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Sample records for interfering rna increases

  1. NBS1 knockdown by small interfering RNA increases ionizing radiation mutagenesis and telomere association in human cells

    NASA Technical Reports Server (NTRS)

    Zhang, Ying; Lim, Chang U K.; Williams, Eli S.; Zhou, Junqing; Zhang, Qinming; Fox, Michael H.; Bailey, Susan M.; Liber, Howard L.

    2005-01-01

    Hypomorphic mutations which lead to decreased function of the NBS1 gene are responsible for Nijmegen breakage syndrome, a rare autosomal recessive hereditary disorder that imparts an increased predisposition to development of malignancy. The NBS1 protein is a component of the MRE11/RAD50/NBS1 complex that plays a critical role in cellular responses to DNA damage and the maintenance of chromosomal integrity. Using small interfering RNA transfection, we have knocked down NBS1 protein levels and analyzed relevant phenotypes in two closely related human lymphoblastoid cell lines with different p53 status, namely wild-type TK6 and mutated WTK1. Both TK6 and WTK1 cells showed an increased level of ionizing radiation-induced mutation at the TK and HPRT loci, impaired phosphorylation of H2AX (gamma-H2AX), and impaired activation of the cell cycle checkpoint regulating kinase, Chk2. In TK6 cells, ionizing radiation-induced accumulation of p53/p21 and apoptosis were reduced. There was a differential response to ionizing radiation-induced cell killing between TK6 and WTK1 cells after NBS1 knockdown; TK6 cells were more resistant to killing, whereas WTK1 cells were more sensitive. NBS1 deficiency also resulted in a significant increase in telomere association that was independent of radiation exposure and p53 status. Our results provide the first experimental evidence that NBS1 deficiency in human cells leads to hypermutability and telomere associations, phenotypes that may contribute to the cancer predisposition seen among patients with this disease.

  2. Lipid nanoparticles for short interfering RNA delivery.

    PubMed

    Leung, Alex K K; Tam, Yuen Yi C; Cullis, Pieter R

    2014-01-01

    The discovery of RNA interference (RNAi) in mammalian cells has created a new class of therapeutics based on the reversible silencing of specific disease-causing genes. This therapeutic potential depends on the ability to deliver inducers of RNAi, such as short-interfering RNA (siRNA) and micro-RNA (miRNA), to cells of target tissues. This chapter reviews various challenges and delivery strategies for siRNA, with a particular focus on the development of lipid nanoparticle (LNP) delivery technologies. Currently, LNP delivery systems are the most advanced technology for systemic delivery of siRNA, with numerous formulations under various stages of clinical trials. We also discuss methods to improve gene silencing potency of LNP-siRNA, as well as application of LNP technologies beyond siRNA to the encapsulation of other nucleic acids such as mRNA and clustered regularly interspaced short palindromic repeats (CRISPR).

  3. Small interfering RNA delivery through positively charged polymer nanoparticles

    NASA Astrophysics Data System (ADS)

    Dragoni, Luca; Ferrari, Raffaele; Lupi, Monica; Cesana, Alberto; Falcetta, Francesca; Ubezio, Paolo; D'Incalci, Maurizio; Morbidelli, Massimo; Moscatelli, Davide

    2016-03-01

    Small interfering RNA (siRNA) is receiving increasing attention with regard to the treatment of many genetic diseases, both acquired and hereditary, such as cancer and diabetes. Being a high molecular weight (MW) polyanion, siRNA is not able to cross a cell membrane, and in addition it is unstable in physiological conditions. Accordingly, a biocompatible nanocarrier able to deliver siRNA into cells is needed. In this work, we synthesized biocompatible positively charged nanoparticles (NPs) following a two-step process that involves ring opening polymerization (ROP) and emulsion free radical polymerization (EFRP). Firstly, we proved the possibility of fine tuning the NPs’ characteristics (e.g. size and surface charge) by changing the synthetic process parameters. Then the capability in loading and delivering undamaged siRNA into a cancer cell cytoplasm has been shown. This latter process occurs through the biodegradation of the polymer constituting the NPs, whose kinetics can be tuned by adjusting the polymer’s MW. Finally, the ability of NPs to carry siRNA inside the cells in order to inhibit their target gene has been demonstrated using green flourescent protein positive cells.

  4. Therapeutic potential of small interfering RNAs/micro interfering RNA in hepatocellular carcinoma.

    PubMed

    Farra, Rossella; Grassi, Mario; Grassi, Gabriele; Dapas, Barbara

    2015-08-14

    Hepatocellular carcinoma (HCC) is the predominant form of primary liver cancer and represents the third leading cause of cancer-related death worldwide. Current available therapeutic approaches are poorly effective, especially for the advanced forms of the disease. In the last year, short double stranded RNA molecules termed small interfering RNAs (siRNAs) and micro interfering RNAs (miRNA), emerged as interesting molecules with potential therapeutic value for HCC. The practical use of these molecules is however limited by the identification of optimal molecular targets and especially by the lack of effective and targeted HCC delivery systems. Here we focus our discussion on the most recent advances in the identification of siRNAs/miRNAs molecular targets and on the development of suitable siRNA/miRNAs delivery systems.

  5. Small interfering RNA-based molecular therapy of cancers

    PubMed Central

    Guo, Wei; Chen, Wangbing; Yu, Wendan; Huang, Wenlin; Deng, Wuguo

    2013-01-01

    RNA interference (RNAi) has become a gold standard for validating gene function in basic life science research and provides a promising therapeutic modality for cancer and other diseases. This mini-review focuses on the potential of small interfering RNAs (siRNAs) in anticancer treatment, including the establishment and screening of cancer-associated siRNA libraries and their applications in anticancer drug target discovery and cancer therapy. This article also describes the current delivery approaches of siRNAs using lipids, polymers, and, in particular, gold nanoparticles to induce significant gene silencing and tumor growth regression. PMID:23327796

  6. Functional Nanostructures for Effective Delivery of Small Interfering RNA Therapeutics

    PubMed Central

    Hong, Cheol Am; Nam, Yoon Sung

    2014-01-01

    Small interfering RNA (siRNA) has proved to be a powerful tool for target-specific gene silencing via RNA interference (RNAi). Its ability to control targeted gene expression gives new hope to gene therapy as a treatment for cancers and genetic diseases. However, siRNA shows poor pharmacological properties, such as low serum stability, off-targeting, and innate immune responses, which present a significant challenge for clinical applications. In addition, siRNA cannot cross the cell membrane for RNAi activity because of its anionic property and stiff structure. Therefore, the development of a safe, stable, and efficient system for the delivery of siRNA therapeutics into the cytoplasm of targeted cells is crucial. Several nanoparticle platforms for siRNA delivery have been developed to overcome the major hurdles facing the therapeutic uses of siRNA. This review covers a broad spectrum of non-viral siRNA delivery systems developed for enhanced cellular uptake and targeted gene silencing in vitro and in vivo and discusses their characteristics and opportunities for clinical applications of therapeutic siRNA. PMID:25285170

  7. Functional nanostructures for effective delivery of small interfering RNA therapeutics.

    PubMed

    Hong, Cheol Am; Nam, Yoon Sung

    2014-01-01

    Small interfering RNA (siRNA) has proved to be a powerful tool for target-specific gene silencing via RNA interference (RNAi). Its ability to control targeted gene expression gives new hope to gene therapy as a treatment for cancers and genetic diseases. However, siRNA shows poor pharmacological properties, such as low serum stability, off-targeting, and innate immune responses, which present a significant challenge for clinical applications. In addition, siRNA cannot cross the cell membrane for RNAi activity because of its anionic property and stiff structure. Therefore, the development of a safe, stable, and efficient system for the delivery of siRNA therapeutics into the cytoplasm of targeted cells is crucial. Several nanoparticle platforms for siRNA delivery have been developed to overcome the major hurdles facing the therapeutic uses of siRNA. This review covers a broad spectrum of non-viral siRNA delivery systems developed for enhanced cellular uptake and targeted gene silencing in vitro and in vivo and discusses their characteristics and opportunities for clinical applications of therapeutic siRNA.

  8. The Drosophila hairpin RNA pathway generates endogenous short interfering RNAs

    PubMed Central

    Okamura, Katsutomo; Chung, Wei-Jen; Ruby, J. Graham; Guo, Huili; Bartel, David P.; Lai, Eric C.

    2009-01-01

    In contrast to microRNAs and Piwi-associated RNAs, short interfering RNAs (siRNAs) are seemingly dispensable for host-directed gene regulation in Drosophila. This notion is based on the fact that mutants lacking the core siRNA-generating enzyme Dicer-2 or the predominant siRNA effector Argonaute 2 are viable, fertile and of relatively normal morphology1,2. Moreover, endogenous Drosophila siRNAs have not yet been identified. Here we report that siRNAs derived from long hairpin RNA genes (hpRNAs) programme Slicer complexes that can repress endogenous target transcripts. The Drosophila hpRNA pathway is a hybrid mechanism that combines canonical RNA interference factors (Dicer-2, Hen1 (known as CG12367) and Argonaute 2) with a canonical microRNA factor (Loquacious) to generate ~21-nucleotide siRNAs. These novel regulatory RNAs reveal unexpected complexity in the sorting of small RNAs, and open a window onto the biological usage of endogenous RNA interference in Drosophila. PMID:18463630

  9. Translocation of Small Interfering RNA and Cholesterol Molecules in Biomembranes

    NASA Astrophysics Data System (ADS)

    Kalia, Rajiv

    2013-03-01

    This presentation will focus on all-atom molecular dynamics (MD) simulation studies of (1) structural and mechanical barriers to translocation of small interfering RNA (siRNA) across a phospholipid bilayer, and (2) flip-flop dynamics of cholesterol (CHOL) molecules across a phospholipid bilayer. In the first case, we find that the siRNA induces a liquid-to-gel phase transformation. In the gel phase we find large compressive lateral stresses in the hydrocarbon chains of lipid molecules, which present a considerable barrier to siRNA passage across the bilayer. In the second case, we study spontaneous CHOL inter-leaflet transport (flip-flop), the effect of this process on mechanical stresses across the bilayer, and the role of CHOL in inducing molecular order in bilayer leaflets. The simulation was run for 15 microseconds and we found 24 CHOL flip-flop events over that duration. On average, a CHOL molecule migrates across the lipid bilayer in about 73 ns after a flip-flop event is triggered. We have calculated diffusion maps and determined free energy surfaces and flip-flop mechanisms for CHOL molecules. Work supported by NSF-OCI-0749360 and NSF-IOS-125317.

  10. Characterization of defective interfering RNAs associated with RNA plant viruses

    SciTech Connect

    Morris, T.J. . School of Biological Sciences); Jackson, A.O. . Dept. of Plant Pathology)

    1993-01-01

    Our lab was the first to describe and characterize a defective interfering RNA (DI RNAs or DIs) in association with a small RNA plant virus. The features of the DIs that we discovered in infections of tomato bushy stunt virus were compatible with the properties of DIs identified in many animal virus infections. Animal virologists have generally recognized the importance of studying DIs because they are invaluable tools for identifying cis-acting sequences important in virus multiplication and because they offer the opportunity to elucidate mechanisms involved in viral persistence and disease attenuation. Hence our discovery offered a comparably valuable tool for use in plant virus studies for the first time. Since the original observation with TBSV, we discovered the second example of plant viral DI RNAs associated with turnip crinkle virus (TCV), and many other reports have now appeared characterizing DI and DI-like RNAs in other plant viral infections. We are seeking to improve our understanding of the mechanisms of DI generation and the precise nature of the RNA sequences necessary for DI replication and encapsidation. We also want to address the nature of the DI mediated symptom attenuation and interference effects in plants, and to determine the feasibility of using transgenic plants constitutively expressing DI RNAs for disease control. The progress made on each of these objectives is summarized along with the proposed experiments for the continuation period.

  11. Evaluation of locked nucleic acid-modified small interfering RNA in vitro and in vivo.

    PubMed

    Mook, Olaf R; Baas, Frank; de Wissel, Marit B; Fluiter, Kees

    2007-03-01

    RNA interference has become widely used as an experimental tool to study gene function. In addition, small interfering RNA (siRNA) may have great potential for the treatment of diseases. Recently, it was shown that siRNA can be used to mediate gene silencing in mouse models. Locally administered siRNAs entered the first clinical trials, but strategies for successful systemic delivery of siRNA are still under development. Challenges still exist about the stability, delivery, and therapeutic efficacy of siRNA. In the present study, we compare the efficacy of two methods of systemic siRNA delivery and the effects of siRNA modifications using locked nucleic acids (LNA) in a xenograft cancer model. Low volume tail vein bolus injections and continuous s.c. delivery using osmotic minipumps yielded similar uptake levels of unmodified siRNA by tumor xenografts. Both routes of administration mediated sequence-specific inhibition of two unrelated targets inside tumor xenografts. Previous studies have shown that LNA can be incorporated into the sense strand of siRNA while the efficacy is retained. Modification of siRNA targeting green fluorescent protein with LNA results in a significant increase in serum stability and thus may be beneficial for clinical applications. We show that minimal 3' end LNA modifications of siRNA are effective in stabilization of siRNA. Multiple LNA modifications in the accompanying strand further increase the stability but negate the efficacy in vitro and in vivo. In vivo, LNA-modified siRNA reduced off-target gene regulation compared with nonmodified siRNA. End-modified siRNA targeting green fluorescent protein provides a good trade-off between stability and efficacy in vivo using the two methods of systemic delivery in the nude mouse model. Therefore, LNA-modified siRNA should be preferred over unmodified siRNA.

  12. Small-interfering RNA (siRNA)-based functional micro- and nanostructures for efficient and selective gene silencing.

    PubMed

    Lee, Soo Hyeon; Chung, Bong Hyun; Park, Tae Gwan; Nam, Yoon Sung; Mok, Hyejung

    2012-07-17

    Because of RNA's ability to encode structure and functional information, researchers have fabricated diverse geometric structures from this polymer at the micro- and nanoscale. With their tunable structures, rigidity, and biocompatibility, novel two-dimensional and three-dimensional RNA structures can serve as a fundamental platform for biomedical applications, including engineered tissues, biosensors, and drug delivery vehicles. The discovery of the potential of small-interfering RNA (siRNA) has underscored the applications of RNA-based micro- and nanostructures in medicine. Small-interfering RNA (siRNA), synthetic double-stranded RNA consisting of approximately 21 base pairs, suppresses problematic target genes in a sequence-specific manner via inherent RNA interference (RNAi) processing. As a result, siRNA offers a potential strategy for treatment of many human diseases. However, due to inefficient delivery to cells and off-target effects, the clinical application of therapeutic siRNA has been very challenging. To address these issues, researchers have studied a variety of nanocarrier systems for siRNA delivery. In this Account, we describe several strategies for efficient siRNA delivery and selective gene silencing. We took advantage of facile chemical conjugation and complementary hybridization to design novel siRNA-based micro- and nanostructures. Using chemical crosslinkers and hydrophobic/hydrophilic polymers at the end of siRNA, we produced various RNA-based structures, including siRNA block copolymers, micelles, linear siRNA homopolymers, and microhydrogels. Because of their increased charge density and flexibility compared with conventional siRNA, these micro- and nanostructures can form polyelectrolyte complexes with poorly charged and biocompatible cationic carriers that are both more condensed and more homogenous than the complexes formed in other carrier systems. In addition, the fabricated siRNA-based structures are linked by cleavable disulfide

  13. Small interfering RNA of alkaline phosphatase inhibits matrix mineralization.

    PubMed

    Kotobuki, Noriko; Matsushima, Asako; Kato, Youichi; Kubo, Yoko; Hirose, Motohiro; Ohgushi, Hajime

    2008-05-01

    To investigate the cascade of matrix mineralization, cells expressing high and low alkaline phosphatase (ALP) were separated from human osteoblast-like (HOS) cells by fluorescence-activated cell sorting with an ALP antibody. After these cells had been recloned from single cells and then cultured under osteogenic conditions, high-ALP-expressing HOS (H-HOS) cells showed matrix mineralization, but low-ALP-expressing HOS (L-HOS) cells did not. The interaction among osteogenic-related genes, such as runt-related transcription factor 2 (RUNX2), collagen type I alpha1 chain (COL1A1), tissue non-specific ALP, and osteocalcin (OCN), is well known as being related to matrix mineralization. Quantitative real-time polymerase chain reaction revealed that the gene expression of ALP was higher in H-HOS cells than in L-HOS, whereas the gene expression of RUNX2, COL1A1, and OCN was lower in H-HOS cells than in L-HOS cells. When small interfering RNAs (siRNAs) of these osteogenic-related genes were introduced into H-HOS cells by transfection, only ALP siRNA inhibited matrix mineralization. Furthermore, the expression of not only the ALP gene, but also the COL1A1 and RUNX2 genes was influenced by the inhibition of ALP, although the expression of OCN was not affected by the inhibition of ALP. We have been able to confirm that the ALP gene is a strong candidate as the trigger of matrix mineralization. These results indicate the usefulness of cloned osteogenic cells in investigating the molecular mechanisms of matrix mineralization, the function of which can be modulated by using a variety of siRNAs.

  14. [In vivo imaging of liposomal small interfering RNA (siRNA) trafficking by positron emission tomography].

    PubMed

    Ando, Hidenori; Yonenaga, Norihito; Asai, Tomohiro; Hatanaka, Kentaro; Koide, Hiroyuki; Tsuzuku, Takuma; Harada, Norihiro; Tsukada, Hideo; Oku, Naoto

    2012-01-01

    In the development of nucleic acid medicines such as small interfering RNA (siRNA) drugs, one problem is how to study the pharmacokinetics and pharmacodynamics, since the precise in vivo behavior of siRNA is hard to detect. In this research, to establish a highly sensitive detection system of siRNA biodistribution in the whole body, the technology of positron imaging was applied. First, a one-step synthetic method in which double-stranded siRNA was directly labeled by a positron emitter, (18)F, was developed. By using [(18)F]-labeled siRNA ([(18)F]-siRNA), the complex of siRNA and polycation liposomes (PCL) containing dicetylphosphate tetraethylenepentamine (TEPA-PCL) was prepared. Then, the biodistribution of the siRNA after intravenous administration to mice was analyzed by planar positron imaging system (PPIS). As a result, whereas naked [(18)F]-siRNA was immediately excreted in mouse bladder after administration, the complex with cationic liposome (CL) was trapped in the lungs. Furthermore, [(18)F]-siRNA carried with PEGylated CL (PL) was distributed throughout the body, suggesting that it circulated in the bloodstream for an extended period of time. Additionally, PET imaging revealed more detailed biodistribution of the siRNA than in vivo imaging system (IVIS) because PET imaging is not affected by the depth variation of target tissues. On the other hand, to induce high accumulation of siRNAs against c-myc, MDM2, and VEGF in tumor tissue, a tumor-targeting probe, RGD peptide, was grafted at the top of PEG chain in PEGylated TEPA-PCL and the effect of the complex on experimental lung metastasis of B16 melanoma was examined. The complex suppressed the progression of tumor. We believe that the positron imaging data would support the development of siRNA agent for clinical use.

