AGO2 antibody. However, unlike in cell lysates, AGO1 and AGO2 associated miRNA profiles in plasma did not correlate, indicating that many cell types contribute to circulating miRNA (given that expression of AGO proteins is tissue specific). Furthermore, AGO-specific miRNA profiles in blood cells differed significantly from miRNAs profiles in plasma indicating that most circulating miRNAs are likely to derive from non-blood cells. Since circulating miRNAs hold great promise as biomarkers for numerous cancers and other diseases, we hypothesize that AGO-specific miRNA profiles might add an additional dimension to circulating miRNA-based diagnostics. PMID:22858679

Distinct anti-oncogenic effect of various microRNAs in different mouse models of liver cancer

PubMed Central

Wu, Heng; Liu, Yan; Wang, XinWei; Calvisi, Diego F.; Song, Guisheng; Chen, Xin

2015-01-01

Deregulation of microRNAs (miRNAs) is a typical feature of human hepatocellular carcinoma (HCC). However, the in vivo relevance of miRNAs along hepatocarcinogenesis remains largely unknown. Here, we show that liver tumors induced in mice by c-Myc overexpression or AKT/Ras co-expression exhibit distinct miRNA expression profiles. Among the downregulated miRNAs, eight (miR-101, miR-107, miR-122, miR-29, miR-365, miR-375, miR-378, and miR-802) were selected and their tumor suppressor activity was determined by overexpressing each of them together with c-Myc or AKT/Ras oncogenes in mouse livers via hydrodynamic transfection. The tumor suppressor activity of these microRNAs was extremely heterogeneous in c-Myc and AKT/Ras mice: while miR-378 had no tumor suppressor activity, miR-107, mir-122, miR-29, miR-365 and miR-802 exhibited weak to moderate tumor suppressor potential. Noticeably, miR-375 showed limited antineoplastic activity against c-Myc driven tumorigenesis, whereas it strongly inhibited AKT/Ras induced hepatocarcinogenesis. Furthermore, miR-101 significantly suppressed both c-Myc and AKT/Ras liver tumor development. Altogether, the present data demonstrate that different oncogenes induce distinct miRNA patterns, whose modulation differently affects hepatocarcinogenesis depending on the driving oncogenes. Finally, our findings support a strong tumor suppressor activity of miR-101 in liver cancer models regardless of the driver oncogenes involved, thus representing a promising therapeutic target in human HCC. PMID:25762642

Strand Displacement Amplification Reaction on Quantum Dot-Encoded Silica Bead for Visual Detection of Multiplex MicroRNAs.

PubMed

Qu, Xiaojun; Jin, Haojun; Liu, Yuqian; Sun, Qingjiang

2018-03-06

The combination of microbead array, isothermal amplification, and molecular signaling enables the continuous development of next-generation molecular diagnostic techniques. Herein we reported the implementation of nicking endonuclease-assisted strand displacement amplification reaction on quantum dots-encoded microbead (Qbead), and demonstrated its feasibility for multiplexed miRNA assay in real sample. The Qbead featured with well-defined core-shell superstructure with dual-colored quantum dots loaded in silica core and shell, respectively, exhibiting remarkably high optical encoding stability. Specially designed stem-loop-structured probes were immobilized onto the Qbead for specific target recognition and amplification. In the presence of low abundance of miRNA target, the target triggered exponential amplification, producing a large quantity of stem-G-quadruplexes, which could be selectively signaled by a fluorescent G-quadruplex intercalator. In one-step operation, the Qbead-based isothermal amplification and signaling generated emissive "core-shell-satellite" superstructure, changing the Qbead emission-color. The target abundance-dependent emission-color changes of the Qbead allowed direct, visual detection of specific miRNA target. This visualization method achieved limit of detection at the subfemtomolar level with a linear dynamic range of 4.5 logs, and point-mutation discrimination capability for precise miRNA analyses. The array of three encoded Qbeads could simultaneously quantify three miRNA biomarkers in ∼500 human hepatoma carcinoma cells. With the advancements in ease of operation, multiplexing, and visualization capabilities, the isothermal amplification-on-Qbead assay could potentially enable the development of point-of-care diagnostics.

Selective targeting of KRAS-Mutant cells by miR-126 through repression of multiple genes essential for the survival of KRAS-Mutant cells

PubMed Central

Hara, Toshifumi; Jones, Matthew F.; Subramanian, Murugan; Li, Xiao Ling; Ou, Oliver; Zhu, Yuelin; Yang, Yuan; Wakefield, Lalage M.; Hussain, S. Perwez; Gaedcke, Jochen; Ried, Thomas; Luo, Ji; Caplen, Natasha J.; Lal, Ashish

2014-01-01

MicroRNAs (miRNAs) regulate the expression of hundreds of genes. However, identifying the critical targets within a miRNA-regulated gene network is challenging. One approach is to identify miRNAs that exert a context-dependent effect, followed by expression profiling to determine how specific targets contribute to this selective effect. In this study, we performed miRNA mimic screens in isogenic KRAS-Wild-type (WT) and KRAS-Mutant colorectal cancer (CRC) cell lines to identify miRNAs selectively targeting KRAS-Mutant cells. One of the miRNAs we identified as a selective inhibitor of the survival of multiple KRAS-Mutant CRC lines was miR-126. In KRAS-Mutant cells, miR-126 over-expression increased the G1 compartment, inhibited clonogenicity and tumorigenicity, while exerting no effect on KRAS-WT cells. Unexpectedly, the miR-126-regulated transcriptome of KRAS-WT and KRAS-Mutant cells showed no significant differences. However, by analyzing the overlap between miR-126 targets with the synthetic lethal genes identified by RNAi in KRAS-Mutant cells, we identified and validated a subset of miR-126-regulated genes selectively required for the survival and clonogenicity of KRAS-Mutant cells. Our strategy therefore identified critical target genes within the miR-126-regulated gene network. We propose that the selective effect of miR-126 on KRAS-Mutant cells could be utilized for the development of targeted therapy for KRAS mutant tumors. PMID:25245095

Sexual dimorphism in the fetal cardiac response to maternal nutrient restriction

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

Muralimanoharan, Sribalasubashini; Li, Cun; Nakayasu, Ernesto S.

Poor maternal nutrition causes intrauterine growth restriction (IUGR); however, its effects on fetal cardiac development are unclear. We have developed a baboon model of moderate maternal undernutrition, leading to IUGR. We hypothesized that IUGR affects fetal cardiac structure and metabolism. Six control pregnant baboons ate ad-libitum (CTRL)) or 70% CTRL from 0.16 of gestation (G). Fetuses were euthanized at C-section at 0.9G under general anesthesia. Male but not female IUGR fetuses showed left ventricular fibrosis inversely correlated with birth weight. Expression of extracellular matrix protein TSP-1 was increased ( SMAD3 and ALK-1 were downregulated in male IUGRs with no differencemore » in females. Autophagy was present in male IUGR evidenced by upregulation of ATG7 expression and lipidation LC3B. Global miRNA expression profiling revealed 56 annotated and novel cardiac miRNAs exclusively dysregulated in female IUGR, and 38 cardiac miRNAs were exclusively dysregulated in males (p<0.05). Fifteen (CTRL) and 23 (IUGR) miRNAs, were differentially expressed between males and. females (p<0.05) suggesting sexual dimorphism, which can be at least partially explained by differential expression of upstream transcription factors (e.g. HNF4α, and NFκB p50). Lipidomics analysis exhibited a net increase in diacylglycerol and plasmalogens, and a decrease in triglycerides and phosphatidylcholines. In summary, IUGR resulting from decreased maternal nutrition is associated with sex-dependent dysregulations in cardiac structure, miRNA expression, and lipid metabolism. If these changes persist postnatally, they may program offspring for higher later life cardiac risk.« less

Placental dysfunction is associated with altered microRNA expression in pregnant women with low folate status

PubMed Central

Mackie, Fiona L.; Lean, Samantha C.; Greenwood, Susan L.; Heazell, Alexander E. P.; Forbes, Karen; Jones, Rebecca L.

2017-01-01

Scope Low maternal folate status during pregnancy increases the risk of delivering small for gestational age (SGA) infants, but the mechanistic link between maternal folate status, SGA, and placental dysfunction is unknown. microRNAs (miRNAs) are altered in pregnancy pathologies and by folate in other systems. We hypothesized that low maternal folate status causes placental dysfunction, mediated by altered miRNA expression. Methods and results A prospective observational study recruited pregnant adolescents and assessed third trimester folate status and placental function. miRNA array, QPCR, and bioinformatics identified placental miRNAs and target genes. Low maternal folate status is associated with higher incidence of SGA infants (28% versus 13%, p < 0.05) and placental dysfunction, including elevated trophoblast proliferation and apoptosis (p < 0.001), reduced amino acid transport (p < 0.01), and altered placental hormones (pregnancy‐associated plasma protein A, progesterone, and human placental lactogen). miR‐222‐3p, miR‐141‐3p, and miR‐34b‐5p were upregulated by low folate status (p < 0.05). Bioinformatics predicted a gene network regulating cell turnover. Quantitative PCR demonstrated that key genes in this network (zinc finger E‐box binding homeobox 2, v‐myc myelocytomatosis viral oncogene homolog (avian), and cyclin‐dependent kinase 6) were reduced (p < 0.05) in placentas with low maternal folate status. Conclusion This study supports that placental dysfunction contributes to impaired fetal growth in women with low folate status and suggests altered placental expression of folate‐sensitive miRNAs and target genes as a mechanistic link. PMID:28105727

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

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.

Identification of microRNAs differentially expressed involved in male flower development.

PubMed

Wang, Zhengjia; Huang, Jianqin; Sun, Zhichao; Zheng, Bingsong

2015-03-01

Hickory (Carya cathayensis Sarg.) is one of the most economically important woody trees in eastern China, but its long flowering phase delays yield. Our understanding of the regulatory roles of microRNAs (miRNAs) in male flower development in hickory remains poor. Using high-throughput sequencing technology, we have pyrosequenced two small RNA libraries from two male flower differentiation stages in hickory. Analysis of the sequencing data identified 114 conserved miRNAs that belonged to 23 miRNA families, five novel miRNAs including their corresponding miRNA*s, and 22 plausible miRNA candidates. Differential expression analysis revealed 12 miRNA sequences that were upregulated in the later (reproductive) stage of male flower development. Quantitative real-time PCR showed similar expression trends as that of the deep sequencing. Novel miRNAs and plausible miRNA candidates were predicted using bioinformatic analysis methods. The miRNAs newly identified in this study have increased the number of known miRNAs in hickory, and the identification of differentially expressed miRNAs will provide new avenues for studies into miRNAs involved in the process of male flower development in hickory and other related trees.

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

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.

Identification and characterization of microRNA in the lung tissue of pigs with different susceptibilities to PCV2 infection.

PubMed

Zhang, Ping; Wang, Liyuan; Li, Yanping; Jiang, Ping; Wang, Yanchao; Wang, Pengfei; Kang, Li; Wang, Yuding; Sun, Yi; Jiang, Yunliang

2018-02-15

Porcine circovirus type 2 (PCV2) is the primary cause of post-weaning multisystemic wasting syndrome (PMWS) and other PCV-associated diseases. According to our previous RNA-sequencing analysis, the differences in the susceptibility to PCV2 infection depended on the genetic differences between the Laiwu (LW) and Yorkshire × Landrace crossbred (YL) pigs, but the cellular microRNA (miRNA) that are differentially expressed between the LW and YL pigs before and after PCV2 infection remain to be determined. In this study, high-throughput sequencing was performed to determine the abundance and differential expression of miRNA in lung tissues from PCV2-infected and PCV2-uninfected LW and YL pigs. In total, 295 known and 95 novel miRNA were identified, and 23 known and 25 novel miRNA were significantly differentially expressed in the PCV2-infected vs. PCV2-uninfected LW pigs and/or the PCV2-infected vs. PCV2-uninfected YL pigs. The expression levels of ssc-miR-122, ssc-miR-192, ssc-miR-451, ssc-miR-486, and ssc-miR-504 were confirmed by quantitative real-time PCR (qRT-PCR). Analysis of the potential targets of the four up-regulated miRNA (i.e., ssc-miR-122, ssc-miR-192, ssc-miR-451 and ssc-miR-486) identified pathways and genes that may be important for disease resistance. Among the up-regulated miRNA, ssc-miR-122 can repress the protein expression and viral DNA replication of PCV2 and down-regulate the expression of the nuclear factor of activated T-cells 5 (NFAT5) and aminopeptidase puromycin sensitive (NPEPPS) by binding to their 3' untranslated region (3'UTR) in PK15 cells. Therefore, ssc-miR-122 may indirectly suppress PCV2 infection by targeting genes related to the host immune system, such as NFAT5 and NPEPPS.

Carica papaya microRNAs are responsive to Papaya meleira virus infection.