  15. Colored petri net modeling of small interfering RNA-mediated messenger RNA degradation

    PubMed Central

    Nickaeen, Niloofar; Moein, Shiva; Heidary, Zarifeh; Ghaisari, Jafar

    2016-01-01

    Background: Mathematical modeling of biological systems is an attractive way for studying complex biological systems and their behaviors. Petri Nets, due to their ability to model systems with various levels of qualitative information, have been wildly used in modeling biological systems in which enough qualitative data may not be at disposal. These nets have been used to answer questions regarding the dynamics of different cell behaviors including the translation process. In one stage of the translation process, the RNA sequence may be degraded. In the process of degradation of RNA sequence, small-noncoding RNA molecules known as small interfering RNA (siRNA) match the target RNA sequence. As a result of this matching, the target RNA sequence is destroyed. Materials and Methods: In this context, the process of matching and destruction is modeled using Colored Petri Nets (CPNs). The model is constructed using CPNs which allow tokens to have a value or type on them. Thus, CPN is a suitable tool to model string structures in which each element of the string has a different type. Using CPNs, long RNA, and siRNA strings are modeled with a finite set of colors. The model is simulated via CPN Tools. Results: A CPN model of the matching between RNA and siRNA strings is constructed in CPN Tools environment. Conclusion: In previous studies, a network of stoichiometric equations was modeled. However, in this particular study, we modeled the mechanism behind the silencing process. Modeling this kind of mechanisms provides us with a tool to examine the effects of different factors such as mutation or drugs on the process. PMID:27376039

  16. Antisense Transcription of Retrotransposons in Drosophila: An Origin of Endogenous Small Interfering RNA Precursors

    PubMed Central

    Russo, Joseph; Harrington, Andrew W.; Steiniger, Mindy

    2016-01-01

    Movement of transposons causes insertions, deletions, and chromosomal rearrangements potentially leading to premature lethality in Drosophila melanogaster. To repress these elements and combat genomic instability, eukaryotes have evolved several small RNA-mediated defense mechanisms. Specifically, in Drosophila somatic cells, endogenous small interfering (esi)RNAs suppress retrotransposon mobility. EsiRNAs are produced by Dicer-2 processing of double-stranded RNA precursors, yet the origins of these precursors are unknown. We show that most transposon families are transcribed in both the sense (S) and antisense (AS) direction in Dmel-2 cells. LTR retrotransposons Dm297, mdg1, and blood, and non-LTR retrotransposons juan and jockey transcripts, are generated from intraelement transcription start sites with canonical RNA polymerase II promoters. We also determined that retrotransposon antisense transcripts are less polyadenylated than sense. RNA-seq and small RNA-seq revealed that Dicer-2 RNA interference (RNAi) depletion causes a decrease in the number of esiRNAs mapping to retrotransposons and an increase in expression of both S and AS retrotransposon transcripts. These data support a model in which double-stranded RNA precursors are derived from convergent transcription and processed by Dicer-2 into esiRNAs that silence both sense and antisense retrotransposon transcripts. Reduction of sense retrotransposon transcripts potentially lowers element-specific protein levels to prevent transposition. This mechanism preserves genomic integrity and is especially important for Drosophila fitness because mobile genetic elements are highly active. PMID:26534950

  17. RNA Interference against Animal Viruses: How Morbilliviruses Generate Extended Diversity To Escape Small Interfering RNA Control

    PubMed Central

    Holz, Carine L.; Albina, Emmanuel; Minet, Cécile; Lancelot, Renaud; Kwiatek, Olivier; Libeau, Geneviève

    2012-01-01

    Viruses are serious threats to human and animal health. Vaccines can prevent viral diseases, but few antiviral treatments are available to control evolving infections. Among new antiviral therapies, RNA interference (RNAi) has been the focus of intensive research. However, along with the development of efficient RNAi-based therapeutics comes the risk of emergence of resistant viruses. In this study, we challenged the in vitro propensity of a morbillivirus (peste des petits ruminants virus), a stable RNA virus, to escape the inhibition conferred by single or multiple small interfering RNAs (siRNAs) against conserved regions of the N gene. Except with the combination of three different siRNAs, the virus systematically escaped RNAi after 3 to 20 consecutive passages. The genetic modifications involved consisted of single or multiple point nucleotide mutations and a deletion of a stretch of six nucleotides, illustrating that this virus has an unusual genomic malleability. PMID:22072768

  18. Detection of small interfering RNA (siRNA) by mass spectrometry procedures in doping controls.

    PubMed

    Thomas, Andreas; Walpurgis, Katja; Delahaut, Philippe; Kohler, Maxie; Schänzer, Wilhelm; Thevis, Mario

    2013-01-01

    Uncovering manipulation of athletic performance via small interfering (si)RNA is an emerging field in sports drug testing. Due to the potential to principally knock down every target gene in the organism by means of the RNA interference pathway, this facet of gene doping has become a realistic scenario. In the present study, two distinct model siRNAs comprising 21 nucleotides were designed as double strands which were perfect counterparts to a sequence of the respective messenger RNA coding the muscle regulator myostatin of Rattus norvegicus. Several modified nucleotides were introduced in both the sense and the antisense strand comprising phosphothioates, 2'-O-methylation, 2'-fluoro-nucleotides, locked nucleic acids and a cholesterol tag at the 3'-end. The model siRNAs were applied to rats at 1 mg/kg (i.v.) and blood as well as urine samples were collected. After isolation of the RNA by means of a RNA purification kit, the target analytes were detected by liquid chromatography - high resolution/high accuracy mass spectrometry (LC-HRMS). Analytes were detected as modified nucleotides after alkaline hydrolysis, as intact oligonucleotide strands (top-down) and by means of denaturing SDS-PAGE analysis. The gel-separated siRNA was further subjected to in-gel hydrolysis with different RNases and subsequent identification of the fragments by untargeted LC-HRMS analysis (bottom-up, 'experimental RNomics'). Combining the results of all approaches, the identification of several 3'-truncated urinary metabolites was accomplished and target analytes were detected up to 24 h after a single administration. Simultaneously collected blood samples yielded no promising results. The methods were validated and found fit-for-purpose for doping controls.

  19. A genome-wide survey of small interfering RNA and microRNA pathway genes in a galling insect.

    PubMed

    Shreve, Jacob T; Shukle, Richard H; Subramanyam, Subhashree; Johnson, Alisha J; Schemerhorn, Brandon J; Williams, Christie E; Stuart, Jeffrey J

    2013-03-01

    Deployment of resistance (R) genes is the most effective control for Hessian fly, Mayetiola destructor (Say); however, deployment of R genes results in an increased frequency of pest genotypes that display virulence to them. RNA interference (RNAi) is a useful reverse genetics tool for studying such insect virulence pathways, but requires a systemic phenotype, which is not found in all species. In an effort to correlate our observed weak RNAi phenotype in M. destructor with a genetic basis, we have aggregated and compared RNAi related genes across M. destructor, three other insect species, and the nematode Caenorhabditis elegans. We report here the annotation of the core genes in the small interfering RNA (siRNA) and microRNA (miRNA) pathways in M. destructor. While most of the miRNA pathway genes were highly conserved across the species studied, the siRNA pathway genes showed increased relative variability in comparison to the miRNA pathway. In particular, the Piwi/Argonaute/Zwille (PAZ) domain of Dicer-2 (DCR-2) had the least amount of sequence similarity of any domain among species surveyed, with a trend of increased conservation in those species with amenable systemic RNAi. A homolog of the systemic interference defective-1 (Sid-1) gene of C. elegans was also not annotated in the M. destructor genome. Indeed, it is of interest that a Sid-1 homolog has not been detected in any dipteran species to date. We hypothesize the sequence architecture of the PAZ domain in the M. destructor DCR-2 protein is related to reduced efficacy of this enzyme and this taken together with the lack of a Sid-1 homolog may account for the weak RNAi response observed to date in this species as well as other dipteran species.

  20. Delivery of small interfering RNA for inhibition of endothelial cell apoptosis by hypoxia and serum deprivation

    SciTech Connect

    Cho, Seung-Woo; Hartle, Lauren; Son, Sun Mi; Yang, Fan; Goldberg, Michael; Xu, Qiaobing; Langer, Robert; Anderson, Daniel G.

    2008-11-07

    RNA interference (RNAi) for anti-angiogenic or pro-apoptotic factors in endothelial cells (ECs) has great potential for the treatment of ischemic diseases by promoting angiogenesis or inhibiting apoptosis. Here, we report the utility of small interfering RNA (siRNA) in inhibiting EC apoptosis induced by tumor necrosis factor-{alpha} (TNF-{alpha}). siRNA was designed and synthesized targeting tumor necrosis factor-{alpha} receptor-1 (TNFR-1) and Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1). Human umbilical vein endothelial cells (HUVECs) were cultured under in vitro hypoxic and serum-deprived conditions to simulate in vivo ischemic conditions. Two days after liposomal delivery of siRNA targeting TNFR-1 and SHP-1, significant silencing of each target (TNFR-1; 76.5% and SHP-1; 97.2%) was detected. Under serum-deprived hypoxic (1% oxygen) conditions, TNF-{alpha} expression in HUVECs increased relative to normoxic (20% oxygen) and serum-containing conditions. Despite enhanced TNF-{alpha} expression, suppression of TNFR-1 or SHP-1 by siRNA delivery not only enhanced expression of angiogenic factors (KDR/Flk-1 and eNOS) and anti-apoptotic factor (Bcl-xL) but also reduced expression of a pro-apoptotic factor (Bax). Transfection of TNFR-1 or SHP-1 siRNA significantly decreased the HUVEC apoptosis while significantly enhancing HUVEC proliferation and capillary formation. The present study demonstrates that TNFR-1 and SHP-1 may be useful targets for the treatment of myocardial or hindlimb ischemia.

  1. Mutant p53 inhibits miRNA biogenesis by interfering with the microprocessor complex.

    PubMed

    Garibaldi, F; Falcone, E; Trisciuoglio, D; Colombo, T; Lisek, K; Walerych, D; Del Sal, G; Paci, P; Bossi, G; Piaggio, G; Gurtner, A

    2016-07-21

    Downregulation of microRNAs (miRNAs) is commonly observed in cancers and promotes tumorigenesis suggesting that miRNAs may function as tumor suppressors. However, the mechanism through which miRNAs are regulated in cancer, and the connection between oncogenes and miRNA biogenesis remain poorly understood. The TP53 tumor-suppressor gene is mutated in half of human cancers resulting in an oncogene with gain-of-function activities. Here we demonstrate that mutant p53 (mutp53) oncoproteins modulate the biogenesis of a subset of miRNAs in cancer cells inhibiting their post-transcriptional maturation. Interestingly, among these miRNAs several are also downregulated in human tumors. By confocal, co-immunoprecipitation and RNA-chromatin immunoprecipitation experiments, we show that endogenous mutp53 binds and sequesters RNA helicases p72/82 from the microprocessor complex, interfering with Drosha-pri-miRNAs association. In agreement with this, the overexpression of p72 leads to an increase of mature miRNAs levels. Moreover, functional experiments demonstrate the oncosuppressive role of mutp53-dependent miRNAs (miR-517a, -519a, -218, -105). Our study highlights a previously undescribed mechanism by which mutp53 interferes with Drosha-p72/82 association leading, at least in part, to miRNA deregulation observed in cancer.

  2. Interaction between cationic agents and small interfering RNA and DNA molecules

    NASA Astrophysics Data System (ADS)

    Unksov, I. N.; Slita, A. V.; Petrova, A. V.; Pereviazko, I.; Bakulev, V. M.; Rolich, V. I.; Bondarenko, A. B.; Kasyanenko, N. A.

    2016-11-01

    Azobenzene containing surfactant AzoTAB was used for investigation of binding in cationic- agent + nucleic acid in NaCl salt aqueous solutions. Two nucleic acids, macromolecular DNA and small interfering RNA, were examined upon the interaction with the surfactant. For DNA the interaction was studied using spectral methods and the methods of viscometry and flow birefringence measurement. For siRNA the possibility of surfactant-based delivery was checked in vitro.

  3. Creation of transgenic rice plants producing small interfering RNA of Rice tungro spherical virus.

    PubMed

    Le, Dung Tien; Chu, Ha Duc; Sasaya, Takahide

    2015-01-01

    Rice tungro spherical virus (RTSV), also known as Rice waika virus, does not cause visible symptoms in infected rice plants. However, the virus plays a critical role in spreading Rice tungro bacilliform virus (RTBV), which is the major cause of severe symptoms of rice tungro disease. Recent studies showed that RNA interference (RNAi) can be used to develop virus-resistance transgenic rice plants. In this report, we presented simple procedures and protocols needed for the creation of transgenic rice plants capable of producing small interfering RNA specific against RTSV sequences. Notably, our study showed that 60 out of 64 individual hygromycin-resistant lines (putative transgenic lines) obtained through transformation carried transgenes designed for producing hairpin double-stranded RNA. Northern blot analyses revealed the presence of small interfering RNA of 21- to 24-mer in 46 out of 56 confirmed transgenic lines. Taken together, our study indicated that transgenic rice plants carrying an inverted repeat of 500-bp fragments encoding various proteins of RTSV can produce small interfering RNA from the hairpin RNA transcribed from that transgene. In light of recent studies with other viruses, it is possible that some of these transgenic rice lines might be resistant to RTSV.

  4. A Convenient In Vivo Model Using Small Interfering RNA Silencing to Rapidly Assess Skeletal Gene Function

    PubMed Central

    Zhang, Wen; Liu, Can; Hai, Bao; Du, Guohong; Wang, Hong; Leng, Huijie; Xu, Yingsheng; Song, Chunli

    2016-01-01

    It is difficult to study bone in vitro because it contains various cell types that engage in cross-talk. Bone biologically links various organs, and it has thus become increasingly evident that skeletal physiology must be studied in an integrative manner in an intact animal. We developed a model using local intraosseous small interfering RNA (siRNA) injection to rapidly assess the effects of a target gene on the local skeletal environment. In this model, 160-g male Sprague-Dawley rats were treated for 1–2 weeks. The left tibia received intraosseous injection of a parathyroid hormone 1 receptor (Pth1r) or insulin-like growth factor 1 receptor (Igf-1r) siRNA transfection complex loaded in poloxamer 407 hydrogel, and the right tibia received the same volume of control siRNA. All the tibias received an intraosseous injection of recombinant human parathyroid hormone (1–34) (rhPTH (1–34)) or insulin-like growth factor-1 (IGF-1). Calcein green and alizarin red were injected 6 and 2 days before euthanasia, respectively. IGF-1R and PTH1R expression levels were detected via RT-PCR assays and immunohistochemistry. Bone mineral density (BMD), microstructure, mineral apposition rates (MARs), and strength were determined by dual-energy X-ray absorptiometry, micro-CT, histology and biomechanical tests. The RT-PCR and immunohistochemistry results revealed that IGF-1R and PTH1R expression levels were dramatically diminished in the siRNA-treated left tibias compared to the right tibias (both p<0.05). Using poloxamer 407 hydrogel as a controlled-release system prolonged the silencing effect of a single dose of siRNA; the mRNA expression levels of IGF-1R were lower at two weeks than at one week (p<0.01). The BMD, bone microstructure parameters, MAR and bone strength were significantly decreased in the left tibias compared to the right tibias (all p<0.05). This simple and convenient local intraosseous siRNA injection model achieved gene silencing with very small quantities of siRNA

  5. Photochemically induced gene silencing using small interfering RNA molecules in combination with lipid carriers.

    PubMed

    Bøe, S; Longva, A S; Hovig, E

    2007-01-01

    Novel strategies for efficient delivery of small interfering RNA (siRNA) molecules with a potential for targeting are required for development of RNA interference (RNAi) therapeutics. Here, we present a strategy that is based on delivery of siRNA molecules through the endocytic pathway, in order to develop a method for site-specific gene silencing. To achieve this, we combined the use of cationic lipids and photochemical internalization (PCI). Using the human S100A4 gene as a model system, we obtained potent gene silencing in four tested human cancer cell lines following PCI induction when using the cationic lipid jetSI-ENDO. Gene silencing was shown at both the RNA and protein levels, with no observed PCI toxicity when using the jetSI reagent and an optimized PCI protocol. This novel induction method opens for in vivo site-specific delivery of siRNA molecules toward a sequence of interest.

  6. Chitosan/interfering RNA nanoparticle mediated gene silencing in disease vector mosquito larvae

    PubMed Central

    Zhang, Xin; Mysore, Keshava; Flannery, Ellen; Michel, Kristin; Severson, David W.; Zhu, Kun Yan

    2015-01-01

    SHORT ABSTRACT Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae. LONG ABSTRACT Vector mosquitoes inflict more human suffering than any other organism—and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field. PMID:25867635

  7. Dermal/transdermal delivery of small interfering RNA and antisense oligonucleotides- advances and hurdles.

    PubMed

    Ita, Kevin

    2017-03-01

    A diverse array of nucleic acids has been studied by several researchers for the management of several diseases. Among these compounds, small interfering RNA and antisense oligonucleotides have attracted considerable attention. Antisense oligonucleotides are synthetic single stranded strings of nucleic acids that bind to RNA and thereby alter or reduce expression of the target RNA while siRNAs, on the other hand, are double-stranded RNA molecules which can hybridize with a specific mRNA sequence and block the translation of numerous genes. One of the main obstacles in the dermal or transdermal delivery of these compounds is their low skin permeability. In this review, various techniques used to enhance the delivery of these molecules into or across the skin are described and in some cases, the correlation between enhanced dermal/transdermal delivery and therapeutic efficacy is highlighted.

  8. Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA.

    PubMed

    Machitani, Mitsuhiro; Sakurai, Fuminori; Wakabayashi, Keisaku; Takayama, Kosuke; Tachibana, Masashi; Mizuguchi, Hiroyuki

    2017-03-17

    RNAi by short hairpin RNA (shRNA) is a powerful tool not only for studying gene functions in various organisms, including mammals, but also for the treatment of severe disorders. However, shRNA-expressing vectors can induce type I interferon (IFN) expression by activation of innate immune responses, leading to off-target effects and unexpected side effects. Several strategies have been developed to prevent type I IFN induction. On the other hand, it has remained unclear whether type I IFNs have effects on shRNA-mediated RNAi. Here, we show that the type I IFNs significantly inhibit shRNA-mediated RNAi. Treatment with recombinant human IFN-α significantly inhibited shRNA-mediated knockdown of target genes, while it did not inhibit small interfering RNA (siRNA)-mediated knockdown. Following treatment with IFN-α, increased and decreased copy numbers of shRNA and its processed form, respectively, were found in the cells transfected with shRNA-expressing plasmids. Dicer protein levels were not altered by IFN-α. These results indicate that type I IFNs inhibit shRNA-mediated RNAi via inhibition of dicer-mediated processing of shRNA to siRNA. Our findings should provide important clues for efficient RNAi-mediated knockdown of target genes in both basic researches and clinical gene therapy.