PubMed

Abreu, Paolla M V; Gaspar, Clicia G; Buss, David S; Ventura, José A; Ferreira, Paulo C G; Fernandes, Patricia M B

2014-01-01

MicroRNAs are implicated in the response to biotic stresses. Papaya meleira virus (PMeV) is the causal agent of sticky disease, a commercially important pathology in papaya for which there are currently no resistant varieties. PMeV has a number of unusual features, such as residence in the laticifers of infected plants, and the response of the papaya to PMeV infection is not well understood. The protein levels of 20S proteasome subunits increase during PMeV infection, suggesting that proteolysis could be an important aspect of the plant defense response mechanism. To date, 10,598 plant microRNAs have been identified in the Plant miRNAs Database, but only two, miR162 and miR403, are from papaya. In this study, known plant microRNA sequences were used to search for potential microRNAs in the papaya genome. A total of 462 microRNAs, representing 72 microRNA families, were identified. The expression of 11 microRNAs, whose targets are involved in 20S and 26S proteasomal degradation and in other stress response pathways, was compared by real-time PCR in healthy and infected papaya leaf tissue. We found that the expression of miRNAs involved in proteasomal degradation increased in response to very low levels of PMeV titre and decreased as the viral titre increased. In contrast, miRNAs implicated in the plant response to biotic stress decreased their expression at very low level of PMeV and increased at high PMeV levels. Corroborating with this results, analysed target genes for this miRNAs had their expression modulated in a dependent manner. This study represents a comprehensive identification of conserved miRNAs inpapaya. The data presented here might help to complement the available molecular and genomic tools for the study of papaya. The differential expression of some miRNAs and identifying their target genes will be helpful for understanding the regulation and interaction of PMeV and papaya.

Carica papaya MicroRNAs Are Responsive to Papaya meleira virus Infection

PubMed Central

Abreu, Paolla M. V.; Gaspar, Clicia G.; Buss, David S.; Ventura, José A.; Ferreira, Paulo C. G.; Fernandes, Patricia M. B.

2014-01-01

MicroRNAs are implicated in the response to biotic stresses. Papaya meleira virus (PMeV) is the causal agent of sticky disease, a commercially important pathology in papaya for which there are currently no resistant varieties. PMeV has a number of unusual features, such as residence in the laticifers of infected plants, and the response of the papaya to PMeV infection is not well understood. The protein levels of 20S proteasome subunits increase during PMeV infection, suggesting that proteolysis could be an important aspect of the plant defense response mechanism. To date, 10,598 plant microRNAs have been identified in the Plant miRNAs Database, but only two, miR162 and miR403, are from papaya. In this study, known plant microRNA sequences were used to search for potential microRNAs in the papaya genome. A total of 462 microRNAs, representing 72 microRNA families, were identified. The expression of 11 microRNAs, whose targets are involved in 20S and 26S proteasomal degradation and in other stress response pathways, was compared by real-time PCR in healthy and infected papaya leaf tissue. We found that the expression of miRNAs involved in proteasomal degradation increased in response to very low levels of PMeV titre and decreased as the viral titre increased. In contrast, miRNAs implicated in the plant response to biotic stress decreased their expression at very low level of PMeV and increased at high PMeV levels. Corroborating with this results, analysed target genes for this miRNAs had their expression modulated in a dependent manner. This study represents a comprehensive identification of conserved miRNAs inpapaya. The data presented here might help to complement the available molecular and genomic tools for the study of papaya. The differential expression of some miRNAs and identifying their target genes will be helpful for understanding the regulation and interaction of PMeV and papaya. PMID:25072834

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.

Targeting Non-Coding RNAs in Plants with the CRISPR-Cas Technology is a Challenge yet Worth Accepting.

PubMed

Basak, Jolly; Nithin, Chandran

2015-01-01

Non-coding RNAs (ncRNAs) have emerged as versatile master regulator of biological functions in recent years. MicroRNAs (miRNAs) are small endogenous ncRNAs of 18-24 nucleotides in length that originates from long self-complementary precursors. Besides their direct involvement in developmental processes, plant miRNAs play key roles in gene regulatory networks and varied biological processes. Alternatively, long ncRNAs (lncRNAs) are a large and diverse class of transcribed ncRNAs whose length exceed that of 200 nucleotides. Plant lncRNAs are transcribed by different RNA polymerases, showing diverse structural features. Plant lncRNAs also are important regulators of gene expression in diverse biological processes. There has been a breakthrough in the technology of genome editing, the CRISPR-Cas9 (clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9) technology, in the last decade. CRISPR loci are transcribed into ncRNA and eventually form a functional complex with Cas9 and further guide the complex to cleave complementary invading DNA. The CRISPR-Cas technology has been successfully applied in model plants such as Arabidopsis and tobacco and important crops like wheat, maize, and rice. However, all these studies are focused on protein coding genes. Information about targeting non-coding genes is scarce. Hitherto, the CRISPR-Cas technology has been exclusively used in vertebrate systems to engineer miRNA/lncRNAs, but it is still relatively unexplored in plants. While briefing miRNAs, lncRNAs and applications of the CRISPR-Cas technology in human and animals, this review essentially elaborates several strategies to overcome the challenges of applying the CRISPR-Cas technology in editing ncRNAs in plants and the future perspective of this field.

Transcriptional, post-transcriptional and chromatin-associated regulation of pri-miRNAs, pre-miRNAs and moRNAs

PubMed Central

Nepal, Chirag; Coolen, Marion; Hadzhiev, Yavor; Cussigh, Delphine; Mydel, Piotr; Steen, Vidar M.; Carninci, Piero; Andersen, Jesper B.; Bally-Cuif, Laure; Müller, Ferenc; Lenhard, Boris

2016-01-01

MicroRNAs (miRNAs) play a major role in the post-transcriptional regulation of target genes, especially in development and differentiation. Our understanding about the transcriptional regulation of miRNA genes is limited by inadequate annotation of primary miRNA (pri-miRNA) transcripts. Here, we used CAGE-seq and RNA-seq to provide genome-wide identification of the pri-miRNA core promoter repertoire and its dynamic usage during zebrafish embryogenesis. We assigned pri-miRNA promoters to 152 precursor-miRNAs (pre-miRNAs), the majority of which were supported by promoter associated post-translational histone modifications (H3K4me3, H2A.Z) and RNA polymerase II (RNAPII) occupancy. We validated seven miR-9 pri-miRNAs by in situ hybridization and showed similar expression patterns as mature miR-9. In addition, processing of an alternative intronic promoter of miR-9–5 was validated by 5′ RACE PCR. Developmental profiling revealed a subset of pri-miRNAs that are maternally inherited. Moreover, we show that promoter-associated H3K4me3, H2A.Z and RNAPII marks are not only present at pri-miRNA promoters but are also specifically enriched at pre-miRNAs, suggesting chromatin level regulation of pre-miRNAs. Furthermore, we demonstrated that CAGE-seq also detects 3′-end processing of pre-miRNAs on Drosha cleavage site that correlates with miRNA-offset RNAs (moRNAs) production and provides a new tool for detecting Drosha processing events and predicting pre-miRNA processing by a genome-wide assay. PMID:26673698

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.