  9. Systematic coarse-grained modeling of complexation between small interfering RNA and polycations

    SciTech Connect

    Wei, Zonghui; Luijten, Erik

    2015-12-28

    All-atom molecular dynamics simulations can provide insight into the properties of polymeric gene-delivery carriers by elucidating their interactions and detailed binding patterns with nucleic acids. However, to explore nanoparticle formation through complexation of these polymers and nucleic acids and study their behavior at experimentally relevant time and length scales, a reliable coarse-grained model is needed. Here, we systematically develop such a model for the complexation of small interfering RNA (siRNA) and grafted polyethyleneimine copolymers, a promising candidate for siRNA delivery. We compare the predictions of this model with all-atom simulations and demonstrate that it is capable of reproducing detailed binding patterns, charge characteristics, and water release kinetics. Since the coarse-grained model accelerates the simulations by one to two orders of magnitude, it will make it possible to quantitatively investigate nanoparticle formation involving multiple siRNA molecules and cationic copolymers.

  10. Systematic coarse-grained modeling of complexation between small interfering RNA and polycations

    PubMed Central

    Wei, Zonghui

    2015-01-01

    All-atom molecular dynamics simulations can provide insight into the properties of polymeric gene-delivery carriers by elucidating their interactions and detailed binding patterns with nucleic acids. However, to explore nanoparticle formation through complexation of these polymers and nucleic acids and study their behavior at experimentally relevant time and length scales, a reliable coarse-grained model is needed. Here, we systematically develop such a model for the complexation of small interfering RNA (siRNA) and grafted polyethyleneimine copolymers, a promising candidate for siRNA delivery. We compare the predictions of this model with all-atom simulations and demonstrate that it is capable of reproducing detailed binding patterns, charge characteristics, and water release kinetics. Since the coarse-grained model accelerates the simulations by one to two orders of magnitude, it will make it possible to quantitatively investigate nanoparticle formation involving multiple siRNA molecules and cationic copolymers. PMID:26723631

  11. Application of biodegradable dendrigraft poly-l-lysine to a small interfering RNA delivery system.

    PubMed

    Kodama, Yukinobu; Kuramoto, Haruka; Mieda, Yukari; Muro, Takahiro; Nakagawa, Hiroo; Kurosaki, Tomoaki; Sakaguchi, Miako; Nakamura, Tadahiro; Kitahara, Takashi; Sasaki, Hitoshi

    2017-01-01

    Dendrigraft poly-l-lysine (DGL), including its central core, consists entirely of lysine, hence it is completely biodegradable. We applied DGL in a small interfering RNA (siRNA) delivery system. Binary complexes with siRNA and DGL had particle sizes of 23-73 nm and ζ-potentials of 34-42 mV. The siRNA-DGL complexes showed significant silencing effects in a mouse colon carcinoma cell line expressing luciferase (Colon26/Luc cells). The siRNA-DGL complexes induced slight cytotoxicity and hematological toxicity at a high charge ratio of DGL to siRNA, probably because of their cationic charges. Therefore, we recharged the siRNA-DGL complexes with γ-polyglutamic acid (γ-PGA), a biodegradable anionic compound, which was reported to reduce the cytotoxicity of cationic complexes. The ternary complexes showed particle sizes of 35-47 nm at a charge ratio of greater than 14 to siRNA with negative charges. Strong silencing effects of the ternary complexes were observed in Colon26/Luc cells without cytotoxicity or hematological toxicity. The cellular uptake and degradation of the binary and ternary complexes were confirmed by fluorescence microscopy. The ternary complexes suppressed luciferase activity in the tumor after direct injection into the tumors of mice bearing Colon26/Luc cells. Thus, a potentially important siRNA delivery system was constructed using biodegradable DGL.

  12. Plasma exosomes can deliver exogenous short interfering RNA to monocytes and lymphocytes

    PubMed Central

    Wahlgren, Jessica; Karlson, Tanya De L.; Brisslert, Mikael; Vaziri Sani, Forugh; Telemo, Esbjörn; Sunnerhagen, Per; Valadi, Hadi

    2012-01-01

    Despite the promise of RNA interference (RNAi) and its potential, e.g. for use in cancer therapy, several technical obstacles must first be overcome. The major hurdle of RNAi-based therapeutics is to deliver nucleic acids across the cell’s plasma membrane. This study demonstrates that exosome vesicles derived from humans can deliver short interfering RNA (siRNA) to human mononuclear blood cells. Exosomes are nano-sized vesicles of endocytic origin that are involved in cell-to-cell communication, i.e. antigen presentation, tolerance development and shuttle RNA (mainly mRNA and microRNA). Having tested different strategies, an optimized method (electroporation) was used to introduce siRNA into human exosomes of various origins. Plasma exosomes (exosomes from peripheral blood) were used as gene delivery vector (GDV) to transport exogenous siRNA to human blood cells. The vesicles effectively delivered the administered siRNA into monocytes and lymphocytes, causing selective gene silencing of mitogen-activated protein kinase 1. These data suggest that human exosomes can be used as a GDV to provide cells with heterologous nucleic acids such as therapeutic siRNAs. PMID:22618874

  13. Recent In Vivo Evidences of Particle-Based Delivery of Small-Interfering RNA (siRNA) into Solid Tumors

    PubMed Central

    2014-01-01

    Small-interfering RNA (siRNA) is both a powerful tool in research and a promising therapeutic platform to modulate expression of disease-related genes. Malignant tumors are attractive disease targets for nucleic acid-based therapies. siRNA directed against oncogenes, and genes driving metastases or angiogenesis have been evaluated in animal models and in some cases, in humans. The outcomes of these studies indicate that drug delivery is a significant limiting factor. This review provides perspectives on in vivo validated nanoparticle-based siRNA delivery systems. Results of recent advances in liposomes and polymeric and inorganic formulations illustrate the need for mutually optimized attributes for performance in systemic circulation, tumor interstitial space, plasma membrane, and endosomes. Physiochemical properties conducive to efficient siRNA delivery are summarized and directions for future research are discussed. PMID:25221632

  14. Transvascular delivery of small interfering RNA to the central nervous system.

    PubMed

    Kumar, Priti; Wu, Haoquan; McBride, Jodi L; Jung, Kyeong-Eun; Kim, Moon Hee; Davidson, Beverly L; Lee, Sang Kyung; Shankar, Premlata; Manjunath, N

    2007-07-05

    A major impediment in the treatment of neurological diseases is the presence of the blood-brain barrier, which precludes the entry of therapeutic molecules from blood to brain. Here we show that a short peptide derived from rabies virus glycoprotein (RVG) enables the transvascular delivery of small interfering RNA (siRNA) to the brain. This 29-amino-acid peptide specifically binds to the acetylcholine receptor expressed by neuronal cells. To enable siRNA binding, a chimaeric peptide was synthesized by adding nonamer arginine residues at the carboxy terminus of RVG. This RVG-9R peptide was able to bind and transduce siRNA to neuronal cells in vitro, resulting in efficient gene silencing. After intravenous injection into mice, RVG-9R delivered siRNA to the neuronal cells, resulting in specific gene silencing within the brain. Furthermore, intravenous treatment with RVG-9R-bound antiviral siRNA afforded robust protection against fatal viral encephalitis in mice. Repeated administration of RVG-9R-bound siRNA did not induce inflammatory cytokines or anti-peptide antibodies. Thus, RVG-9R provides a safe and noninvasive approach for the delivery of siRNA and potentially other therapeutic molecules across the blood-brain barrier.

  15. Small Interfering RNA Pathway Modulates Initial Viral Infection in Midgut Epithelium of Insect after Ingestion of Virus

    PubMed Central

    Lan, Hanhong; Chen, Hongyan; Liu, Yuyan; Jiang, Chaoyang; Mao, Qianzhuo; Jia, Dongsheng; Chen, Qian

    2015-01-01

    ABSTRACT Numerous viruses are transmitted in a persistent manner by insect vectors. Persistent viruses establish their initial infection in the midgut epithelium, from where they disseminate to the midgut visceral muscles. Although propagation of viruses in insect vectors can be controlled by the small interfering RNA (siRNA) antiviral pathway, whether the siRNA pathway can control viral dissemination from the midgut epithelium is unknown. Infection by a rice virus (Southern rice black streaked dwarf virus [SRBSDV]) of its incompetent vector (the small brown planthopper [SBPH]) is restricted to the midgut epithelium. Here, we show that the siRNA pathway is triggered by SRBSDV infection in continuously cultured cells derived from the SBPH and in the midgut of the intact insect. Knockdown of the expression of the core component Dicer-2 of the siRNA pathway by RNA interference strongly increased the ability of SRBSDV to propagate in continuously cultured SBPH cells and in the midgut epithelium, allowing viral titers in the midgut epithelium to reach the threshold (1.99 × 109 copies of the SRBSDV P10 gene/μg of midgut RNA) needed for viral dissemination into the SBPH midgut muscles. Our results thus represent the first elucidation of the threshold for viral dissemination from the insect midgut epithelium. Silencing of Dicer-2 further facilitated the transmission of SRBSDV into rice plants by SBPHs. Taken together, our results reveal the new finding that the siRNA pathway can control the initial infection of the insect midgut epithelium by a virus, which finally affects the competence of the virus's vector. IMPORTANCE Many viral pathogens that cause significant global health and agricultural problems are transmitted via insect vectors. The first bottleneck in viral infection, the midgut epithelium, is a principal determinant of the ability of an insect species to transmit a virus. Southern rice black streaked dwarf virus (SRBSDV) is restricted exclusively to the

  16. Development of RNA interference-based therapeutics and application of multi-target small interfering RNAs.

    PubMed

    Li, Tiejun; Wu, Meihua; Zhu, York Yuanyuan; Chen, Jianxin; Chen, Li

    2014-08-01

    RNA interference (RNAi) has been proven in recent years to be a newly advanced and powerful tool for development of therapeutic agents toward various unmet medical needs such as cancer, in particular, a great attention has been paid to the development of antineoplastic agents. Recent success in clinical trials related to RNAi-based therapeutics on cancer and ocular disease has validated that small interfering RNAs (siRNAs) constitute a new promising class of therapeutics. Currently, a great wealth of multi-target based siRNA structural modifications is available for promoting siRNA-mediated gene silencing with low side effects. Here, the latest developments in RNAi-based therapeutics and novel structural modifications described for siRNAs--in particular multi-target siRNAs--are reviewed.

  17. A novel albumin nanocomplex containing both small interfering RNA and gold nanorods for synergetic anticancer therapy

    NASA Astrophysics Data System (ADS)

    Choi, Jin-Ha; Hwang, Hai-Jin; Shin, Seung Won; Choi, Jeong-Woo; Um, Soong Ho; Oh, Byung-Keun

    2015-05-01

    Therapeutic nanocomplexes have been extensively developed for the effective treatment of aggressive cancers because of their outstanding versatility, easy manipulation, and low cytotoxicity. In this study, we describe the synthesis of a novel bovine serum albumin (BSA)-based nanocomplex harboring both Bcl-2-specific small interfering RNA (siRNA) and gold (Au) nanorods (siRNA and rods encapsulated in BSA; SREB) with the aim of developing a targeted breast cancer therapeutic. The SREB complexes contained 2 × 105 siRNA molecules and eight Au nanorods per BSA complex and were successively functionalized with polyethylene glycol (PEG) and anti-ErbB-2 antibodies to facilitate active targeting. The synergetic therapeutic activity originating from the two components effectively induced cell death (~80% reduction in viability compared with control cells) in target breast cancer cells after a single dose of laser irradiation. Intracellular SREB nanocomplex decomposition by proteolytic enzymes resulted in simultaneous RNA interference and thermal ablation, thus leading to apoptosis in the targeted cancer cells. Moreover, these therapeutic effects were sustained for approximately 72 hours. The intrinsic biocompatibility, multifunctionality, and potent in vitro anticancer properties of these SREB nanocomplexes indicate that they have great therapeutic potential for in vivo targeted cancer therapy, in addition to other areas of nanomedicine.Therapeutic nanocomplexes have been extensively developed for the effective treatment of aggressive cancers because of their outstanding versatility, easy manipulation, and low cytotoxicity. In this study, we describe the synthesis of a novel bovine serum albumin (BSA)-based nanocomplex harboring both Bcl-2-specific small interfering RNA (siRNA) and gold (Au) nanorods (siRNA and rods encapsulated in BSA; SREB) with the aim of developing a targeted breast cancer therapeutic. The SREB complexes contained 2 × 105 siRNA molecules and eight Au

  18. Intravaginal gene silencing using biodegradable polymer nanoparticles densely loaded with small-interfering RNA

    NASA Astrophysics Data System (ADS)

    Woodrow, Kim A.; Cu, Yen; Booth, Carmen J.; Saucier-Sawyer, Jennifer K.; Wood, Monica J.; Mark Saltzman, W.

    2009-06-01

    Vaginal instillation of small-interfering RNA (siRNA) using liposomes has led to silencing of endogenous genes in the genital tract and protection against challenge from infectious disease. Although siRNA lipoplexes are easily formulated, several of the most effective transfection agents available commercially may be toxic to the mucosal epithelia and none are able to provide controlled or sustained release. Here, we demonstrate an alternative approach using nanoparticles composed entirely of FDA-approved materials. To render these materials effective for gene silencing, we developed novel approaches to load them with high amounts of siRNA. A single dose of siRNA-loaded nanoparticles to the mouse female reproductive tract caused efficient and sustained gene silencing. Knockdown of gene expression was observed proximal (in the vaginal lumen) and distal (in the uterine horns) to the site of topical delivery. In addition, nanoparticles penetrated deep into the epithelial tissue. This is the first report demonstrating that biodegradable polymer nanoparticles are effective delivery vehicles for siRNA to the vaginal mucosa.

  19. Development of a software tool and criteria evaluation for efficient design of small interfering RNA

    SciTech Connect

    Chaudhary, Aparna; Srivastava, Sonam; Garg, Sanjeev

    2011-01-07

    Research highlights: {yields} The developed tool predicted siRNA constructs with better thermodynamic stability and total score based on positional and other criteria. {yields} Off-target silencing below score 30 were observed for the best siRNA constructs for different genes. {yields} Immunostimulation and cytotoxicity motifs considered and penalized in the developed tool. {yields} Both positional and compositional criteria were observed to be important. -- Abstract: RNA interference can be used as a tool for gene silencing mediated by small interfering RNAs (siRNA). The critical step in effective and specific RNAi processing is the selection of suitable constructs. Major design criteria, i.e., Reynolds's design rules, thermodynamic stability, internal repeats, immunostimulatory motifs were emphasized and implemented in the siRNA design tool. The tool provides thermodynamic stability score, GC content and a total score based on other design criteria in the output. The viability of the tool was established with different datasets. In general, the siRNA constructs produced by the tool had better thermodynamic score and positional properties. Comparable thermodynamic scores and better total scores were observed with the existing tools. Moreover, the results generated had comparable off-target silencing effect. Criteria evaluations with additional criteria were achieved in WEKA.

  20. SIRT1 inhibits EV71 genome replication and RNA translation by interfering with the viral polymerase and 5'UTR RNA.

    PubMed

    Han, Yang; Wang, Lvyin; Cui, Jin; Song, Yu; Luo, Zhen; Chen, Junbo; Xiong, Ying; Zhang, Qi; Liu, Fang; Ho, Wenzhe; Liu, Yingle; Wu, Kailang; Wu, Jianguo

    2016-12-15

    Enterovirus 71 (EV71) possesses a single-stranded positive RNA genome that contains a single open reading frame (ORF) flanked by a 5' untranslated region (5'UTR) and a polyadenylated 3'UTR. Here, we demonstrated that EV71 activates the production of silent mating type information regulation 2 homolog 1 (SIRT1), a histone deacetylase (HDAC). EV71 further stimulates SIRT1 sumoylation and deacetylase activity, and enhances SIRT1 translocation from the nucleus to the cytoplasm. More interestingly, activated SIRT1 subsequently binds with the EV71 3D(pol) protein (a viral RNA-dependent RNA polymerase, RdRp) to repress the acetylation and RdRp activity of 3D(pol), resulting in the attenuation of viral genome replication. Moreover, SIRT1 interacts with the cloverleaf structure of the EV71 RNA 5'UTR to inhibit viral RNA transcription, and binds to the internal ribosome entry site (IRES) of the EV71 5'UTR to attenuate viral RNA translation. Thus, EV71 stimulates SIRT1 production and activity, which in turn represses EV71 genome replication by inhibiting viral polymerase, and attenuates EV71 RNA transcription and translation by interfering with viral RNA. These results uncover a new function of SIRT1 and reveal a new mechanism underlying the regulation of EV71 replication.

  1. Promoted adipogenesis of rat mesenchymal stem cells by transfection of small interfering RNA complexed with a cationized dextran.

    PubMed

    Nagane, Kentaro; Jo, Jun-ichiro; Tabata, Yasuhiko

    2010-01-01

    The objective of this study is to investigate the possibility of small interfering RNA (siRNA) complexed with a cationized dextran of nonviral carrier to biologically modify the adipogenesis extent of bone marrow-derived mesenchymal stem cells (MSC). Spermine was chemically introduced to the hydroxyl groups of dextran to prepare the cationized dextran (spermine-dextran). The spermine-dextran could form a complex with siRNA, and the physicochemical properties were changed by the molecular weight of dextran, the molar percentage of spermine introduced to dextran, and the molar ratio of nitrogen molecule of spermine-dextran to the phosphorous ones of siRNA (N/P ratio). The gene expression level of luciferase or green fluorescence protein was significantly suppressed by transfection with the complex of spermine-dextran and siRNA. The gene expression level by the complex decreased with an increase in the extent of complex internalized. Biochemical experiments revealed that culture in an adipogenic differentiation medium allowed MSC to differentiate into adipogenic cells. However, upon culturing with siRNA of anti-transcription coactivator containing PDZ-binding motif (TAZ) for osteogenic differentiation complexed with the spermine-dextran, the adipogenesis of MSC was further promoted. It is concluded that the spemine-dextran was a promising nonviral carrier to suppress the expression level of differentiation gene, resulting in the modification of cell differentiation direction.

  2. Inhibition of pathologic immunoglobulin free light chain production by small interfering RNA molecules

    PubMed Central

    Phipps, Jonathan E.; Kestler, Daniel P.; Foster, James S.; Kennel, Stephen J.; Donnell, Robert; Weiss, Deborah T.; Solomon, Alan; Wall, Jonathan S.

    2010-01-01

    Objectives Morbidity and mortality occurring in patients with multiple myeloma, AL amyloidosis, and light chain deposition disease can result from the pathologic deposition of monoclonal Ig light chains (LCs) in kidneys and other organs. To reduce synthesis of such components, therapy for these disorders typically has involved anti-plasma cell agents; however, this approach is not always effective and can have adverse consequences. We have investigated another means to achieve this objective; namely, RNA interference (RNAi). Materials and Methods SP2/O mouse myeloma cells were stably transfected with a construct encoding a λ6 LC (Wil) under control of the CMV promoter, while λ2-producing myeloma cell line RPMI 8226 was purchased from the ATCC. Both were treated with small interfering RNA (siRNA) directed specifically to the V, J, or C portions of the molecules and then analyzed by ELISA, flow cytometry and real time PCR. Results Transfected cells were found to constitutively express detectable quantities of mRNA and protein Wil and, after exposure to siRNAs, an ~40% reduction in mRNA and LC production was evidenced at 48 hours. An even greater effect was seen with the 8226 cells. Conclusion Our results have shown that RNAi can markedly reduce LC synthesis and provide the basis for testing the therapeutic potential of this strategy using in vivo experimental models of multiple myeloma. PMID:20637260

  3. Design, simplified cloning, and in-silico analysis of multisite small interfering RNA-targeting cassettes

    PubMed Central

    Baghban-Kohnehrouz, Bahram; Nayeri, Shahnoush

    2016-01-01

    Multiple gene silencing is being required to target and tangle metabolic pathways in eukaryotes and researchers have to develop a subtle method for construction of RNA interference (RNAi) cassettes. Although, several vectors have been developed due to different screening and cloning strategies but still some potential limitations remain to be dissolved. Here, we worked out a simple cloning strategy to develop multisite small interfering RNA (siRNA) cassette from different genes by two cloning steps. In this method, effective siRNA sites in the target messenger RNAs (mRNAs) were determined using in silico analysis and consecutively arranged to reduce length of inverted repeats. Here, we used one-step (polymerase chain reaction) PCR by designed long primer sets covering the selected siRNA sites. Rapid screening, cost-effective and shorten procedure are advantages of this method compare to PCR classic cloning. Validity of constructs was confirmed by optimal centroid secondary structures with high stability in plants. PMID:27844018

  4. In vivo efficacy and off-target effects of locked nucleic acid (LNA) and unlocked nucleic acid (UNA) modified siRNA and small internally segmented interfering RNA (sisiRNA) in mice bearing human tumor xenografts.

    PubMed

    Mook, Orf; Vreijling, Jeroen; Wengel, Suzy L; Wengel, Jesper; Zhou, Chuanzheng; Chattopadhyaya, Jyoti; Baas, Frank; Fluiter, Kees

    2010-07-01

    The clinical use of small interfering RNA (siRNA) is hampered by poor uptake by tissues and instability in circulation. In addition, off-target effects pose a significant additional problem for therapeutic use of siRNA. Chemical modifications of siRNA have been reported to increase stability and reduce off-target effects enabling possible therapeutic use of siRNA. Recently a large scale direct comparison of the impact of 21 different types of novel chemical modifications on siRNA efficiency and cell viability was published.1 It was found that several types of chemical modifications could enhance siRNA activity beyond that of an unmodified siRNA in vitro. In addition, a novel siRNA design, termed small internally segmented interfering RNA (sisiRNA), composed of an intact antisense strand and segmented guide strand stabilized using LNA was shown to be effective in cell based assays. In the present study we examined the in vivo efficacy of the LNA and UNA modified siRNA and sisiRNA in a mouse model bearing human tumor xenografts. We studied the biodistribution and efficacy of target knockdown in the mouse model. In addition we used whole genome profiling to assess the off-target effects in the liver of the mouse and the tumor xenografts. We report that LNA and UNA modified siRNA and sisiRNA improve the efficacy in target knockdown as compared with unmodified siRNA in the tumor xenografts without formulation. However, the level of off-target gene regulation in both the tumor and the liver correlated with the increase in efficacy in target knockdown, unless the seed region of the siRNA was modified.

  5. CCR5 small interfering RNA ameliorated joint inflammation in rats with adjuvant-induced arthritis.

    PubMed

    Duan, Hongmei; Yang, Pingting; Fang, Fang; Ding, Shuang; Xiao, Weiguo

    2014-12-01

    Rheumatoid arthritis (RA) is a systemic inflammatory disease. C-C chemokine receptor type 5 (CCR5) is found in inflamed synovium of RA patients and is necessary for formation of RA. We aimed to check whether delivery of CCR5-specific small interfering RNA (siRNA) via electroporation suppresses local inflammation in arthritis rats. Vectors encoding siRNA that target CCR5 or negative control siRNA were constructed for gene silencing and the silencing effects of suppressing CCR5 expression in synovium examined by western blot. The vector with strongest effect was delivered into the knee joint of adjuvant-induced arthritis (AIA) rats by the in vivo electroporation method 7, 10, 13, and 16 days after immunization with Complete Freund's adjuvant. During an observation of 28 days, behavior, paw swelling, arthritis and histopathologic scoring were estimated. The expression level of CCR5 in synovium was evaluated by western blot and real-time PCR. Anti-CCR5 D1 siRNA was effectively inhibited CCR5 expression in vitro. Moreover, delivery of the siRNA into inflammatory joint also suppressed the expression of CCR5 in vivo and markedly suppressed paw swelling and inflammation. Local electroporation of anti-CCR5 siRNA into the left inflamed joints could achieve the silencing of CCR5 gene and alleviate local inflammation just in the knee joint injected with siRNA other than the opposite joint. Inhibition of CCR5 expression may provide a potential for treatment of RA.

  6. Efficacy of RNA interference knockdown using aerosolized short interfering RNAs bound to nanoparticles in three diverse aphid species.

    PubMed

    Thairu, M W; Skidmore, I H; Bansal, R; Nováková, E; Hansen, T E; Li-Byarlay, H; Wickline, S A; Hansen, A K

    2017-03-17

    RNA interference (RNAi) has emerged as a promising method for validating gene function; however, its utility in nonmodel insects has proven problematic, with delivery methods being one of the main obstacles. This study investigates a novel method of RNAi delivery in aphids, the aerosolization of short interfering RNA (siRNA)-nanoparticle complexes. By using nanoparticles as a siRNA carrier, the likelihood of cellular uptake is increased, when compared to methods previously used in insects. To determine the efficacy of this RNAi delivery system, siRNAs were aerosolized with and without nanoparticles in three aphid species: Acyrthosiphon pisum, Aphis glycines and Schizaphis graminum. The genes targeted for knockdown were carotene dehydrogenase (tor), which is important for pigmentation in Ac. pisum, and branched chain-amino acid transaminase (bcat), which is essential in the metabolism of branched-chain amino acids in all three aphid species. Overall, we observed modest gene knockdown of tor in Ac. pisum and moderate gene knockdown of bcat in Ap. glycines along with its associated phenotype. We also determined that the nanoparticle emulsion significantly increased the efficacy of gene knockdown. Overall, these results suggest that the aerosolized siRNA-nanoparticle delivery method is a promising new high-throughput and non-invasive RNAi delivery method in some aphid species.

  7. Rapid delivery of small interfering RNA by biosurfactant MEL-A-containing liposomes

    SciTech Connect

    Inoh, Yoshikazu; Furuno, Tadahide; Hirashima, Naohide; Kitamoto, Dai; Nakanishi, Mamoru

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer We use MEL-A-containing cationic liposomes for siRNA delivery. Black-Right-Pointing-Pointer MEL-A-containing cationic liposomes can efficiently and rapidly deliver siRNA into the cytoplasm. Black-Right-Pointing-Pointer Rapid delivery of siRNA is due to the membrane fusion between liposomes and plasma membrane. -- Abstract: The downregulation of gene expression by RNA interference holds great potential for genetic analysis and gene therapy. However, a more efficient delivery system for small interfering RNA (siRNA) into the target cells is required for wide fields such as cell biology, physiology, and clinical application. Non-viral vectors are stronger candidates than viral vectors because they are safer and easier to prepare. We have previously used a new method for gene transfection by combining cationic liposomes with the biosurfactant mannosylerythritol lipid-A (MEL-A). The novel MEL-A-containing cationic liposomes rapidly delivered DNA (plasmids and oligonucleotides) into the cytosol and nucleus through membrane fusion between liposomes and the plasma membrane, and consequently, enhanced the gene transfection efficiency. In this study, we determined the efficiency of MEL-A-containing cationic liposomes for siRNA delivery. We observed that exogenous and endogenous protein expression was suppressed by approximately 60% at 24 h after brief (30 min) incubation of target cells with MEL-A-containing cationic liposome/siRNA complexes. Confocal microscopic analysis showed that suppression of protein expression was caused by rapid siRNA delivery into the cytosol. We found that the MEL-A-containing cationic liposomes directly delivered siRNA into the cytoplasm by the membrane fusion in addition to endocytotic pathway whereas Lipofectamine Trade-Mark-Sign RNAiMax delivered siRNA only by the endocytotic pathway. It seems that the ability to rapidly and directly deliver siRNA into the cytosol using MEL-A-containing cationic

  8. Characterization of a defective interfering RNA that contains a mosaic of a plant viral genome

    SciTech Connect

    Morris, T.J.; Jackson, A.O.

    1991-01-01

    Our lab was the first to describe and characterize a defective interfering RNA (DI RNAs or DIs) in association with a small RNA plant virus. The features of the DIs that we discovered in infections of tomato bushy stunt virus were compatible with the properties of DIs identified in many animal virus infections. Animal virologists have generally recognized the importance of studying DIs because they are invaluable tools for identifying cis-acting sequences important in virus multiplication and because they offer the opportunity to elucidate mechanisms involved in viral persistence and disease attenuation. Hence our discovery offered a comparably valuable tool for use in plant virus studies for the first time. Since then, we have also discovered the second example of plant viral DI RNAs associated with turnip crinkle virus (TCV), a virus structurally related to TBSV. We proposed a thorough characterization of this unique class of symptom modulating RNAs with the overall objective of identifying viral RNA nucleotide, sequences involved in such fundamental processes as virus replication and encapsidation as well as the degree of symptom expression resulting from the viral-DI-host interaction. The proposed research focused on the molecular characterization of the DI RNAs and the helper virus. We had demonstrated that the DIs were collinear deletion mutants of the genome of a cherry strain of tomato bushy stunt virus (TBSV). We had also shown that these low molecular weight RNAs interfered with the helper plant virus and modulated disease expression by preventing the development of a lethal necrotic disease in susceptible host plants. We also suggested that by exploring the mechanisms associated with the symptom attenuation effect, we might be able to devise novel strategies useful for engineering viral disease resistance.

  9. Systemic delivery of small interfering RNA by use of targeted polycation liposomes for cancer therapy.

    PubMed

    Kenjo, Eriya; Asai, Tomohiro; Yonenaga, Norihito; Ando, Hidenori; Ishii, Takayuki; Hatanaka, Kentaro; Shimizu, Kosuke; Urita, Yugo; Dewa, Takehisa; Nango, Mamoru; Tsukada, Hideo; Oku, Naoto

    2013-01-01

    Novel polycation liposomes decorated with cyclic(Cys-Arg-Gly-Asp-D-Phe) peptide (cyclicRGD)-polyethylene glycol (PEG) (RGD-PEG-polycation liposomes (PCL)) were previously developed for cancer therapy based on RNA interference. Here, we demonstrate the in vivo delivery of small interfering RNA (siRNA) to tumors by use of RGD-PEG-PCL in B16F10 melanoma-bearing mice. Pharmacokinetic data obtained by positron emission tomography showed that cholesterol-conjugated siRNA formulated in RGD-PEG-PCL markedly accumulated in the tumors. Delivered by RGD-PEG-PCL, a therapeutic cocktail of siRNAs composed of cholesterol-conjugated siRNAs for c-myc, MDM2, and vascular endothelial growth factor (VEGF) were able to significantly inhibit the growth of B16F10 melanoma both in vitro and in vivo. These data suggest that targeted delivery of siRNAs by use of RGD-PEG-PCL has considerable potential for cancer treatment.

  10. Delivery of antiviral small interfering RNA with gold nanoparticles inhibits dengue virus infection in vitro

    PubMed Central

    Paul, Amber M.; Shi, Yongliang; Acharya, Dhiraj; Douglas, Jessica R.; Cooley, Amanda; Anderson, John F.; Huang, Faqing

    2014-01-01

    Dengue virus (DENV) infection in humans can cause flu-like illness, life-threatening haemorrhagic fever or even death. There is no specific anti-DENV therapeutic or approved vaccine currently available, partially due to the possibility of antibody-dependent enhancement reaction. Small interfering RNAs (siRNAs) that target specific viral genes are considered a promising therapeutic alternative against DENV infection. However, in vivo, siRNAs are vulnerable to degradation by serum nucleases and rapid renal excretion due to their small size and anionic character. To enhance siRNA delivery and stability, we complexed anti-DENV siRNAs with biocompatible gold nanoparticles (AuNPs) and tested them in vitro. We found that cationic AuNP–siRNA complexes could enter Vero cells and significantly reduce DENV serotype 2 (DENV-2) replication and infectious virion release under both pre- and post-infection conditions. In addition, RNase-treated AuNP–siRNA complexes could still inhibit DENV-2 replication, suggesting that AuNPs maintained siRNA stability. Collectively, these results demonstrated that AuNPs were able to efficiently deliver siRNAs and control infection in vitro, indicating a novel anti-DENV strategy. PMID:24828333

  11. In Vivo Magnetic Resonance Imaging of Small Interfering RNA Nanodelivery to Pancreatic Islets.

    PubMed

    Wang, Ping; Moore, Anna

    2016-01-01

    Pancreatic islet transplantation is a promising therapeutic approach for type 1 diabetes.However, recent advances in islet transplantation are limited by significant graft loss after transplantation. Multiple immunological and nonimmunological factors contribute to this loss. Novel therapies that could target the core reasons for the islet graft loss are desperately needed. Small interfering RNA can be used to inhibit the expression of virtually any gene with single-nucleotide specificity including genes responsible for islet damage. Applying adequate delivery of siRNA molecules to pancreatic islets prior to transplantation holds a great potential for improving the survival of islet grafts. Noninvasive imaging provides means for monitoring the survival of transplanted islets in real time. Here, we summarize the approach that has been developed to deliver siRNA to pancreatic islets in conjunction with tracking of the graft outcome by in vivo magnetic resonance imaging (MRI). We synthesize a nano-sized theranostic agent consisting of magnetic nanoparticles (MN), a reporter for MRI, labeled with Cy5.5 dye for near-infrared fluorescence (NIRF) imaging, and conjugated to siRNA molecule targeting genes that are harmful to islet grafts. Pre-labeling of islets by MN-Cy5.5-siRNA allowed us to monitor the survival of transplanted islet grafts by MRI and NIRF imaging and resulted in efficient silencing of the target genes in vivo. This novel approach combines a therapeutic effect provided by RNA interference technology with in vivo MR imaging and is expected to significantly improve the outcome of islet transplantation in patients with type 1 diabetes.

  12. Suppression of Breast Cancer Cell Migration by Small Interfering RNA Delivered by Polyethylenimine-Functionalized Graphene Oxide

    NASA Astrophysics Data System (ADS)

    Huang, Yuan-Pin; Hung, Chao-Ming; Hsu, Yi-Chiang; Zhong, Cai-Yan; Wang, Wan-Rou; Chang, Chi-Chang; Lee, Mon-Juan

    2016-05-01

    The carbon-based nanomaterial graphene can be chemically modified to associate with various molecules such as chemicals and biomolecules and developed as novel carriers for drug and gene delivery. In this study, a nonviral gene transfection reagent was produced by functionalizing graphene oxide (GO) with a polycationic polymer, polyethylenimine (PEI), to increase the biocompatibility of GO and to transfect small interfering RNA (siRNA) against C-X-C chemokine receptor type 4 (CXCR4), a biomarker associated with cancer metastasis, into invasive breast cancer cells. PEI-functionalized GO (PEI-GO) was a homogeneous aqueous solution that remained in suspension during storage at 4 °C for at least 6 months. The particle size of PEI-GO was 172 ± 4.58 and 188 ± 5.00 nm at 4 and 25 °C, respectively, and increased slightly to 262 ± 17.6 nm at 37 °C, but remained unaltered with time. Binding affinity of PEI-GO toward siRNA was assessed by electrophoretic mobility shift assay (EMSA), in which PEI-GO and siRNA were completely associated at a PEI-GO:siRNA weight ratio of 2:1 and above. The invasive breast cancer cell line, MDA-MB-231, was transfected with PEI-GO in complex with siRNAs against CXCR4 (siCXCR4). Suppression of the mRNA and protein expression of CXCR4 by the PEI-GO/siCXCR4 complex was confirmed by real-time PCR and western blot analysis. In addition, the metastatic potential of MDA-MB-231 cells was attenuated by the PEI-GO/siCXCR4 complex as demonstrated in wound healing assay. Our results suggest that PEI-GO is effective in the delivery of siRNA and may contribute to targeted gene therapy to suppress cancer metastasis.

  13. Characterization of defective interfering RNAs associated with RNA plant viruses. Progress report

    SciTech Connect

    Morris, T.J.; Jackson, A.O.

    1993-04-01

    Our lab was the first to describe and characterize a defective interfering RNA (DI RNAs or DIs) in association with a small RNA plant virus. The features of the DIs that we discovered in infections of tomato bushy stunt virus were compatible with the properties of DIs identified in many animal virus infections. Animal virologists have generally recognized the importance of studying DIs because they are invaluable tools for identifying cis-acting sequences important in virus multiplication and because they offer the opportunity to elucidate mechanisms involved in viral persistence and disease attenuation. Hence our discovery offered a comparably valuable tool for use in plant virus studies for the first time. Since the original observation with TBSV, we discovered the second example of plant viral DI RNAs associated with turnip crinkle virus (TCV), and many other reports have now appeared characterizing DI and DI-like RNAs in other plant viral infections. We are seeking to improve our understanding of the mechanisms of DI generation and the precise nature of the RNA sequences necessary for DI replication and encapsidation. We also want to address the nature of the DI mediated symptom attenuation and interference effects in plants, and to determine the feasibility of using transgenic plants constitutively expressing DI RNAs for disease control. The progress made on each of these objectives is summarized along with the proposed experiments for the continuation period.