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 in brain development

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

Petri, Rebecca; Malmevik, Josephine; Fasching, Liana

2014-02-01

MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. In the brain, a large number of miRNAs are expressed and there is a growing body of evidence demonstrating that miRNAs are essential for brain development and neuronal function. Conditional knockout studies of the core components in the miRNA biogenesis pathway, such as Dicer and DGCR8, have demonstrated a crucial role for miRNAs during the development of the central nervous system. Furthermore, mice deleted for specific miRNAs and miRNA-clusters demonstrate diverse functional roles for different miRNAs during the development of different brain structures. miRNAs havemore » been proposed to regulate cellular functions such as differentiation, proliferation and fate-determination of neural progenitors. In this review we summarise the findings from recent studies that highlight the importance of miRNAs in brain development with a focus on the mouse model. We also discuss the technical limitations of current miRNA studies that still limit our understanding of this family of non-coding RNAs and propose the use of novel and refined technologies that are needed in order to fully determine the impact of specific miRNAs in brain development. - Highlights: • miRNAs are essential for brain development and neuronal function. • KO of Dicer is embryonically lethal. • Conditional Dicer KO results in defective proliferation or increased apoptosis. • KO of individual miRNAs or miRNA families is necessary to determine function.« less

Deregulation of microRNA-181c in cerebrospinal fluid of patients with clinically isolated syndrome is associated with early conversion to relapsing-remitting multiple sclerosis.

PubMed

Ahlbrecht, Jonas; Martino, Filippo; Pul, Refik; Skripuletz, Thomas; Sühs, Kurt-Wolfram; Schauerte, Celina; Yildiz, Özlem; Trebst, Corinna; Tasto, Lars; Thum, Sabrina; Pfanne, Angelika; Roesler, Romy; Lauda, Florian; Hecker, Michael; Zettl, Uwe K; Tumani, Hayrettin; Thum, Thomas; Stangel, Martin

2016-08-01

MiRNA-181c, miRNA-633 and miRNA-922 have been reported to be deregulated in multiple sclerosis. To investigate the association between miRNA-181c, miRNA-633 and miRNA-922 and conversion from clinically isolated syndrome (CIS) to relapsing-remitting multiple sclerosis (RRMS); and to compare microRNAs in cerebrospinal fluid (CSF) and serum with regard to dysfunction of the blood-CSF barrier. CSF and serum miRNA-181c, miRNA-633 and miRNA-922 were retrospectively determined by quantitative real-time polymerase chain reaction in CIS patients with (CIS-RRMS) and without (CIS-CIS) conversion to RRMS within 1 year. Thirty of 58 CIS patients developed RRMS. Cerebrospinal fluid miRNA-922, serum miRNA-922 and cerebrospinal fluid miRNA-181c were significantly higher in CIS-RRMS compared to CIS-CIS (P=0.027, P=0.048, P=0.029, respectively). High levels of cerebrospinal fluid miRNA-181c were independently associated with conversion from CIS to RRMS in multivariate Cox regression analysis (hazard ratio 2.99, 95% confidence interval 1.41-6.34, P=0.005). A combination of high cerebrospinal fluid miRNA-181c, younger age and more than nine lesions on magnetic resonance imaging showed the highest specificity (96%) and positive predictive value (94%) for conversion from CIS to RRMS. MiRNA-181c was higher in serum than in cerebrospinal fluid (P <0.001), while miRNA-633 and miRNA-922 were no different in cerebrospinal fluid and serum. Cerebrospinal fluid/serum albumin quotients did not correlate with microRNAs in cerebrospinal fluid (all P>0.711). Cerebrospinal fluid miRNA-181c might serve as a biomarker for early conversion to RRMS. Moreover, our data suggest an intrathecal origin of microRNAs detected in the cerebrospinal fluid. © The Author(s), 2015.

miRNA*: a passenger stranded in RNA-induced silencing complex?

PubMed

Mah, S M; Buske, C; Humphries, R K; Kuchenbauer, F

2010-01-01

Processing of the pre-microRNA (pre-miRNA) through Dicer1 generates a miRNA duplex, consisting of a miRNA and miRNA* strand (also termed guide strand and passenger strand, respectively). Despite the general consensus that miRNA*s have no regulatory activity, recent publications have provided evidence that the abundance, possible function, and physiological relevance of miRNA*s have been underestimated. This review provides an account of our current understanding of miRNA* origination and activity, mounting evidence for their unique functions and regulatory mechanisms, and examples of specific miRNA*s from the literature.

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.

Monitoring the Spatiotemporal Activities of miRNAs in Small Animal Models Using Molecular Imaging Modalities

PubMed Central

Baril, Patrick; Ezzine, Safia; Pichon, Chantal

2015-01-01

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression by binding mRNA targets via sequence complementary inducing translational repression and/or mRNA degradation. A current challenge in the field of miRNA biology is to understand the functionality of miRNAs under physiopathological conditions. Recent evidence indicates that miRNA expression is more complex than simple regulation at the transcriptional level. MiRNAs undergo complex post-transcriptional regulations such miRNA processing, editing, accumulation and re-cycling within P-bodies. They are dynamically regulated and have a well-orchestrated spatiotemporal localization pattern. Real-time and spatio-temporal analyses of miRNA expression are difficult to evaluate and often underestimated. Therefore, important information connecting miRNA expression and function can be lost. Conventional miRNA profiling methods such as Northern blot, real-time PCR, microarray, in situ hybridization and deep sequencing continue to contribute to our knowledge of miRNA biology. However, these methods can seldom shed light on the spatiotemporal organization and function of miRNAs in real-time. Non-invasive molecular imaging methods have the potential to address these issues and are thus attracting increasing attention. This paper reviews the state-of-the-art of methods used to detect miRNAs and discusses their contribution in the emerging field of miRNA biology and therapy. PMID:25749473

Monitoring the spatiotemporal activities of miRNAs in small animal models using molecular imaging modalities.