  14. Delivery of Small Interfering RNA by Peptide-Targeted Mesoporous Silica Nanoparticle-Supported Lipid Bilayers

    PubMed Central

    Ashley, Carlee E.; Carnes, Eric C.; Epler, Katharine E.; Padilla, David P.; Phillips, Genevieve K.; Castillo, Robert E.; Wilkinson, Dan C.; Wilkinson, Brian S.; Burgard, Cameron A.; Sewell, Robin M.; Townson, Jason L.; Chackerian, Bryce; Willman, Cheryl L.; Peabody, David S.; Wharton, Walker; Brinker, C. Jeffrey

    2012-01-01

    The therapeutic potential of small interfering RNAs (siRNAs) is severely limited by the availability of delivery platforms that protect siRNA from degradation, deliver it to the target cell with high specificity and efficiency, and promote its endosomal escape and cytosolic dispersion. Here we report that mesoporous silica nanoparticle-supported lipid bilayers (or ‘protocells’), exhibit multiple properties that overcome many of the limitations of existing delivery platforms. Protocells have a 10- to 100-fold greater capacity for siRNA than corresponding lipid nanoparticles and are markedly more stable when incubated under physiological conditions. Protocells loaded with a cocktail of siRNAs bind to cells in a manner dependent on the presence of an appropriate targeting peptide and, through an endocytic pathway followed by endosomal disruption, promote delivery of the silencing nucleotides to the cytoplasm. The expression of each of the genes targeted by the siRNAs was shown to be repressed at the protein level, resulting in a potent induction of growth arrest and apoptosis. Incubation of control cells that lack expression of the antigen recognized by the targeting peptide with siRNA-loaded protocells induced neither repression of protein expression nor apoptosis, indicating the precise specificity of cytotoxic activity. In terms of loading capacity, targeting capabilities, and potency of action, protocells provide unique attributes as a delivery platform for therapeutic oligonucleotides. PMID:22309035

  15. Delivery of small interfering RNA by peptide-targeted mesoporous silica nanoparticle-supported lipid bilayers.

    PubMed

    Ashley, Carlee E; Carnes, Eric C; Epler, Katharine E; Padilla, David P; Phillips, Genevieve K; Castillo, Robert E; Wilkinson, Dan C; Wilkinson, Brian S; Burgard, Cameron A; Kalinich, Robin M; Townson, Jason L; Chackerian, Bryce; Willman, Cheryl L; Peabody, David S; Wharton, Walker; Brinker, C Jeffrey

    2012-03-27

    The therapeutic potential of small interfering RNAs (siRNAs) is severely limited by the availability of delivery platforms that protect siRNA from degradation, deliver it to the target cell with high specificity and efficiency, and promote its endosomal escape and cytosolic dispersion. Here we report that mesoporous silica nanoparticle-supported lipid bilayers (or "protocells") exhibit multiple properties that overcome many of the limitations of existing delivery platforms. Protocells have a 10- to 100-fold greater capacity for siRNA than corresponding lipid nanoparticles and are markedly more stable when incubated under physiological conditions. Protocells loaded with a cocktail of siRNAs bind to cells in a manner dependent on the presence of an appropriate targeting peptide and, through an endocytic pathway followed by endosomal disruption, promote delivery of the silencing nucleotides to the cytoplasm. The expression of each of the genes targeted by the siRNAs was shown to be repressed at the protein level, resulting in a potent induction of growth arrest and apoptosis. Incubation of control cells that lack expression of the antigen recognized by the targeting peptide with siRNA-loaded protocells induced neither repression of protein expression nor apoptosis, indicating the precise specificity of cytotoxic activity. In terms of loading capacity, targeting capabilities, and potency of action, protocells provide unique attributes as a delivery platform for therapeutic oligonucleotides.

  16. MicroRNA-Targeted and Small Interfering RNA–Mediated mRNA Degradation Is Regulated by Argonaute, Dicer, and RNA-Dependent RNA Polymerase in Arabidopsis[W][OA

    PubMed Central

    Ronemus, Michael; Vaughn, Matthew W.; Martienssen, Robert A.

    2006-01-01

    ARGONAUTE1 (AGO1) of Arabidopsis thaliana mediates the cleavage of microRNA (miRNA)-targeted mRNAs, and it has also been implicated in the posttranscriptional silencing of transgenes and the maintenance of chromatin structure. Mutations in AGO1 severely disrupt plant development, indicating that miRNA function and possibly other aspects of RNA interference are essential for maintaining normal patterns of gene expression. Using microarrays, we found that 1 to 6% of genes display significant expression changes in several alleles of ago1 at multiple developmental stages, with the majority showing higher levels. Several classes of known miRNA targets increased markedly in ago1, whereas others showed little or no change. Cleavage of mRNAs within miRNA-homologous sites was reduced but not abolished in an ago1 -null background, indicating that redundant slicer activity exists in Arabidopsis. Small interfering RNAs and larger 30- to 60-nucleotide RNA fragments corresponding to highly upregulated miRNA target genes accumulated in wild-type plants but not in ago1, the RNA-dependent RNA polymerase mutants rdr2 and rdr6, or the Dicer-like mutants dcl1 and dcl3. Both sense and antisense RNAs corresponding to these miRNA targets accumulated in the ago1 and dcl1 backgrounds. These results indicate that a subset of endogenous mRNA targets of RNA interference may be regulated through a mechanism of second-strand RNA synthesis and degradation initiated by or in addition to miRNA-mediated cleavage. PMID:16798886

  17. An Atelocollagen Coating for Efficient Local Gene Silencing by Using Small Interfering RNA.

    PubMed

    Koenig, Olivia; Nothdurft, Dimitrios; Perle, Nadja; Neumann, Bernd; Behring, Andreas; Degenkolbe, Ilka; Walker, Tobias; Schlensak, Christian; Wendel, Hans Peter; Nolte, Andrea

    2017-03-17

    In the last decades, many efforts have been made to counteract adverse effects after stenting atherosclerotic coronary arteries. A breakthrough in better vascular wall regeneration was noted in the new era of drug-eluting stents. A novel personalized approach is the development of gene-eluting stents promising an alteration in gene expression involved in regeneration. We investigated a coating system consisting of the polymer atelocollagen (ATCOL) and a specific small interfering RNA (siRNA) for intercellular adhesion molecule-1 (ICAM-1) found on the surface of defective endothelial cells (ECs). We demonstrated very high cell viability, in which EA.hy926 grew on 0.008% or 0.032% ATCOL layers. Additionally, hemocompatibility assays proved the biocompatibility of this coating. The highest transfection efficiency with EA.hy926 was achieved with 5 μg siRNA immobilized in ATCOL after 2 days. The release of fluorescent-labeled siRNA was about 9 days. Long-term knockdown of ICAM-1 was analyzed by flow cytometry, revealing that the coating with 0.008% ATCOL and 5 μg siICAM-1 provoked gene silencing up to 8 days. 5'-RNA ligase-mediated rapid amplification of cDNA ends PCR (RLM-RACE-PCR) demonstrated the specificity of our established ATCOL gene-silencing coating, meaning that our coating is well suited for further investigations in in vivo studies. Herein, we would like to demonstrate that our ATCOL is well-suited for better artery wall regeneration after stent implantation.

  18. Ultradeformable cationic liposomes for delivery of small interfering RNA (siRNA) into human primary melanocytes.

    PubMed

    Geusens, B; Lambert, J; De Smedt, S C; Buyens, K; Sanders, N N; Van Gele, M

    2009-02-10

    The aim of this work was to develop a system that can deliver siRNA into cells present in the human epidermis. More specifically, we wanted to block the expression of a specific Myosin Va exon F containing isoform that is physiologically involved in melanosome transport in human melanocytes. Therefore, we prepared and investigated the capacity of ultradeformable cationic liposomes (UCLs) to deliver siRNA in hard-to-transfect human primary melanocytes. UCLs were formulated from different w:w ratios (6:1, 8:1 and 10:1) of the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and the edge activator sodium cholate. Subsequently, UCL/siRNA complexes were prepared and their particle size, surface charge, deformability, cytotoxicity, transfection efficiency and long-term stability were tested. The best results were obtained with UCLs composed of a DOTAP/NaChol ratio of 6:1 (w:w) which are promising for future in vivo experiments.

  19. Poly(glycerol methacrylate)-based degradable nanoparticles for delivery of small interfering RNA.

    PubMed

    Morsi, Noha G; Ali, Shimaa M; Elsonbaty, Sherouk S; Afifi, Ahmed A; Hamad, Mostafa A; Gao, Hui; Elsabahy, Mahmoud

    2017-04-07

    Nucleic acids therapeutic efficiency is generally limited by their low stability and intracellular bioavailability, and by the toxicity of the carriers used to deliver them to the target sites. Aminated poly(glycerol methacrylate) polymers are biodegradable and pH-sensitive polymers that have been used previously to deliver antisense oligonucleotide and show high transfection efficiency. The purpose of this study is to compare the efficiency and toxicity of aminated linear poly(glycerol methacrylate) (ALT) biodegradable polymer to the most commonly used cationic degradable (i.e. chitosan) and non-degradable (i.e. polyethylenimine (PEI)) polymers for delivery of short interfering RNA (siRNA). ALT, PEI and chitosan polymers were able to form nanosized particles with siRNA. Size, size-distribution and zeta-potential were measured over a wide range of nitrogen-to-phosphate (N/P) ratios, and the stability of the formed nanoparticles in saline and upon freeze-drying was also assessed. No significant cytotoxicity at the range of the tested concentrations of ALT and chitosan nanoparticles was observed, whereas the non-degradable PEI showed significant toxicity in huh-7 hepatocyte-derived carcinoma cell line. The safety profiles of the degradable polymers (ALT and chitosan) over non-degradable PEI were demonstrated in vitro and in vivo. In addition, ALT nanoparticles were able to deliver siRNA in vivo with significantly higher efficiency than chitosan nanoparticles. The results in the present study give evidence of the great implications of ALT nanoparticles in biomedical applications due to their biocompatibility, low cytotoxicity, high stability and simple preparation method.

  20. Caenorhabditis elegans RSD-2 and RSD-6 promote germ cell immortality by maintaining small interfering RNA populations.

    PubMed

    Sakaguchi, Aisa; Sarkies, Peter; Simon, Matt; Doebley, Anna-Lisa; Goldstein, Leonard D; Hedges, Ashley; Ikegami, Kohta; Alvares, Stacy M; Yang, Liwei; LaRocque, Jeannine R; Hall, Julie; Miska, Eric A; Ahmed, Shawn

    2014-10-14

    Germ cells are maintained in a pristine non-aging state as they proliferate over generations. Here, we show that a novel function of the Caenorhabditis elegans RNA interference proteins RNAi spreading defective (RSD)-2 and RSD-6 is to promote germ cell immortality at high temperature. rsd mutants cultured at high temperatures became progressively sterile and displayed loss of small interfering RNAs (siRNAs) that target spermatogenesis genes, simple repeats, and transposons. Desilencing of spermatogenesis genes occurred in late-generation rsd mutants, although defective spermatogenesis was insufficient to explain the majority of sterility. Increased expression of repetitive loci occurred in both germ and somatic cells of late-generation rsd mutant adults, suggesting that desilencing of many heterochromatic segments of the genome contributes to sterility. Nuclear RNAi defective (NRDE)-2 promotes nuclear silencing in response to exogenous double-stranded RNA, and our data imply that RSD-2, RSD-6, and NRDE-2 function in a common transgenerational nuclear silencing pathway that responds to endogenous siRNAs. We propose that RSD-2 and RSD-6 promote germ cell immortality at stressful temperatures by maintaining transgenerational epigenetic inheritance of endogenous siRNA populations that promote genome silencing.

  1. Small interfering RNA therapy against carbohydrate sulfotransferase 15 inhibits cardiac remodeling in rats with dilated cardiomyopathy.

    PubMed

    Watanabe, Kenichi; Arumugam, Somasundaram; Sreedhar, Remya; Thandavarayan, Rajarajan A; Nakamura, Takashi; Nakamura, Masahiko; Harima, Meilei; Yoneyama, Hiroyuki; Suzuki, Kenji

    2015-07-01

    Carbohydrate sulfotransferase 15 (CHST15) is a sulfotransferase responsible for biosynthesis of chondroitin sulfate E (CS-E), which plays important roles in numerous biological events such as biosynthesis of proinflammatory cytokines. However, the effects of CHST15 siRNA in rats with chronic heart failure (CHF) after experimental autoimmune myocarditis (EAM) have not yet been investigated. CHF was elicited in Lewis rats by immunization with cardiac myosin, and after immunization, the rats were divided into two groups and treated with either CHST15 siRNA (2μg/week) or vehicle. Age matched normal rats without immunizations were also included in this study. After 7weeks of treatment, we investigated the effects of CHST15 siRNA on cardiac function, proinflammatory cytokines, and cardiac remodeling in EAM rats. Myocardial functional parameters measured by hemodynamic and echocardiographic studies were significantly improved by CHST15 siRNA treatment in rats with CHF compared with that of vehicle-treated CHF rats. CHST15 siRNA significantly reduced cardiac fibrosis, and hypertrophy and its marker molecules (left ventricular (LV) mRNA expressions of transforming growth factor beta1, collagens I and III, and atrial natriuretic peptide) compared with vehicle-treated CHF rats. CHF-induced increased myocardial mRNA expressions of proinflammatory cytokines [interleukin (IL)-6, IL-1β], monocyte chemoattractant protein-1, and matrix metalloproteinases (MMP-2 and -9), and CHST15 were also suppressed by the treatment with CHST15 siRNA. Western blotting study has confirmed the results obtained from mRNA analysis as CHST15 siRNA treated rats expressed reduced levels of inflammatory and cardiac remodeling marker proteins. Our results demonstrate for the first time, that CHST15 siRNA treatment significantly improved LV function and ameliorated the progression of cardiac remodeling in rats with CHF after EAM.

  2. Effective Small Interfering RNA Therapy to Treat CLCN7-dependent Autosomal Dominant Osteopetrosis Type 2

    PubMed Central

    Capulli, Mattia; Maurizi, Antonio; Ventura, Luca; Rucci, Nadia; Teti, Anna

    2015-01-01

    In about 70% of patients affected by autosomal dominant osteopetrosis type 2 (ADO2), osteoclast activity is reduced by heterozygous mutations of the CLCN7 gene, encoding the ClC-7 chloride/hydrogen antiporter. CLCN7G215R-, CLCN7R767W-, and CLCN7R286W-specific siRNAs silenced transfected mutant mRNA/EGFP in HEK293 cells, in RAW264.7 cells and in human osteoclasts, with no change of CLCN7WT mRNA and no effect of scrambled siRNA on the mutant transcripts. Osteoclasts from Clcn7G213R ADO2 mice showed reduced bone resorption, a condition rescued by Clcn7G213R-specific siRNA. Treatment of ADO2 mice with Clcn7G213R-specific siRNA induced increase of bone resorption variables and decrease of trabecular bone mass, leading to an overall improvement of the osteopetrotic bone phenotype. Treatment did not induce overt adverse effects and was effective also with siRNAs specific for other mutants. These results demonstrate that a siRNA-based experimental treatment of ADO2 is feasible, and underscore a translational impact for future strategy to cure this therapeutically neglected form of osteopetrosis. PMID:26325626

  3. Small interfering RNA pathway modulates persistent infection of a plant virus in its insect vector.

    PubMed

    Lan, Hanhong; Wang, Haitao; Chen, Qian; Chen, Hongyan; Jia, Dongsheng; Mao, Qianzhuo; Wei, Taiyun

    2016-02-11

    Plant reoviruses, rhabdoviruses, tospoviruses, and tenuiviruses are transmitted by insect vectors in a persistent-propagative manner. How such persistent infection of plant viruses in insect vectors is established and maintained remains poorly understood. In this study, we used rice gall dwarf virus (RGDV), a plant reovirus, and its main vector leafhopper Recilia dorsalis as a virus-insect system to determine how the small interference (siRNA) pathway modulates persistent infection of a plant virus in its insect vector. We showed that a conserved siRNA antiviral response was triggered by the persistent replication of RGDV in cultured leafhopper cells and in intact insects, by appearance of virus-specific siRNAs, primarily 21-nt long, and the increased expression of siRNA pathway core components Dicer-2 and Argonaute-2. Silencing of Dicer-2 using RNA interference strongly suppressed production of virus-specific siRNAs, promoted viral accumulation, and caused cytopathological changes in vitro and in vivo. When the viral accumulation level rose above a certain threshold of viral genome copy (1.32 × 10(14) copies/μg insect RNA), the infection of the leafhopper by RGDV was lethal rather than persistent. Taken together, our results revealed a new finding that the siRNA pathway in insect vector can modulate persistent infection of plant viruses.

  4. Connexin-specific cell-to-cell transfer of short interfering RNA by gap junctions

    PubMed Central

    Valiunas, V; Polosina, YY; Miller, H; Potapova, IA; Valiuniene, L; Doronin, S; Mathias, RT; Robinson, RB; Rosen, MR; Cohen, IS; Brink, PR

    2005-01-01

    The purpose of this study was to determine whether oligonucleotides the size of siRNA are permeable to gap junctions and whether a specific siRNA for DNA polymerase β (pol β) can move from one cell to another via gap junctions, thus allowing one cell to inhibit gene expression in another cell directly. To test this hypothesis, fluorescently labelled oligonucleotides (morpholinos) 12, 16 and 24 nucleotides in length were synthesized and introduced into one cell of a pair using a patch pipette. These probes moved from cell to cell through gap junctions composed of connexin 43 (Cx43). Moreover, the rate of transfer declined with increasing length of the oligonucleotide. To test whether siRNA for pol β was permeable to gap junctions we used three cell lines: (1) NRK cells that endogenously express Cx43; (2) Mβ16tsA cells, which express Cx32 and Cx26 but not Cx43; and (3) connexin-deficient N2A cells. NRK and Mβ16tsA cells were each divided into two groups, one of which was stably transfected to express a small hairpin RNA (shRNA), which gives rise to siRNA that targets pol β. These two pol β knockdown cell lines (NRK-kcdc and Mβ16tsA-kcdc) were co-cultured with labelled wild type, NRK-wt or Mβ16tsA-wt cells or N2A cells. The levels of pol β mRNA and protein were determined by semiquantitative RT-PCR and immunoblotting. Co-culture of Mβ16tsA-kcdc cells with Mβ16tsA-wt, N2A or NRK-wt cells had no effect on pol β levels in these cells. Similarly, co-culture of NRK-kcdc with N2A cells had no effect on pol β levels in the N2A cells. In contrast, co-culture of NRK-kcdc with NRK-wt cells resulted in a significant reduction in pol β in the wt cells. The inability of Mβ16tsA-kcdc cells to transfer siRNA is consistent with the fact that oligonucleotides of the 12 nucleotide length were not permeable to Cx32/Cx26 channels. This suggested that Cx43 but not Cx32/Cx26 channels allowed the cell-to-cell movement of the siRNA. These results support the novel hypothesis

  5. Small interfering RNA (siRNA)-mediated knockdown of macrophage migration inhibitory factor (MIF) suppressed cyclin D1 expression and hepatocellular carcinoma cell proliferation.

    PubMed

    Huang, Xiao-Hui; Jian, Wei-Hua; Wu, Zhao-Feng; Zhao, Jie; Wang, Hua; Li, Wen; Xia, Jin-Tang

    2014-07-30

    Macrophage migration inhibitory factor (MIF), a proinflammatory and immunoregulatory chemokine, plays important roles in cancer-related biological processes. However, few studies have focused on the clinical relevance of MIF and cyclin D1 expression in hepatocellular carcinoma cells (HCCs). In this study, MIF and cyclin D1 expression levels in HCC tissues and cell lines were significantly upregulated compared with adjacent normal tissues or a normal liver cell line. In HCC specimens, MIF expression positively correlated with cyclin D1 expression. Additionally, MIF and cyclin D1 expression positively correlated with tumor size. MIF knockdown inhibited the proliferation of PLC and HepG2 cells and promoted apoptosis. However, small interfering RNA (siRNA) against MIF did not influence the cell cycle in these cells. In an in vivo xenograft model, MIF knockdown reduced the tumor growth rate. The expression levels of Bcl-2, p-caspase-3, BIM and Bax were upregulated, while the expression levels of cyclin D1, p-Akt and p-ERK were downregulated in MIF-knockdown cells. These findings indicate that MIF siRNA reduces proliferation and increases apoptosis in HCC cells. MIF knockdown inhibits the expression of growth-related proteins and induces the expression of apoptosis-related proteins, supporting a role for MIF as a novel therapeutic target for HCC.

  6. Down-regulation of survivin expression by small interfering RNA induces pancreatic cancer cell apoptosis and enhances its radiosensitivity

    PubMed Central

    Guan, Hai-Tao; Xue, Xing-Huan; Dai, Zhi-Jun; Wang, Xi-Jing; Li, Ang; Qin, Zhao-Yin

    2006-01-01

    AIM: To investigate the inhibitory effect of small interfering RNA (siRNA) on the expression of survivin in pancreatic cancer cell line PC-2 and the role of siRNA in inducing PC-2 cell apoptosis and enhancing its radiosensitivity. METHODS: A siRNA plasmid expression vector against survivin was constructed and transfected into PC-2 cells with LipofectamineTM 2000. The down regulation of survivin expression was detected by semi-quantitive RT-PCR and immunohistochemical SP method and the role of siRNA in inducing PC-2 cell apoptosis and enhancing its radiosensitivity was detected by flow cytometry. RESULTS: The sequence-specific siRNA efficiently and specifically down-regulated the expression of survivin at both mRNA and protein levels. The expression inhibition ratio was 81.25% at mRNA level detected by semi-quantitive RT-PCR and 74.24% at protein level detected by immunohistochemical method. Forty-eight hours after transfection,apoptosis was induced in 7.03% cells by siRNA and in 14.58% cells by siRNA combined with radiation. CONCLUSION: The siRNA plasmid expression vector against survivin can inhibit the expression of survivin in PC-2 cells efficiently and specifically. Inhibiting the expression of survivin can induce apoptosis of PC-2 cells and enhance its radiosensitivity significantly. RNAi against survivin is of potential value in gene therapy of pancreatic cancer. PMID:16718816

  7. In silico reconstruction of viral genomes from small RNAs improves virus-derived small interfering RNA profiling.

    PubMed

    Vodovar, Nicolas; Goic, Bertsy; Blanc, Hervé; Saleh, Maria-Carla

    2011-11-01

    RNA interference (RNAi) is the essential component of antiviral immunity in invertebrates and plants. One of the landmarks of the antiviral RNAi response is the production of virus-derived small interfering RNA (vsiRNA) from viral double-stranded RNA (dsRNA). vsiRNAs constitute a fragmented image of the viral genome sequence that results from Dicer cleavage. vsiRNA sequence profiling is used extensively as a surrogate to study the antiviral RNAi response by determining the nature of the viral dsRNA molecules exposed to and processed by the RNAi machinery. The accuracy of these profiles depends on the actual viral genome sequence used as a reference to align vsiRNA reads, and the interpretation of inaccurate profiles can be misleading. Using Flock house virus and Drosophila melanogaster as a model RNAi-competent organism, we show accurate reconstruction of full-length virus reference sequence from vsiRNAs and prediction of the structure of defective interfering particles (DIs). We developed a Perl script, named Paparazzi, that reconstitutes viral genomes through an iterative alignment/consensus call procedure using a related reference sequence as scaffold. As prevalent DI-derived reads introduce artifacts during reconstruction, Paparazzi eliminates DI-specific reads to improve the quality of the reconstructed genome. Paparazzi constitutes a promising alternative to Sanger sequencing in this context and an effective tool to study antiviral RNAi mechanisms by accurately quantifying vsiRNA along the replicating viral genome. We further discuss Paparazzi as a companion tool for virus discovery as it provides full-length genome sequences and corrects for potential artifacts of assembly.

  8. Noninvasive Imaging of Lipid Nanoparticle–Mediated Systemic Delivery of Small-Interfering RNA to the Liver

    PubMed Central

    Tao, Weikang; Davide, Joseph P; Cai, Mingmei; Zhang, Guo-Jun; South, Victoria J; Matter, Andrea; Ng, Bruce; Zhang, Ye; Sepp-Lorenzino, Laura

    2010-01-01

    Mouse models with liver-specific expression of firefly luciferase were developed that enable a noninvasive and longitudinal assessment of small-interfering RNA (siRNA)–mediated gene silencing in hepatocytes of live animals via bioluminescence imaging. Using these models, a set of lipid nanoparticles (LNPs) with different compositions of cationic lipids, polyethylene glycol (PEG), and cholesterol, were tested for their abilities in delivering a luciferase siRNA to the liver via systemic administration. A dose-dependent luciferase knockdown by LNP/siRNA assemblies was measured by in vivo bioluminescence imaging, which correlated well with the results from parallel ex vivo analyses of luciferase mRNA and protein levels in the liver. RNA interference (RNAi)–mediated target silencing was further confirmed by the detection of RNAi-specific target mRNA cleavage. A single dose of LNP02L at 3 mg/kg (siRNA) caused 90% reduction of luciferase expression and the target repression lasted for at least 10 days. With identical components, LNPs containing 2% PEG are more potent than those with 5.4% PEG. Our results demonstrate that these liver-luciferase mouse models provide a powerful tool for a high-throughput evaluation of hepatic delivery platforms by noninvasive imaging and that the molar ratio of PEG lipid can affect the efficacy of LNPs in silencing liver targets via systemic administration. PMID:20628357

  9. Noninvasive imaging of lipid nanoparticle-mediated systemic delivery of small-interfering RNA to the liver.

    PubMed

    Tao, Weikang; Davide, Joseph P; Cai, Mingmei; Zhang, Guo-Jun; South, Victoria J; Matter, Andrea; Ng, Bruce; Zhang, Ye; Sepp-Lorenzino, Laura

    2010-09-01

    Mouse models with liver-specific expression of firefly luciferase were developed that enable a noninvasive and longitudinal assessment of small-interfering RNA (siRNA)-mediated gene silencing in hepatocytes of live animals via bioluminescence imaging. Using these models, a set of lipid nanoparticles (LNPs) with different compositions of cationic lipids, polyethylene glycol (PEG), and cholesterol, were tested for their abilities in delivering a luciferase siRNA to the liver via systemic administration. A dose-dependent luciferase knockdown by LNP/siRNA assemblies was measured by in vivo bioluminescence imaging, which correlated well with the results from parallel ex vivo analyses of luciferase mRNA and protein levels in the liver. RNA interference (RNAi)-mediated target silencing was further confirmed by the detection of RNAi-specific target mRNA cleavage. A single dose of LNP02L at 3 mg/kg (siRNA) caused 90% reduction of luciferase expression and the target repression lasted for at least 10 days. With identical components, LNPs containing 2% PEG are more potent than those with 5.4% PEG. Our results demonstrate that these liver-luciferase mouse models provide a powerful tool for a high-throughput evaluation of hepatic delivery platforms by noninvasive imaging and that the molar ratio of PEG lipid can affect the efficacy of LNPs in silencing liver targets via systemic administration.

  10. Biodistribution of Small Interfering RNA at the Organ and Cellular Levels after Lipid Nanoparticle-mediated Delivery

    PubMed Central

    Shi, Bin; Keough, Ed; Matter, Andrea; Leander, Karen; Young, Stephanie; Carlini, Ed; Sachs, Alan B.; Tao, Weikang; Abrams, Marc; Howell, Bonnie; Sepp-Lorenzino, Laura

    2011-01-01

    Chemically stabilized small interfering RNA (siRNA) can be delivered systemically by intravenous injection of lipid nanoparticles (LNPs) in rodents and primates. The biodistribution and kinetics of LNP–siRNA delivery in mice at organ and cellular resolution have been studied using immunofluorescence (IF) staining and quantitative polymerase chain reaction (qPCR). At 0.5 and 2 hr post tail vein injection of Cy5-labeled siRNA encapsulated in LNP, the organ rank-order of siRNA levels is liver > spleen > kidney, with only negligible accumulation in duodenum, lung, heart, and brain. Similar conclusions were drawn by using qPCR to measure tissue siRNA levels as a secondary end point. siRNA levels in these tissues decreased by more than 10-fold after 24 hr. Within the liver, LNPs delivered siRNA to hepatocytes, Kupffer cells, and sinusoids in a time-dependent manner, as revealed by IF staining and signal quantitation methods established using OPERA/Columbus software. siRNA first accumulated in liver sinusoids and trafficked to hepatocytes by 2 hr post dose, corresponding to the onset of target mRNA silencing. Fluorescence in situ hybridization methods were used to detect both strands of siRNA in fixed tissues. Collectively, the authors have implemented a platform to evaluate biodistribution of siRNA across cell types and across tissues in vivo, with the objective of elucidating the pharmacokinetic and pharmacodynamic relationship to guide optimization of delivery vehicles. PMID:21804077

  11. On the use of short-interfering RNA to study alcohol-related genes.

    PubMed

    Adams, Christopher A; Zawada, W Michael

    2008-01-01

    One strategy to determine the contribution of individual genes to the development of complex traits and behaviors such as alcohol consumption is to reduce or completely eliminate the activity of those genes in the cells or organism under investigation and then study the effects of this modification. But how can a single specific gene be inactivated? One strategy that has been developed in recent years is the use of small, artificially generated molecules called short-interfering RNAs (siRNAs). This article briefly describes the principles of this strategy and presents some initial results obtained with this approach.

  12. P-SAMS: a web site for plant artificial microRNA and synthetic trans-acting small interfering RNA design

    PubMed Central

    Fahlgren, Noah; Hill, Steven T.; Carrington, James C.; Carbonell, Alberto

    2016-01-01

    Summary: The Plant Small RNA Maker Site (P-SAMS) is a web tool for the simple and automated design of artificial miRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) for efficient and specific targeted gene silencing in plants. P-SAMS includes two applications, P-SAMS amiRNA Designer and P-SAMS syn-tasiRNA Designer. The navigation through both applications is wizard-assisted, and the job runtime is relatively short. Both applications output the sequence of designed small RNA(s), and the sequence of the two oligonucleotides required for cloning into ‘B/c’ compatible vectors. Availability and implementation: The P-SAMS website is available at http://p-sams.carringtonlab.org. Contact: acarbonell@ibmcp.upv.es or nfahlgren@danforthcenter.org PMID:26382195

  13. Zwitterionic poly(carboxybetaine)-based cationic liposomes for effective delivery of small interfering RNA therapeutics without accelerated blood clearance phenomenon.

    PubMed

    Li, Yan; Liu, Ruiyuan; Shi, Yuanjie; Zhang, Zhenzhong; Zhang, Xin

    2015-01-01

    For efficient delivery of small interfering RNA (siRNA) to the target diseased site in vivo, it is important to design suitable vehicles to control the blood circulation of siRNA. It has been shown that surface modification of cationic liposome/siRNA complexes (lipoplexes) with polyethylene glycol (PEG) could enhance the circulation time of lipoplexes. However, the first injection of PEGylated lipoplexes in vivo induces accelerated blood clearance and enhances hepatic accumulation of the following injected PEGylated lipoplexes, which is known as the accelerated blood clearance (ABC) phenomenon. Herein, we developed zwitterionic poly(carboxybetaine) (PCB) modified lipoplexes for the delivery of siRNA therapeutics, which could avoid protein adsorption and enhance the stability of lipoplexes as that for PEG. Quite different from the PEGylation, the PCBylated lipoplexes could avoid ABC phenomenon, which extended the blood circulation time and enhanced the tumor accumulation of lipoplexes in vivo. After accumulation in tumor site, the PCBylation could promote the cellular uptake and endosomal/lysosomal escape of lipoplexes due to its unique chemical structure and pH-sensitive ability. With excellent tumor accumulation, cellular uptake and endosomal/lysosomal escape abilities, the PCBylated lipoplexes significantly inhibited tumor growth and induced tumor cell apoptosis.

  14. Zwitterionic Poly(carboxybetaine)-based Cationic Liposomes for Effective Delivery of Small Interfering RNA Therapeutics without Accelerated Blood Clearance Phenomenon

    PubMed Central

    Li, Yan; Liu, Ruiyuan; Shi, Yuanjie; Zhang, Zhenzhong; Zhang, Xin

    2015-01-01

    For efficient delivery of small interfering RNA (siRNA) to the target diseased site in vivo, it is important to design suitable vehicles to control the blood circulation of siRNA. It has been shown that surface modification of cationic liposome/siRNA complexes (lipoplexes) with polyethylene glycol (PEG) could enhance the circulation time of lipoplexes. However, the first injection of PEGylated lipoplexes in vivo induces accelerated blood clearance and enhances hepatic accumulation of the following injected PEGylated lipoplexes, which is known as the accelerated blood clearance (ABC) phenomenon. Herein, we developed zwitterionic poly(carboxybetaine) (PCB) modified lipoplexes for the delivery of siRNA therapeutics, which could avoid protein adsorption and enhance the stability of lipoplexes as that for PEG. Quite different from the PEGylation, the PCBylated lipoplexes could avoid ABC phenomenon, which extended the blood circulation time and enhanced the tumor accumulation of lipoplexes in vivo. After accumulation in tumor site, the PCBylation could promote the cellular uptake and endosomal/lysosomal escape of lipoplexes due to its unique chemical structure and pH-sensitive ability. With excellent tumor accumulation, cellular uptake and endosomal/lysosomal escape abilities, the PCBylated lipoplexes significantly inhibited tumor growth and induced tumor cell apoptosis. PMID:25825598

  15. Phytophthora have distinct endogenous small RNA populations that include short interfering and microRNAs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In eukaryotes, RNA silencing pathways utilize 20–30-nucleotide small RNAs to regulate gene expression, specify and maintain chromatin structure, and repress viruses and mobile genetic elements. RNA silencing was likely present in the common ancestor of modern eukaryotes, but most research has focuse...

  16. Effects of small interfering RNA targeting sphingosine kinase-1 gene on the animal model of Alzheimer's disease.

    PubMed

    Zhang, Yuan; Yu, Qian; Lai, Tian-bao; Yang, Yang; Li, Gang; Sun, Sheng-gang

    2013-06-01

    Alzheimer's disease (AD) is an age-related, progressive neurodegenerative disorder that occurs gradually and results in memory, behavior, and personality changes. Abnormal sphingolipid metabolism was reported in AD previously. This study aimed to investigate whether sphK1 could exacerbate the accumulation of amyloid protein (Aβ) and sharpen the learning and memory ability of the animal model of AD using siRNA interference. An adenovirus vector expressing small interfering RNA (siRNA) against the sphK1 gene (sphK1-siRNA) was designed, and the effects of sphK1-siRNA on the APP/PS1 mouse four weeks after treatment with sphK1-siRNA hippocampal injection were examined. SphK1 protein expression was confirmed by using Western blotting and ceramide content coupled with S1P secretion was evaluated by enzyme-linked immunosorbent assay (ELISA). Aβ load was detected by immunohistochemical staining and ELISA. Morris water maze was adopted to test the learning and memory ability of the APP/PS1 mice. A significant difference in the expression of sphK1 protein and mRNA was observed between the siRNA group and the control group. Aβ load in transfected mice was accelerated in vivo, with significant aggravation of the learning and memory ability. The sphK1 gene modulation in the Aβ load and the learning and memory ability in the animal model of AD may be important for the treatment of AD.

  17. Efficient Protection and Transfection of Small Interfering RNA by Cationic Shell-Crosslinked Knedel-Like Nanoparticles

    PubMed Central

    Shen, Yuefei; Fang, Huafeng; Zhang, Ke; Shrestha, Ritu; Wooley, Karen L.

    2013-01-01

    Despite the great potential of small interfering RNA (siRNA) as a therapeutic agent, progress in this area has been hampered by a lack of efficient biocompatible transfection agents. Recently, cationic shell-crosslinked knedel-like nanoparticles (cSCKs) were found to possess lower cytotoxicity and better transfection ability for phosphorothioate ODNs and plasmid DNA than the commonly used cationic lipid-based agent Lipofectamine. To determine the usefulness of cSCKs for siRNA transfection, a small library of cSCKs with varying percentage of primary and tertiary amines was assessed for its ability to bind to siRNA, inhibit siRNA degradation in human serum, and to transfect HeLa and mouse macrophage cell lines. The silencing efficiency in HeLa cells was greatest with the cSCK with 100% primary amines (pa100) as determined by their viability following transfection with cytotoxic and non-cytotoxic siRNAs. cSCK-pa100 showed greater silencing efficiency than Lipofectamine 2000 in the HeLa cells, as well in 293T and human bronchial epithelial (HEK) cells, but was comparable in human bronchial epithelial (BEAS-2B) cells and human mammary epithelial (MCF10a) cells. cSCK-pa100 also showed greater silencing of iNOS expression than Lipofectamine 2000 in a mouse macrophage cell line, and provided greater protection from serum degradation, demonstrating its potential usefulness as an siRNA transfection agent. The siRNA silencing of iNOS at lower concentrations of siRNA could be enhanced by complexation with the fusogenic GALA peptide, which was shown to enhance endosomal escape following uptake. PMID:23557117

  18. Cloned Defective Interfering Influenza RNA and a Possible Pan-Specific Treatment of Respiratory Virus Diseases.

    PubMed

    Dimmock, Nigel J; Easton, Andrew J

    2015-07-08

    Defective interfering (DI) genomes are characterised by their ability to interfere with the replication of the virus from which they were derived, and other genetically compatible viruses. DI genomes are synthesized by nearly all known viruses and represent a vast natural reservoir of antivirals that can potentially be exploited for use in the clinic. This review describes the application of DI virus to protect from virus-associated diseases in vivo using as an example a highly active cloned influenza A DI genome and virus that protects broadly in preclinical trials against different subtypes of influenza A and against non-influenza A respiratory viruses. This influenza A-derived DI genome protects by two totally different mechanisms: molecular interference with influenza A replication and by stimulating innate immunity that acts against non-influenza A viruses. The review considers what is needed to develop DI genomes to the point of entry into clinical trials.