PubMed

Baril, Patrick; Ezzine, Safia; Pichon, Chantal

2015-03-04

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression by binding mRNA targets via sequence complementary inducing translational repression and/or mRNA degradation. A current challenge in the field of miRNA biology is to understand the functionality of miRNAs under physiopathological conditions. Recent evidence indicates that miRNA expression is more complex than simple regulation at the transcriptional level. MiRNAs undergo complex post-transcriptional regulations such miRNA processing, editing, accumulation and re-cycling within P-bodies. They are dynamically regulated and have a well-orchestrated spatiotemporal localization pattern. Real-time and spatio-temporal analyses of miRNA expression are difficult to evaluate and often underestimated. Therefore, important information connecting miRNA expression and function can be lost. Conventional miRNA profiling methods such as Northern blot, real-time PCR, microarray, in situ hybridization and deep sequencing continue to contribute to our knowledge of miRNA biology. However, these methods can seldom shed light on the spatiotemporal organization and function of miRNAs in real-time. Non-invasive molecular imaging methods have the potential to address these issues and are thus attracting increasing attention. This paper reviews the state-of-the-art of methods used to detect miRNAs and discusses their contribution in the emerging field of miRNA biology and therapy.

MiRNA-miRNA synergistic network: construction via co-regulating functional modules and disease miRNA topological features.

PubMed

Xu, Juan; Li, Chuan-Xing; Li, Yong-Sheng; Lv, Jun-Ying; Ma, Ye; Shao, Ting-Ting; Xu, Liang-De; Wang, Ying-Ying; Du, Lei; Zhang, Yun-Peng; Jiang, Wei; Li, Chun-Quan; Xiao, Yun; Li, Xia

2011-02-01

Synergistic regulations among multiple microRNAs (miRNAs) are important to understand the mechanisms of complex post-transcriptional regulations in humans. Complex diseases are affected by several miRNAs rather than a single miRNA. So, it is a challenge to identify miRNA synergism and thereby further determine miRNA functions at a system-wide level and investigate disease miRNA features in the miRNA-miRNA synergistic network from a new view. Here, we constructed a miRNA-miRNA functional synergistic network (MFSN) via co-regulating functional modules that have three features: common targets of corresponding miRNA pairs, enriched in the same gene ontology category and close proximity in the protein interaction network. Predicted miRNA synergism is validated by significantly high co-expression of functional modules and significantly negative regulation to functional modules. We found that the MFSN exhibits a scale free, small world and modular architecture. Furthermore, the topological features of disease miRNAs in the MFSN are distinct from non-disease miRNAs. They have more synergism, indicating their higher complexity of functions and are the global central cores of the MFSN. In addition, miRNAs associated with the same disease are close to each other. The structure of the MFSN and the features of disease miRNAs are validated to be robust using different miRNA target data sets.

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

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.

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.

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.

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.

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

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.

MicroRNA expression profiling of human breast cancer identifies new markers of tumor subtype.

PubMed

Blenkiron, Cherie; Goldstein, Leonard D; Thorne, Natalie P; Spiteri, Inmaculada; Chin, Suet-Feung; Dunning, Mark J; Barbosa-Morais, Nuno L; Teschendorff, Andrew E; Green, Andrew R; Ellis, Ian O; Tavaré, Simon; Caldas, Carlos; Miska, Eric A

2007-01-01

MicroRNAs (miRNAs), a class of short non-coding RNAs found in many plants and animals, often act post-transcriptionally to inhibit gene expression. Here we report the analysis of miRNA expression in 93 primary human breast tumors, using a bead-based flow cytometric miRNA expression profiling method. Of 309 human miRNAs assayed, we identify 133 miRNAs expressed in human breast and breast tumors. We used mRNA expression profiling to classify the breast tumors as luminal A, luminal B, basal-like, HER2+ and normal-like. A number of miRNAs are differentially expressed between these molecular tumor subtypes and individual miRNAs are associated with clinicopathological factors. Furthermore, we find that miRNAs could classify basal versus luminal tumor subtypes in an independent data set. In some cases, changes in miRNA expression correlate with genomic loss or gain; in others, changes in miRNA expression are likely due to changes in primary transcription and or miRNA biogenesis. Finally, the expression of DICER1 and AGO2 is correlated with tumor subtype and may explain some of the changes in miRNA expression observed. This study represents the first integrated analysis of miRNA expression, mRNA expression and genomic changes in human breast cancer and may serve as a basis for functional studies of the role of miRNAs in the etiology of breast cancer. Furthermore, we demonstrate that bead-based flow cytometric miRNA expression profiling might be a suitable platform to classify breast cancer into prognostic molecular subtypes.

MicroRNA therapeutics: towards a new era for the management of cancer and other diseases.

PubMed

Rupaimoole, Rajesha; Slack, Frank J

2017-03-01

In just over two decades since the discovery of the first microRNA (miRNA), the field of miRNA biology has expanded considerably. Insights into the roles of miRNAs in development and disease, particularly in cancer, have made miRNAs attractive tools and targets for novel therapeutic approaches. Functional studies have confirmed that miRNA dysregulation is causal in many cases of cancer, with miRNAs acting as tumour suppressors or oncogenes (oncomiRs), and miRNA mimics and molecules targeted at miRNAs (antimiRs) have shown promise in preclinical development. Several miRNA-targeted therapeutics have reached clinical development, including a mimic of the tumour suppressor miRNA miR-34, which reached phase I clinical trials for treating cancer, and antimiRs targeted at miR-122, which reached phase II trials for treating hepatitis. In this article, we describe recent advances in our understanding of miRNAs in cancer and in other diseases and provide an overview of current miRNA therapeutics in the clinic. We also discuss the challenge of identifying the most efficacious therapeutic candidates and provide a perspective on achieving safe and targeted delivery of miRNA therapeutics.

A whole-mount in situ hybridization method for microRNA detection in Caenorhabditis elegans

PubMed Central

Andachi, Yoshiki; Kohara, Yuji

2016-01-01

Whole-mount in situ hybridization (WISH) is an outstanding method to decipher the spatiotemporal expression patterns of microRNAs (miRNAs) and provides important clues for elucidating their functions. The first WISH method for miRNA detection was developed in zebrafish. Although this method was quickly adapted for other vertebrates and fruit flies, WISH analysis has not been successfully used to detect miRNAs in Caenorhabditis elegans. Here, we show a novel WISH method for miRNA detection in C. elegans. Using this method, mir-1 miRNA was detected in the body-wall muscle where the expression and roles of mir-1 miRNA have been previously elucidated. Application of the method to let-7 family miRNAs, let-7, mir-48, mir-84, and mir-241, revealed their distinct but partially overlapping expression patterns, indicating that miRNAs sharing a short common sequence were distinguishably detected. In pash-1 mutants that were depleted of mature miRNAs, signals of mir-48 miRNA were greatly reduced, suggesting that mature miRNAs were detected by the method. These results demonstrate the validity of WISH to detect mature miRNAs in C. elegans. PMID:27154969

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

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

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.