  19. SIRT1 inhibits EV71 genome replication and RNA translation by interfering with the viral polymerase and 5′UTR RNA

    PubMed Central

    Han, Yang; Wang, Lvyin; Cui, Jin; Song, Yu; Luo, Zhen; Chen, Junbo; Xiong, Ying; Zhang, Qi; Liu, Fang; Ho, Wenzhe; Liu, Yingle; Wu, Jianguo

    2016-01-01

    ABSTRACT Enterovirus 71 (EV71) possesses a single-stranded positive RNA genome that contains a single open reading frame (ORF) flanked by a 5′ untranslated region (5′UTR) and a polyadenylated 3′UTR. Here, we demonstrated that EV71 activates the production of silent mating type information regulation 2 homolog 1 (SIRT1), a histone deacetylase (HDAC). EV71 further stimulates SIRT1 sumoylation and deacetylase activity, and enhances SIRT1 translocation from the nucleus to the cytoplasm. More interestingly, activated SIRT1 subsequently binds with the EV71 3Dpol protein (a viral RNA-dependent RNA polymerase, RdRp) to repress the acetylation and RdRp activity of 3Dpol, resulting in the attenuation of viral genome replication. Moreover, SIRT1 interacts with the cloverleaf structure of the EV71 RNA 5′UTR to inhibit viral RNA transcription, and binds to the internal ribosome entry site (IRES) of the EV71 5′UTR to attenuate viral RNA translation. Thus, EV71 stimulates SIRT1 production and activity, which in turn represses EV71 genome replication by inhibiting viral polymerase, and attenuates EV71 RNA transcription and translation by interfering with viral RNA. These results uncover a new function of SIRT1 and reveal a new mechanism underlying the regulation of EV71 replication. PMID:27875274

  20. Bridging small interfering RNA with giant therapeutic outcomes using nanometric liposomes.

    PubMed

    Singh, Yuvraj; Tomar, Sandeep; Khan, Shariq; Meher, Jaya Gopal; Pawar, Vivek K; Raval, Kavit; Sharma, Komal; Singh, Pankaj K; Chaurasia, Mohini; Surendar Reddy, B; Chourasia, Manish K

    2015-12-28

    The scope of RNAi based therapeutics is unquestionable. However, if we dissect the current trend of clinical trials for afore mentioned drug class, some stark trends appear: 1) naked siRNA only exerts influence in topical mode whilst systemic delivery requires a carrier and 2) even after two decades of extensive efforts, not even a single siRNA containing product is commercially available. It was therefore felt that a perspective simplifying the unique intricacies of working with a merger of siRNA and liposomes from a pharmaceutical viewpoint could draw the attention of a wider array of interested researchers. We begin from the beginning and attempt to conduit the gap between theoretical logic and experimental/actual constraints. This, in turn could stimulate the next generation of investigators, gearing them to tackle the conundrum, which is siRNA delivery.

  1. Inhibition of vascular endothelial growth factor by small interfering RNA upregulates differentiation, maturation and function of dendritic cells

    PubMed Central

    WANG, HAIYAN; ZHANG, LUPING; ZHANG, SHAOYAN; LI, YANNIAN

    2015-01-01

    This study aimed to investigate the effects of vascular endothelial growth factor (VEGF) secreted by MCF-7 breast cancer cells on the differentiation, maturation and function of dendritic cells (DCs). Small interfering RNAs (siRNAs) directed against the VEGF gene were designed and transfected into MCF-7 breast cancer cells at an optimal concentration (100 nmol/l) using cationic liposome transfection reagent, whereas the control group was transfected with only transfection reagent. Western blot analysis and ELISA were used to determine VEGF protein expression and VEGF concentration, respectively. Mononuclear cells were cultured with the culture supernatants from primary MCF-7 cells (control group) and siRNA-treated MCF-7 cells (siRNA group). The DC phenotypes, including CD1a, CD80, CD83, CD86 and HLA-DR, were evaluated by flow cytometry. The MTT assay was used to assess the cytotoxicity of DC-mediated tumor-specific cytotoxic T lymphocytes (CTLs) against MCF-7 cells in the two different culture supernatants. The VEGF-targeted constructed siRNA inhibited VEGF expression in MCF-7 cells. Cultivation with the culture supernatants from MCF-7 cells treated with siRNA affected DC morphology. DCs in the siRNA group exhibited a significantly higher expression of CD86, CD80, CD83 and HLA-DR compared to the cells in the control group, whereas the expression of CD1a in the siRNA group was significantly lower compared to that in the control group. The cytotoxic activity of CTLs mediated by DCs was significantly altered by siRNA transfection. These results indicated that VEGF may play a significant role in tumor development, progression and immunosuppression. PMID:25452786

  2. Characterization of a defective interfering RNA that contains a mosaic of a plant viral genome. Final report

    SciTech Connect

    Morris, T.J.; Jackson, A.O.

    1991-12-31

    Our lab was the first to describe and characterize a defective interfering RNA (DI RNAs or DIs) in association with a small RNA plant virus. The features of the DIs that we discovered in infections of tomato bushy stunt virus were compatible with the properties of DIs identified in many animal virus infections. Animal virologists have generally recognized the importance of studying DIs because they are invaluable tools for identifying cis-acting sequences important in virus multiplication and because they offer the opportunity to elucidate mechanisms involved in viral persistence and disease attenuation. Hence our discovery offered a comparably valuable tool for use in plant virus studies for the first time. Since then, we have also discovered the second example of plant viral DI RNAs associated with turnip crinkle virus (TCV), a virus structurally related to TBSV. We proposed a thorough characterization of this unique class of symptom modulating RNAs with the overall objective of identifying viral RNA nucleotide, sequences involved in such fundamental processes as virus replication and encapsidation as well as the degree of symptom expression resulting from the viral-DI-host interaction. The proposed research focused on the molecular characterization of the DI RNAs and the helper virus. We had demonstrated that the DIs were collinear deletion mutants of the genome of a cherry strain of tomato bushy stunt virus (TBSV). We had also shown that these low molecular weight RNAs interfered with the helper plant virus and modulated disease expression by preventing the development of a lethal necrotic disease in susceptible host plants. We also suggested that by exploring the mechanisms associated with the symptom attenuation effect, we might be able to devise novel strategies useful for engineering viral disease resistance.

  3. Excessive production and extreme editing of human metapneumovirus defective interfering RNA is associated with type I IFN induction.

    PubMed

    van den Hoogen, Bernadette G; van Boheemen, Sander; de Rijck, Jonneke; van Nieuwkoop, Stefan; Smith, Derek J; Laksono, Brigitta; Gultyaev, Alexander; Osterhaus, Albert D M E; Fouchier, Ron A M

    2014-08-01

    Type I IFN production is one of the hallmarks of host innate immune responses upon virus infection. Whilst most respiratory viruses carry IFN antagonists, reports on human metapneumovirus (HMPV) have been conflicting. Using deep sequencing, we have demonstrated that HMPV particles accumulate excessive amounts of defective interfering RNA (DIs) rapidly upon in vitro passage, and that these are associated with IFN induction. Importantly, the DIs were edited extensively; up to 70% of the original A and T residues had mutated to G or C, respectively. Such high editing rates of viral RNA have not, to our knowledge, been reported before. Bioinformatics and PCR assays indicated that adenosine deaminase acting on RNA (ADAR) was the most likely editing enzyme. HMPV thus has an unusually high propensity to generate DIs, which are edited at an unprecedented high frequency. The conflicting published data on HMPV IFN induction and antagonism are probably explained by DIs in virus stocks. The interaction of HMPV DIs with the RNA-editing machinery and IFN responses warrants further investigation.

  4. Interference of hepatitis C virus RNA replication by short interfering RNAs

    NASA Astrophysics Data System (ADS)

    Kapadia, Sharookh B.; Brideau-Andersen, Amy; Chisari, Francis V.

    2003-02-01

    Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, which can lead to the development of liver cirrhosis and hepatocellular carcinoma. Current therapy of patients with chronic HCV infection includes treatment with IFN in combination with ribavirin. Because most treated patients do not resolve the infection, alternative treatment is essential. RNA interference (RNAi) is a recently discovered antiviral mechanism present in plants and animals that induces double-stranded RNA degradation. Using a selectable subgenomic HCV replicon cell culture system, we have shown that RNAi can specifically inhibit HCV RNA replication and protein expression in Huh-7 cells that stably replicate the HCV genome, and that this antiviral effect is independent of IFN. These results suggest that RNAi may represent a new approach for the treatment of persistent HCV infection.

  5. Comprehensive Annotation of Physcomitrella patens Small RNA Loci Reveals That the Heterochromatic Short Interfering RNA Pathway Is Largely Conserved in Land Plants[OPEN

    PubMed Central

    Coruh, Ceyda; Cho, Sung Hyun; Shahid, Saima; Liu, Qikun; Wierzbicki, Andrzej; Axtell, Michael J.

    2015-01-01

    Many plant small RNAs are sequence-specific negative regulators of target mRNAs and/or chromatin. In angiosperms, the two most abundant endogenous small RNA populations are usually 21-nucleotide microRNAs (miRNAs) and 24-nucleotide heterochromatic short interfering RNAs (siRNAs). Heterochromatic siRNAs are derived from repetitive regions and reinforce DNA methylation at targeted loci. The existence and extent of heterochromatic siRNAs in other land plant lineages has been unclear. Using small RNA-sequencing (RNA-seq) of the moss Physcomitrella patens, we identified 1090 loci that produce mostly 23- to 24-nucleotide siRNAs. These loci are mostly in intergenic regions with dense DNA methylation. Accumulation of siRNAs from these loci depends upon P. patens homologs of DICER-LIKE3 (DCL3), RNA-DEPENDENT RNA POLYMERASE2, and the largest subunit of DNA-DEPENDENT RNA POLYMERASE IV, with the largest subunit of a Pol V homolog contributing to expression at a smaller subset of the loci. A MINIMAL DICER-LIKE (mDCL) gene, which lacks the N-terminal helicase domain typical of DCL proteins, is specifically required for 23-nucleotide siRNA accumulation. We conclude that heterochromatic siRNAs, and their biogenesis pathways, are largely identical between angiosperms and P. patens, with the notable exception of the P. patens-specific use of mDCL to produce 23-nucleotide siRNAs. PMID:26209555

  6. Filamentous pathogen effectors interfering with small RNA silencing in plant hosts.

    PubMed

    Ye, Wenwu; Ma, Wenbo

    2016-08-01

    Filamentous eukaryotic pathogens including fungi and oomycetes are major threats of plant health. During the co-evolutionary arms race with the hosts, these pathogens have evolved a large repertoire of secreted virulence proteins, called effectors, to facilitate colonization and infection. Many effectors are believed to directly manipulate targeted processes inside the host cells; and a fundamental function of the effectors is to dampen immunity. Recent evidence suggests that the destructive oomycete pathogens in the genus Phytophthora encode RNA silencing suppressors. These effectors play an important virulence role during infection, likely through their inhibitory effect on host small RNA-mediated defense.

  7. Impact of CHK2-small interfering RNA on CpG ODN7909-enhanced radiosensitivity in lung cancer A549 cells

    PubMed Central

    Chen, Wei; Liu, Xiaoqun; Qiao, Tiankui; Yuan, Sujuan

    2012-01-01

    Objective To investigate the impact of checkpoint kinase 2 (CHK2)-small interfering RNA (CHK2-siRNA) on the enhancement of radiosensitivity by CpG oligodeoxynucleotide (ODN) 7909 in lung cancer A549 cells. Methods The A549 cells were randomly divided into five groups: control, CpG, X-ray, CpG + X-ray, and CHK2-siRNA + CpG + X-ray. Cell colonization was observed using inverted microscopy. Cell cycle and apoptosis were analyzed by flow cytometry. CHK2 expression was detected by Western blot. CHK2-siRNA was adopted to silence the expression of CHK2. Results The level of CHK2 phosphorylation was higher in the CpG + X-ray group than in the X-ray group. Increases in G2/mitotic (M) phase arrest and apoptosis and a decrease of cell survival rate in the CpG + X-ray group were statistically significant (P < 0.05) when compared with the CHK2-siRNA + CpG + X-ray group in which the expression of CHK2 was obviously inhibited. The combination of CpG ODN7909 and X-ray irradiation was found to enhance the mitotic death of A549 cells. The sensitization enhancement ratio of mean death dose (D0) was 1.42 in the CpG + X-ray group, which was higher than that of the CHK2-siRNA + CpG + X-ray group, in which D0 was 1.05. Conclusion To a certain extent, the impact of a combination of CpG ODN7909 and X-ray on G2/M phase arrest, apoptosis, and rate of cell survival was attenuated by CHK2-siRNA in human lung adenocarcinoma A549 cells, indicating that increased phosphorylation of CHK2 might be a radiosensitive pathway. PMID:23233807

  8. Effect of base modifications on structure, thermodynamic stability, and gene silencing activity of short interfering RNA

    PubMed Central

    Sipa, Katarzyna; Sochacka, Elzbieta; Kazmierczak-Baranska, Julia; Maszewska, Maria; Janicka, Magdalena; Nowak, Genowefa; Nawrot, Barbara

    2007-01-01

    A series of nucleobase-modified siRNA duplexes containing “rare” nucleosides, 2-thiouridine (s2U), pseudouridine (Ψ), and dihydrouridine (D), were evaluated for their thermodynamic stability and gene silencing activity. The duplexes with modified units at terminal positions exhibited similar stability as the nonmodified reference. Introduction of the s2U or Ψ units into the central part of the antisense strand resulted in duplexes with higher melting temperatures (Tm). In contrary, D unit similarly like wobble base pair led to the less stable duplexes (ΔTm 3.9 and 6.6°C, respectively). Gene-silencing activity of siRNA duplexes directed toward enhanced green fluorescent protein or beta-site APP cleaving enzyme was tested in a dual fluorescence assay. The duplexes with s2U and Ψ units at their 3′-ends and with a D unit at their 5′-ends (with respect to the guide strands) were the most potent gene expression inhibitors. Duplexes with s2U and Ψ units at their 5′-ends were by 50% less active than the nonmodified counterpart. Those containing a D unit or wobble base pair in the central domain had the lowest Tm, disturbed the A-type helical structure, and had more than three times lower activity than their nonmodified congener. Activity of siRNA containing the wobble base pair could be rescued by placing the thio-nucleoside at the position 3′-adjacent to the mutation site. Thermally stable siRNA molecules containing several s2U units in the antisense strand were biologically as potent as their native counterparts. The present results provide a new chemical tool for modulation of siRNA gene-silencing activity. PMID:17585051

  9. A small interfering RNA targeting Lnk accelerates bone fracture healing with early neovascularization.

    PubMed

    Kawakami, Yohei; Ii, Masaaki; Matsumoto, Tomoyuki; Kawamoto, Atsuhiko; Kuroda, Ryosuke; Akimaru, Hiroshi; Mifune, Yutaka; Shoji, Taro; Fukui, Tomoaki; Asahi, Michio; Kurosaka, Masahiro; Asahara, Takayuki

    2013-09-01

    Lnk, an intracellular adapter protein, is expressed in hematopoietic cell lineages, which has recently been proved as an essential inhibitory signaling molecule for stem cell self-renewal in the stem cell factor-c-Kit signaling pathway with enhanced hematopoietic and osteogenic reconstitution in Lnk-deficient mice. Moreover, the therapeutic potential of hematopoietic stem/endothelial progenitor cells (EPCs) for fracture healing has been demonstrated with mechanistic insight into vasculogenesis/angiogenesis and osteogenesis enhancement in the fracture sites. We report here, Lnk siRNA-transfected endothelial commitment of c-kit+/Sca-1+/lineage- subpopulations of bone marrow cells have high EPC colony-forming capacity exhibiting endothelial markers, VE-Cad, VEGF and Ang-1. Lnk siRNA-transfected osteoblasts also show highly osteoblastic capacity. In vivo, locally transfected Lnk siRNA could successfully downregulate the expression of Lnk at the fracture site up to 1 week, and radiological and histological examination showed extremely accelerated fracture healing in Lnk siRNA-transfected mice. Moreover, Lnk siRNA-transfected mice exhibited sufficient therapeutic outcomes with intrinstic enhancement of angiogenesis and osteogenesis, specifically, the mice demonstrated better blood flow recovery in the sites of fracture. In our series of experiments, we clarified that a negatively regulated Lnk system contributed to a favorable circumstance for fracture healing by enhancing vasculogenesis/angiogenesis and osteogenesis. These findings suggest that downregulation of Lnk system may have the clinical potential for faster fracture healing, which contributes to the reduction of delayed unions or non-unions.

  10. Short Interfering RNA Inhibits Rift Valley Fever Virus Replication and Degradation of Protein Kinase R in Human Cells.

    PubMed

    Faburay, Bonto; Richt, Juergen A

    2016-01-01

    Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen causing severe outbreaks in humans and livestock in sub-Saharan Africa and the Arabian Peninsula. Human infections are characterized by fever, sometimes leading to encephalitis, retinitis, hemorrhagic fever, and occasionally death. There are currently no fully licensed vaccines or effective therapies for human use. Gene silencing mediated by double-stranded short interfering RNA (siRNA) is a sequence-specific, highly conserved mechanism in eukaryotes, which serves as an antiviral defense mechanism. Here, we demonstrate that siRNA duplexes directed against the RVFV nucleoprotein can effectively inhibit RVFV replication in human (MRC5 cells) and African green monkey cells (Vero E6 cells). Using these cells, we demonstrate that individual or complex siRNAs, targeting the RVFV nucleoprotein gene completely abrogate viral protein expression and prevent degradation of the host innate antiviral factor, protein kinase R (PKR). Importantly, pre-treatment of cells with the nucleoprotein-specific siRNAs markedly reduces the virus titer. The antiviral effect of the siRNAs was not attributable to interferon or the interferon response effector molecule, PKR. Thus, the antiviral activity of RVFV nucleoprotein-specific siRNAs may provide novel therapeutic strategy against RVFV infections in animals and humans.

  11. A Defective Interfering Influenza RNA Inhibits Infectious Influenza Virus Replication in Human Respiratory Tract Cells: A Potential New Human Antiviral

    PubMed Central

    Smith, Claire M.; Scott, Paul D.; O’Callaghan, Christopher; Easton, Andrew J.; Dimmock, Nigel J.

    2016-01-01

    Defective interfering (DI) viruses arise during the replication of influenza A virus and contain a non-infective version of the genome that is able to interfere with the production of infectious virus. In this study we hypothesise that a cloned DI influenza A virus RNA may prevent infection of human respiratory epithelial cells with infection by influenza A. The DI RNA (244/PR8) was derived by a natural deletion process from segment 1 of influenza A/PR/8/34 (H1N1); it comprises 395 nucleotides and is packaged in the DI virion in place of a full-length genome segment 1. Given intranasally, 244/PR8 DI virus protects mice and ferrets from clinical influenza caused by a number of different influenza A subtypes and interferes with production of infectious influenza A virus in cells in culture. However, evidence that DI influenza viruses are active in cells of the human respiratory tract is lacking. Here we show that 244/PR8 DI RNA is replicated by an influenza A challenge virus in human lung diploid fibroblasts, bronchial epithelial cells, and primary nasal basal cells, and that the yield of challenge virus is significantly reduced in a dose-dependent manner indicating that DI influenza virus has potential as a human antiviral. PMID:27556481

  12. A Defective Interfering Influenza RNA Inhibits Infectious Influenza Virus Replication in Human Respiratory Tract Cells: A Potential New Human Antiviral.

    PubMed

    Smith, Claire M; Scott, Paul D; O'Callaghan, Christopher; Easton, Andrew J; Dimmock, Nigel J

    2016-08-22

    Defective interfering (DI) viruses arise during the replication of influenza A virus and contain a non-infective version of the genome that is able to interfere with the production of infectious virus. In this study we hypothesise that a cloned DI influenza A virus RNA may prevent infection of human respiratory epithelial cells with infection by influenza A. The DI RNA (244/PR8) was derived by a natural deletion process from segment 1 of influenza A/PR/8/34 (H1N1); it comprises 395 nucleotides and is packaged in the DI virion in place of a full-length genome segment 1. Given intranasally, 244/PR8 DI virus protects mice and ferrets from clinical influenza caused by a number of different influenza A subtypes and interferes with production of infectious influenza A virus in cells in culture. However, evidence that DI influenza viruses are active in cells of the human respiratory tract is lacking. Here we show that 244/PR8 DI RNA is replicated by an influenza A challenge virus in human lung diploid fibroblasts, bronchial epithelial cells, and primary nasal basal cells, and that the yield of challenge virus is significantly reduced in a dose-dependent manner indicating that DI influenza virus has potential as a human antiviral.