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

Random walks on mutual microRNA-target gene interaction network improve the prediction of disease-associated microRNAs.

PubMed

Le, Duc-Hau; Verbeke, Lieven; Son, Le Hoang; Chu, Dinh-Toi; Pham, Van-Huy

2017-11-14

MicroRNAs (miRNAs) have been shown to play an important role in pathological initiation, progression and maintenance. Because identification in the laboratory of disease-related miRNAs is not straightforward, numerous network-based methods have been developed to predict novel miRNAs in silico. Homogeneous networks (in which every node is a miRNA) based on the targets shared between miRNAs have been widely used to predict their role in disease phenotypes. Although such homogeneous networks can predict potential disease-associated miRNAs, they do not consider the roles of the target genes of the miRNAs. Here, we introduce a novel method based on a heterogeneous network that not only considers miRNAs but also the corresponding target genes in the network model. Instead of constructing homogeneous miRNA networks, we built heterogeneous miRNA networks consisting of both miRNAs and their target genes, using databases of known miRNA-target gene interactions. In addition, as recent studies demonstrated reciprocal regulatory relations between miRNAs and their target genes, we considered these heterogeneous miRNA networks to be undirected, assuming mutual miRNA-target interactions. Next, we introduced a novel method (RWRMTN) operating on these mutual heterogeneous miRNA networks to rank candidate disease-related miRNAs using a random walk with restart (RWR) based algorithm. Using both known disease-associated miRNAs and their target genes as seed nodes, the method can identify additional miRNAs involved in the disease phenotype. Experiments indicated that RWRMTN outperformed two existing state-of-the-art methods: RWRMDA, a network-based method that also uses a RWR on homogeneous (rather than heterogeneous) miRNA networks, and RLSMDA, a machine learning-based method. Interestingly, we could relate this performance gain to the emergence of "disease modules" in the heterogeneous miRNA networks used as input for the algorithm. Moreover, we could demonstrate that RWRMTN is stable, performing well when using both experimentally validated and predicted miRNA-target gene interaction data for network construction. Finally, using RWRMTN, we identified 76 novel miRNAs associated with 23 disease phenotypes which were present in a recent database of known disease-miRNA associations. Summarizing, using random walks on mutual miRNA-target networks improves the prediction of novel disease-associated miRNAs because of the existence of "disease modules" in these networks.

Dicer Cooperates with p53 to Suppress DNA Damage and Skin Carcinogenesis in Mice

PubMed Central

Lyle, Stephen; Hoover, Kathleen; Colpan, Cansu; Zhu, Zhiqing; Matijasevic, Zdenka; Jones, Stephen N.

2014-01-01

Dicer is required for the maturation of microRNA, and loss of Dicer and miRNA processing has been found to alter numerous biological events during embryogenesis, including the development of mammalian skin and hair. We have previously examined the role of miRNA biogenesis in mouse embryonic fibroblasts and found that deletion of Dicer induces cell senescence regulated, in part, by the p53 tumor suppressor. Although Dicer and miRNA molecules are thought to have either oncogenic or tumor suppressing roles in various types of cancer, a role for Dicer and miRNAs in skin carcinogenesis has not been established. Here we show that perinatal ablation of Dicer in the skin of mice leads to loss of fur in adult mice, increased epidermal cell proliferation and apoptosis, and the accumulation of widespread DNA damage in epidermal cells. Co-ablation of Dicer and p53 did not alter the timing or extent of fur loss, but greatly reduced survival of Dicer-skin ablated mice, as these mice developed multiple and highly aggressive skin carcinomas. Our results describe a new mouse model for spontaneous basal and squamous cell tumorigenesis. Furthermore, our findings reveal that loss of Dicer in the epidermis induces extensive DNA damage, activation of the DNA damage response and p53-dependent apoptosis, and that Dicer and p53 cooperate to suppress mammalian skin carcinogenesis. PMID:24979267

Development of a miRNA surface-enhanced Raman scattering assay using benchtop and handheld Raman systems

NASA Astrophysics Data System (ADS)

Schechinger, Monika; Marks, Haley; Locke, Andrea; Choudhury, Mahua; Cote, Gerard

2018-01-01

DNA-functionalized nanoparticles, when paired with surface-enhanced Raman spectroscopy (SERS), can rapidly detect microRNA. However, widespread use of this approach is hindered by drawbacks associated with large and expensive benchtop Raman microscopes. MicroRNA-17 (miRNA-17) has emerged as a potential epigenetic indicator of preeclampsia, a condition that occurs during pregnancy. Biomarker detection using an SERS point-of-care device could enable prompt diagnosis and prevention as early as the first trimester. Recently, strides have been made in developing portable Raman systems for field applications. An SERS assay for miRNA-17 was assessed and translated from traditional benchtop Raman microscopes to a handheld system. Three different photoactive molecules were compared as potential Raman reporter molecules: a chromophore, malachite green isothiocyanate (MGITC), a fluorophore, tetramethylrhodamine isothiocyanate, and a polarizable small molecule 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB). For the benchtop Raman microscope, the DTNB-labeled assay yielded the greatest sensitivity under 532-nm laser excitation, but the MGITC-labeled assay prevailed at 785 nm. Conversely, DTNB was preferable for the miniaturized 785-nm Raman system. This comparison showed significant SERS enhancement variation in response to 1-nM miRNA-17, implying that the sensitivity of the assay may be more heavily dependent on the excitation wavelength, instrumentation, and Raman reporter chosen than on the plasmonic coupling from DNA/miRNA-mediated nanoparticle assemblies.

Downregulation of carnitine acyl-carnitine translocase by miRNAs 132 and 212 amplifies glucose-stimulated insulin secretion.

PubMed

Soni, Mufaddal S; Rabaglia, Mary E; Bhatnagar, Sushant; Shang, Jin; Ilkayeva, Olga; Mynatt, Randall; Zhou, Yun-Ping; Schadt, Eric E; Thornberry, Nancy A; Muoio, Deborah M; Keller, Mark P; Attie, Alan D

2014-11-01

We previously demonstrated that micro-RNAs (miRNAs) 132 and 212 are differentially upregulated in response to obesity in two mouse strains that differ in their susceptibility to obesity-induced diabetes. Here we show the overexpression of miRNAs 132 and 212 enhances insulin secretion (IS) in response to glucose and other secretagogues including nonfuel stimuli. We determined that carnitine acyl-carnitine translocase (CACT; Slc25a20) is a direct target of these miRNAs. CACT is responsible for transporting long-chain acyl-carnitines into the mitochondria for β-oxidation. Small interfering RNA-mediated knockdown of CACT in β-cells led to the accumulation of fatty acyl-carnitines and enhanced IS. The addition of long-chain fatty acyl-carnitines promoted IS from rat insulinoma β-cells (INS-1) as well as primary mouse islets. The effect on INS-1 cells was augmented in response to suppression of CACT. A nonhydrolyzable ether analog of palmitoyl-carnitine stimulated IS, showing that β-oxidation of palmitoyl-carnitine is not required for its stimulation of IS. These studies establish a link between miRNA-dependent regulation of CACT and fatty acyl-carnitine-mediated regulation of IS. © 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Drosha Promotes Splicing of a Pre-microRNA-like Alternative Exon

PubMed Central

Havens, Mallory A.; Reich, Ashley A.; Hastings, Michelle L.

2014-01-01

The ribonuclease III enzyme Drosha has a central role in the biogenesis of microRNA (miRNA) by binding and cleaving hairpin structures in primary RNA transcripts into precursor miRNAs (pre-miRNAs). Many miRNA genes are located within protein-coding host genes and cleaved by Drosha in a manner that is coincident with splicing of introns by the spliceosome. The close proximity of splicing and pre-miRNA biogenesis suggests a potential for co-regulation of miRNA and host gene expression, though this relationship is not completely understood. Here, we describe a cleavage-independent role for Drosha in the splicing of an exon that has a predicted hairpin structure resembling a Drosha substrate. We find that Drosha can cleave the alternatively spliced exon 5 of the eIF4H gene into a pre-miRNA both in vitro and in cells. However, the primary role of Drosha in eIF4H gene expression is to promote the splicing of exon 5. Drosha binds to the exon and enhances splicing in a manner that depends on RNA structure but not on cleavage by Drosha. We conclude that Drosha can function like a splicing enhancer and promote exon inclusion. Our results reveal a new mechanism of alternative splicing regulation involving a cleavage-independent role for Drosha in splicing. PMID:24786770

Modulation of Atlantic salmon miRNome response to sea louse infestation.

PubMed

Valenzuela-Muñoz, Valentina; Novoa, Beatriz; Figueras, Antonio; Gallardo-Escárate, Cristian

2017-11-01

MicroRNAs are non-coding RNA that plays a crucial role in post-transcriptional regulation and immune system regulation. On other hand, sea lice are prevalent parasites that affect salmon farming, generating different degrees of immune suppression depending on the salmon and sea louse species. Caligus rogercresseyi for example, which affects the salmon industry in Chile, decreases Th1 response, macrophage activation, TLR-mediated response and iron regulation in infected fish. In this study, we explore Atlantic salmon miRNome during infestation by C. rogercresseyi. Using small RNA sequencing, we annotated 1718 miRNAs for skin and head kidney from infected Atlantic salmon. The most abundant families identified were mir-10, mir-21, mir-30, mir-181 and let7. Significant differences were found between tissue, with 1404 annotated miRNA in head kidney and 529 in skin. Differential analysis of transcript expression indicated that at an early stage of infestation miRNA expression was higher in head kidney than in skin tissue, revealing tissue-specific expression patterns. In parallel, miRNA target prediction using 3'UTRs from highly regulated immune-related genes and iron metabolism showed that mir-140-4 and mir-181a-2-5 modulate the expression of TLR22 and Aminolevulinic acid synthase, respectively. This study contributes knowledge about the immune response of Atlantic salmon during infestation with sea lice. Copyright © 2017 Elsevier Ltd. All rights reserved.

Selection of reference genes for miRNA qRT-PCR under abiotic stress in grapevine.

PubMed

Luo, Meng; Gao, Zhen; Li, Hui; Li, Qin; Zhang, Caixi; Xu, Wenping; Song, Shiren; Ma, Chao; Wang, Shiping

2018-03-13

Grapevine is among the fruit crops with high economic value, and because of the economic losses caused by abiotic stresses, the stress resistance of Vitis vinifera has become an increasingly important research area. Among the mechanisms responding to environmental stresses, the role of miRNA has received much attention recently. qRT-PCR is a powerful method for miRNA quantitation, but the accuracy of the method strongly depends on the appropriate reference genes. To determine the most suitable reference genes for grapevine miRNA qRT-PCR, 15 genes were chosen as candidate reference genes. After eliminating 6 candidate reference genes with unsatisfactory amplification efficiency, the expression stability of the remaining candidate reference genes under salinity, cold and drought was analysed using four algorithms, geNorm, NormFinder, deltaCt and Bestkeeper. The results indicated that U6 snRNA was the most suitable reference gene under salinity and cold stresses; whereas miR168 was the best for drought stress. The best reference gene sets for salinity, cold and drought stresses were miR160e + miR164a, miR160e + miR168 and ACT + UBQ + GAPDH, respectively. The selected reference genes or gene sets were verified using miR319 or miR408 as the target gene.

MicroRNA biogenesis and function in plants.

PubMed

Chen, Xuemei

2005-10-31

A microRNA (miRNA) is a 21-24 nucleotide RNA product of a non-protein-coding gene. Plants, like animals, have a large number of miRNA-encoding genes in their genomes. The biogenesis of miRNAs in Arabidopsis is similar to that in animals in that miRNAs are processed from primary precursors by at least two steps mediated by RNAse III-like enzymes and that the miRNAs are incorporated into a protein complex named RISC. However, the biogenesis of plant miRNAs consists of an additional step, i.e., the miRNAs are methylated on the ribose of the last nucleotide by the miRNA methyltransferase HEN1. The high degree of sequence complementarity between plant miRNAs and their target mRNAs has facilitated the bioinformatic prediction of miRNA targets, many of which have been subsequently validated. Plant miRNAs have been predicted or confirmed to regulate a variety of processes, such as development, metabolism, and stress responses. A large category of miRNA targets consists of genes encoding transcription factors that play important roles in patterning the plant form.

Identification and profiling of conserved and novel microRNAs involved in oil and oleic acid production during embryogenesis in Carya cathayensis Sarg.

PubMed

Wang, Zhengjia; Huang, Ruiming; Sun, Zhichao; Zhang, Tong; Huang, Jianqin

2017-05-01

MicroRNAs (miRNAs) are important regulators of plant development and fruit formation. Mature embryos of hickory (Carya cathayensis Sarg.) nuts contain more than 70% oil (comprising 90% unsaturated fatty acids), along with a substantial amount of oleic acid. To understand the roles of miRNAs involved in oil and oleic acid production during hickory embryogenesis, three small RNA libraries from different stages of embryogenesis were constructed. Deep sequencing of these three libraries identified 95 conserved miRNAs with 19 miRNA*s, 7 novel miRNAs (as well as their corresponding miRNA*s), and 26 potentially novel miRNAs. The analysis identified 15 miRNAs involved in oil and oleic acid production that are differentially expressed during embryogenesis in hickory. Among them, nine miRNA sequences, including eight conserved and one novel, were confirmed by qRT-PCR. In addition, 145 target genes of the novel miRNAs were predicted using a bioinformatic approach. Our results provide a framework for better understanding the roles of miRNAs during embryogenesis in hickory.

Genome-wide analysis of miRNAs in Carya cathayensis.

PubMed

Sun, Zhi-Chao; Zhang, Liang-Sheng; Wang, Zheng-Jia

2017-11-29

MicroRNA (miRNA) plays an important role in plant development regulation. Hickory is an economically important plant in which the amount of flowering determines its production. Here, 51 conserved miRNAs, which belong to 16 families and 195 novel miRNAs were identified in hickory genome. For each conserved miRNA family, we used sequences from hickory and other plants to construct a phylogenetic tree, which shows that each family has members in hickory. Some of the conserved miRNA families (i.e., miR167 and miR397) have more members in hickory than in other plants because of gene expansion. MiR166 exhibited tandem duplication with three copies being observed. Many members of these conserved miRNA families were detected in hickory flowers, and the expression patterns of target genes were opposite to those of the related miRNAs, indicating that miRNAs may have important functions in floral regulation of hickory. Taken together, a comprehensive analysis was conducted to identify miRNAs produced in hickory flower organs, demonstrating functional conservation and diversity of miRNA families among hickory, Arabidopsis, grape, and poplar.

MicroRNAs: A Puzzling Tool in Cancer Diagnostics and Therapy.

PubMed

D'Angelo, Barbara; Benedetti, Elisabetta; Cimini, Annamaria; Giordano, Antonio

2016-11-01

MicroRNAs (miRNAs) constitute a dominating class of small RNAs that regulate diverse cellular functions. Due the pivotal role of miRNAs in biological processes, a deregulated miRNA expression is likely involved in human cancers. MicroRNAs possess tumor suppressor capability, as well as display oncogenic characteristics. Interestingly, miRNAs exist in various biological fluids as circulating entities. Changes in the profile of circulating miRNAs are indicative of pathophysiological conditions in human cancer. This concept has led to consider circulating miRNAs valid biomarkers in cancer diagnostics. Furthermore, current research promotes the use of miRNAs as a target in cancer therapy. However, miRNAs are an evolving research field. Although miRNAs have been demonstrated to be potentially valuable tools both in cancer diagnosis and treatment, a greater effort should be made to improve our understanding of miRNAs biology. This review describes the biology of microRNAs, emphasizing on the use of miRNAs in cancer diagnostics and therapy. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Dissecting microRNA dysregulation in age-related macular degeneration: new targets for eye gene therapy.

PubMed

Askou, Anne Louise; Alsing, Sidsel; Holmgaard, Andreas; Bek, Toke; Corydon, Thomas J

2018-02-01

MicroRNAs (miRNAs) are key regulators of gene expression in humans. Overexpression or depletion of individual miRNAs is associated with human disease. Current knowledge suggests that the retina is influenced by miRNAs and that dysregulation of miRNAs as well as alterations in components of the miRNA biogenesis machinery are involved in retinal diseases, including age-related macular degeneration (AMD). Furthermore, recent studies have indicated that the vitreous has a specific panel of circulating miRNAs and that this panel varies according to the specific pathological stress experienced by the retinal cells. MicroRNA (miRNA) profiling indicates subtype-specific miRNA profiles for late-stage AMD highlighting the importance of proper miRNA regulation in AMD. This review will describe the function of important miRNAs involved in inflammation, oxidative stress and pathological neovascularization, the key molecular mechanisms leading to AMD, and focus on dysregulated miRNAs as potential therapeutic targets in AMD. © 2017 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

Analysis of expression of microRNAs and genes involved in the control of key signaling mechanisms that support or inhibit development of brain tumors of different grades.

PubMed

Koshkin, Philip Alexandrovich; Chistiakov, Dimitry Alexandrovich; Nikitin, Alexey Georgievich; Konovalov, Alexander Nikolaevich; Potapov, Alexander Alexandrovich; Usachev, Dmitry Yrevich; Pitskhelauri, David Ilich; Kobyakov, Gregory Lvovich; Shishkina, Lyudmila Valentinovna; Chekhonin, Vladimir Pavlovich

2014-03-20

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules involved in the regulation of key biological processes. Different miRNAs with pro-oncogenic and anti-oncogenic properties have been identified in glioblastomas. We decided to analyze expression profiles of 10 mature miRNAs (miR-7-1, miR-10а, miR-17, miR-20а, miR-21, miR-23а, miR-26а, miR-137, and miR-222) in post-surgery glioma specimens of different grades in order to find whether the expression level correlates with tumor grades. We also measured expression of six key genes such as PTEN, p21/CDKN1A, MDR1, ABCG2, BAX, and BCL-2 involved in the regulation of critical glioma signaling pathways to establish the effect of miRNAs on these signaling mechanisms. Using RT-PCR, we performed expression analysis of 25 tumor fresh samples (grades II-IV). We found gradual increase in miR-21 and miR-23a levels in all tumor grades whereas miR-7 and miR-137 were significantly down-regulated depending on the glioma grade. MDR, ABCG2, and p21/CDKN1A levels were significantly up-regulated while expression of PTEN was down-regulated in tumor samples compared to the normal brain tissue. These observations provide new insights into molecular pathogenic mechanisms of glioma progression and suggest about a potential value of miRNAs as a putative diagnostic marker of brain tumors. Copyright © 2014 Elsevier B.V. All rights reserved.