  13. Short Interfering RNA Inhibits Rift Valley Fever Virus Replication and Degradation of Protein Kinase R in Human Cells

    PubMed Central

    Faburay, Bonto; Richt, Juergen A.

    2016-01-01

    Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen causing severe outbreaks in humans and livestock in sub-Saharan Africa and the Arabian Peninsula. Human infections are characterized by fever, sometimes leading to encephalitis, retinitis, hemorrhagic fever, and occasionally death. There are currently no fully licensed vaccines or effective therapies for human use. Gene silencing mediated by double-stranded short interfering RNA (siRNA) is a sequence-specific, highly conserved mechanism in eukaryotes, which serves as an antiviral defense mechanism. Here, we demonstrate that siRNA duplexes directed against the RVFV nucleoprotein can effectively inhibit RVFV replication in human (MRC5 cells) and African green monkey cells (Vero E6 cells). Using these cells, we demonstrate that individual or complex siRNAs, targeting the RVFV nucleoprotein gene completely abrogate viral protein expression and prevent degradation of the host innate antiviral factor, protein kinase R (PKR). Importantly, pre-treatment of cells with the nucleoprotein-specific siRNAs markedly reduces the virus titer. The antiviral effect of the siRNAs was not attributable to interferon or the interferon response effector molecule, PKR. Thus, the antiviral activity of RVFV nucleoprotein-specific siRNAs may provide novel therapeutic strategy against RVFV infections in animals and humans. PMID:27933051

  14. Efficient delivery of small interfering RNA into injured spinal cords in rats by photomechanical waves

    NASA Astrophysics Data System (ADS)

    Ando, Takahiro; Sato, Shunichi; Toyooka, Terushige; Kobayashi, Hiroaki; Nawashiro, Hiroshi; Ashida, Hiroshi; Obara, Minoru

    2011-03-01

    In the central nervous system, lack of axonal regeneration leads to permanent functional disabilities. In spinal cord injury (SCI), the over-expressions of intermediate filament (IF) proteins, such as glial fibrillary acidic protein (GFAP) and vimentin, are mainly involved in glial scar formation; these proteins work as both physical and biochemical barriers to axonal regeneration. Thus, silencing of these IF proteins would be an attractive strategy to treat SCI. In this study, we first attempted to deliver fluorescent probe-labeled siRNAs into injured spinal cords in rats by applying photomechanical waves (PMWs) to examine the capability of PMWs as a tool for siRNA delivery. Intense fluorescence from siRNAs was observed in much broader regions in the spinal cords with PMW application when compared with those with siRNA injection alone. Based on this result, we delivered siRNAs for GFAP and vimentin into injured spinal tissues in rats by applying PMWs. The treatment resulted in efficient silencing of the proteins at five days after SCI and a decrease of the cavity area in the injured tissue at three weeks after SCI when compared with those with siRNA injection alone. These results demonstrate the capability of PMWs for efficient delivery of siRNAs into injured spinal cords and treatment of SCIs.

  15. Delivery of Small Interfering RNA to Inhibit Vascular Endothelial Growth Factor in Zebrafish Using Natural Brain Endothelia Cell-Secreted Exosome Nanovesicles for the Treatment of Brain Cancer.

    PubMed

    Yang, Tianzhi; Fogarty, Brittany; LaForge, Bret; Aziz, Salma; Pham, Thuy; Lai, Leanne; Bai, Shuhua

    2017-03-01

    Although small interfering RNA (siRNA) holds great therapeutic promise, its delivery to the disease site remains a paramount obstacle. In this study, we tested whether brain endothelial cell-derived exosomes could deliver siRNA across the blood-brain barrier (BBB) in zebrafish. Natural exosomes were isolated from brain endothelial bEND.3 cell culture media and vascular endothelial growth factor (VEGF) siRNA was loaded in exosomes with the assistance of a transfection reagent. While fluorescence-activated cell flow cytometry and immunocytochemistry staining studies indicated that wild-type exosomes significantly increased the uptake of fluorescence-labeled siRNA in the autologous brain endothelial cells, decreased fluorescence intensity was observed in the cells treated with the tetraspanin CD63 antibody-blocked exosome-delivered formulation (p < 0.05). In the transport study, exosomes also enhanced the permeability of rhodamine 123 in a co-cultured monolayer of brain endothelial bEND.3 cell and astrocyte. Inhibition at the expression of VEGF RNA and protein levels was observed in glioblastoma-astrocytoma U-87 MG cells treated with exosome-delivered siRNAs. Imaging results showed that exosome delivered more siRNAs across the BBB in Tg(fli1:GFP) zebrafish. In a xenotransplanted brain tumor model, exosome-delivered VEGF siRNAs decreased the fluorescence intensity of labeled cancer cells in the brain of zebrafish. Brain endothelial cell-derived exosomes could be potentially used as a natural carrier for the brain delivery of exogenous siRNA.

  16. Genome-wide screening for components of small interfering RNA (siRNA) and micro-RNA (miRNA) pathways in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae).

    PubMed

    Xu, H-J; Chen, T; Ma, X-F; Xue, J; Pan, P-L; Zhang, X-C; Cheng, J-A; Zhang, C-X

    2013-12-01

    The brown planthopper (BPH), Nilaparvata lugens, is a major rice pest in Asia, and accumulated evidence indicates that this species is susceptible to RNA interference (RNAi); however, the mechanism underlying RNAi and parental RNAi has not yet been determined. We comprehensively investigated the repertoire of core genes involved in small interfering RNA (siRNA) and micro-RNA (miRNA) pathways in the BPH by comparing its newly assembled transcriptome and genome with those of Drosophila melanogaster, Tribolium castaneum and Caenorhabditis elegans. Our analysis showed that the BPH possesses one drosha and two Dicer (dcr) genes, three dsRNA-binding motif protein genes, two Argonaute (ago) genes, two Eri-1-like genes (eri-1), and a Sid-1-like gene (sid-1). Additionally, we report for first time that parental RNAi might occur in this species, and siRNA pathway and Sid-1 were required for high efficiency of systemic RNAi triggered by exogenous dsRNA. Furthermore, our results also demonstrated that the miRNA pathway was involved in BPH metamorphosis as depletion of the ago1 or dcr1 gene severely impaired ecdysis. The BPH might be a good model system to study the molecular mechanism of systemic RNAi in hemimetabolous insects, and RNAi has potential to be developed to control this pest in agricultural settings.

  17. Ribozyme-enhanced single-stranded Ago2-processed interfering RNA triggers efficient gene silencing with fewer off-target effects

    PubMed Central

    Shang, Renfu; Zhang, Fengjuan; Xu, Beiying; Xi, Hairui; Zhang, Xue; Wang, Weihua; Wu, Ligang

    2015-01-01

    Short-hairpin RNAs (shRNAs) are widely used to produce small-interfering RNAs (siRNAs) for gene silencing. Here we design an alternative siRNA precursor, named single-stranded, Argonaute 2 (Ago2)-processed interfering RNA (saiRNA), containing a 16–18 bp stem and a loop complementary to the target transcript. The introduction of a self-cleaving ribozyme derived from hepatitis delta virus to the 3′ end of the transcribed saiRNA dramatically improves its silencing activity by generating a short 3′ overhang that facilitates the efficient binding of saiRNA to Ago2. The same ribozyme also enhances the activity of Dicer-dependent shRNAs. Unlike a classical shRNA, the strand-specific cleavage of saiRNA by Ago2 during processing eliminates the passenger strand and prevents the association of siRNA with non-nucleolytic Ago proteins. As a result, off-target effects are reduced. In addition, saiRNA exhibits less competition with the biogenesis of endogenous miRNAs. Therefore, ribozyme-enhanced saiRNA provides a reliable tool for RNA interference applications. PMID:26455506

  18. Laser‐Triggered Small Interfering RNA Releasing Gold Nanoshells against Heat Shock Protein for Sensitized Photothermal Therapy

    PubMed Central

    Wang, Zhaohui; Li, Siwen; Zhang, Min; Ma, Yi; Liu, Yuxi; Gao, Weidong; Zhang, Jiaqi

    2016-01-01

    The resistance of cancer cells to photothermal therapy is closely related to the overexpression of heat shock proteins (HSPs), which are abnormally upregulated when cells are under lethal stresses. Common strategies that use small molecule inhibitors against HSPs to enhance hyperthermia effect lack spatial and temporal control of drug release, leading to unavoidable systemic toxicity. Herein, a versatile photothermal platform is developed which is composed of a hollow gold nanoshell core densely packed with small interfering RNAs against heat shock protein 70 (Hsp70). Upon near infrared light irradiation, the small interfering RNAs can detach from gold surface specifically and escape from endosomes for Hsp70 silencing. Meanwhile, the temperature increases for hyperthermia therapy due to the high photothermal efficiency of the nanoshells. Efficient downregulation of Hsp70 after light activation is achieved in vitro and in vivo. Ultimately, the light‐controlled dual functional nanosystem, with the effects of Hsp70 silencing and temperature elevation, results in sensitized photothermal therapy in nude mice model under mild temperature. This strategy smartly combines the localized photothermal therapy with controlled Hsp70 silencing, and has great potential for clinical translation with a simple and easily controlled structure. PMID:28251053

  19. Laser-Triggered Small Interfering RNA Releasing Gold Nanoshells against Heat Shock Protein for Sensitized Photothermal Therapy.

    PubMed

    Wang, Zhaohui; Li, Siwen; Zhang, Min; Ma, Yi; Liu, Yuxi; Gao, Weidong; Zhang, Jiaqi; Gu, Yueqing

    2017-02-01

    The resistance of cancer cells to photothermal therapy is closely related to the overexpression of heat shock proteins (HSPs), which are abnormally upregulated when cells are under lethal stresses. Common strategies that use small molecule inhibitors against HSPs to enhance hyperthermia effect lack spatial and temporal control of drug release, leading to unavoidable systemic toxicity. Herein, a versatile photothermal platform is developed which is composed of a hollow gold nanoshell core densely packed with small interfering RNAs against heat shock protein 70 (Hsp70). Upon near infrared light irradiation, the small interfering RNAs can detach from gold surface specifically and escape from endosomes for Hsp70 silencing. Meanwhile, the temperature increases for hyperthermia therapy due to the high photothermal efficiency of the nanoshells. Efficient downregulation of Hsp70 after light activation is achieved in vitro and in vivo. Ultimately, the light-controlled dual functional nanosystem, with the effects of Hsp70 silencing and temperature elevation, results in sensitized photothermal therapy in nude mice model under mild temperature. This strategy smartly combines the localized photothermal therapy with controlled Hsp70 silencing, and has great potential for clinical translation with a simple and easily controlled structure.

  20. Drosophila oncogene Gas41 is an RNA interference modulator that intersects heterochromatin and the small interfering RNA pathway.

    PubMed

    Gandhi, Sumit G; Bag, Indira; Sengupta, Saswati; Pal-Bhadra, Manika; Bhadra, Utpal

    2015-01-01

    Glioma amplified sequence41 (Gas41) is a highly conserved putative transcription factor that is frequently abundant in human gliomas. Gas41 shows oncogenic activity by promoting cell growth and viability. In the present study, we show that Gas41 is required for proper functioning of RNA interference (RNAi) machinery in the nuclei, although three basic structural domains of RNAi components PAZ, PIWI and dsRNA with respect to binding are absent in the structural sequences. Variations of structural domains are highly conserved among prokaryotes and eukaryotes. Gas41 interacts with cytological RNase III enzyme Dicer1 both biochemically and genetically. However, Drosophila Gas41 functions as chromatin remodeler and interacts with different heterochromatin markers and repeat-induced transgene silencing by modulating position effect variegation. We also show that transcriptional inactive Gas41 mutant interferes with the functional assembly of heterochromatin-associated proteins, dimethylated lysine 9 of histone H3 and heterochromatic protein 1 in developing embryos. A reduction of heterochromatic markers is accompanied by the mini-w promoter sequence in Gas41 mutants. These findings suggest that Drosophila Gas41 guides the repeat associated gene silencing and the Dicer1 interaction, thereby depicting a new role for Gas41. Gas41 is a critical RNAi component. In Drosophila, Gas41 plays a dual role. On the one hand, it appears to participate with Dicer 1 in the RNAi pathway and, alternatively, it also participates in repeat-induced gene silencing by accumulating heterochromatin proteins at the mini-w array promoters. Therefore, it represents an intriguing and apparently paradoxical new finding in RNA technology with respect to the process of heterochromatin gene silencing.

  1. Susceptibility of PTEN-positive metastatic tumors to small interfering RNA targeting the mammalian target of rapamycin.

    PubMed

    Koide, Hiroyuki; Asai, Tomohiro; Kato, Hiroki; Yonenaga, Norihito; Yokota, Masafumi; Ando, Hidenori; Dewa, Takehisa; Nango, Mamoru; Maeda, Noriyuki; Oku, Naoto

    2015-01-01

    PTEN-positive tumors are not susceptible to the treatment with rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR). Here, we determined the susceptibility of PTEN-positive cells to small interfering RNA for mTOR (si-mTOR) by using a novel liposomal delivery system. We prepared dicetyl phosphate-tetraethylenepentamine-based polycation liposomes (TEPA-PCL) decorated with polyethylene glycol (PEG) grafting Ala-Pro-Arg-Pro-Gly (APRPG), a VRGFR-1-targeting peptide. APRPG-PEG-decorated TEPA-PCL carrying si-mTOR (APRPG-TEPA-PCL/si-mTOR) had an antiproliferative effect against B16F10 murine melanoma cells (PTEN-positive) and significantly inhibited both the proliferation and tube formation of mouse 2H-11 endothelial-like cells (PTEN-positive). APRPG-TEPA-PCL/si-mTOR treatment did not induce Akt phosphorylation (Ser473) in either B16F10 or 2H-11 cells although there was strong phosphorylation of Akt in response to rapamycin treatment. Intravenous injection of APRPG-TEPA-PCL/si-mTOR significantly suppressed the tumor growth compared with rapamycin treatment in mice bearing B16F10 melanoma. These findings suggest that APRPG-TEPA-PCL/si-mTOR is useful for the treatment of PTEN-positive tumors.

  2. Suppression of NHE1 by small interfering RNA inhibits HIF-1α-induced angiogenesis in vitro via modulation of calpain activity.

    PubMed

    Mo, Xian-Gang; Chen, Qing-Wei; Li, Xing-Sheng; Zheng, Min-Ming; Ke, Da-Zhi; Deng, Wei; Li, Gui-Qiong; Jiang, Jin; Wu, Zhi-Qin; Wang, Li; Wang, Peng; Yang, Yan; Cao, Guang-Yi

    2011-03-01

    Hypoxia-inducible factor-1 (HIF-1) orchestrates angiogenesis under hypoxic conditions mainly due to increased expression of such target genes as vascular endothelial growth factor (VEGF). Na+/H+exchanger-1 (NHE1), a potential HIF target gene product, plays a pivotal role in proliferation, survival, migration, adhesion and so on. However, it is unknown whether NHE1 is involved in HIF-1α-induced angiogenesis. This present study demonstrated that the expression of NHE1 was much higher in human umbilical vein endothelial cells (HUVECs) infected with adenovirus encoding HIF-1α (rAd-HIF) than with vacuum adenovirus (vAd). HIF-1α also increased the expression of VEGF, the expression and activity of calpains, and the intracellular pH. Moreover, small interfering RNA targeting NHE1 (NHE1 siRNA) dramatically decreased the expression of NHE1 and thus lowered the intracellular pH, and it also attenuated the protein expression of calpain-2 but not calpain-1, resulting in the lower calpain activity. Furthermore, HIF-1α enhanced the proliferation, migration and Matrigel tube formation, which were inhibited by NHE1 siRNA. Finally, the inhibitory effect of NHE1 siRNA was reversed by VEGF and the reversibility of the later was abrogated by the calpain inhibitor ALLM. In conclusion, the findings have revealed that NHE1 might participate in HIF-1-induced angiogenesis due, at least in part, to the alteration of the calpain activity, suggesting that NHE1 as well as calpains might represent a potential target of controlling angiogenesis in response to the hypoxic stress under various pathological conditions.

  3. A genome-wide survey of small interfering RNA and micro RNA pathway genes in a galling insect

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mayetiola destructor (Say), Hessian fly, is a significant pest of wheat in most production regions worldwide. Deployment of resistance (R) genes is the most effective control for this pest; however, deployment of R genes results in an increased frequency of pest genotypes that display virulence to t...

  4. Small interfering RNA library screen of human kinases and phosphatases identifies polo-like kinase 1 as a promising new target for the treatment of pediatric rhabdomyosarcomas.

    PubMed

    Hu, Kaiji; Lee, Cathy; Qiu, Dexin; Fotovati, Abbas; Davies, Alastair; Abu-Ali, Samah; Wai, Daniel; Lawlor, Elizabeth R; Triche, Timothy J; Pallen, Catherine J; Dunn, Sandra E

    2009-11-01

    Rhabdomyosarcoma, consisting of alveolar (aRMS) and embryonal (eRMS) subtypes, is the most common type of sarcoma in children. Currently, there are no targeted drug therapies available for rhabdomyosarcoma. In searching for new molecular therapeutic targets, we carried out genome-wide small interfering RNA (siRNA) library screens targeting human phosphatases (n = 206) and kinases (n = 691) initially against an aRMS cell line, RH30. Sixteen phosphatases and 50 kinases were identified based on growth inhibition after 72 hours. Inhibiting polo-like kinase 1 (PLK1) had the most remarkable impact on growth inhibition (approximately 80%) and apoptosis on all three rhabdomyosarcoma cell lines tested, namely, RH30, CW9019 (aRMS), and RD (eRMS), whereas there was no effect on normal muscle cells. The loss of PLK1 expression and subsequent growth inhibition correlated with decreased p-CDC25C and Cyclin B1. Increased expression of WEE 1 was also noted. The induction of apoptosis after PLK1 silencing was confirmed by increased p-H2AX, propidium iodide uptake, and chromatin condensation, as well as caspase-3 and poly(ADP-ribose) polymerase cleavage. Pediatric Ewing's sarcoma (TC-32), neuroblastoma (IMR32 and KCNR), and glioblastoma (SF188) models were also highly sensitive to PLK1 inhibition. Finally, based on cDNA microarray analyses, PLK1 mRNA was overexpressed (>1.5 fold) in 10 of 10 rhabdomyosarcoma cell lines and in 47% and 51% of primary aRMS (17 of 36 samples) and eRMS (21 of 41 samples) tumors, respectively, compared with normal muscles. Similarly, pediatric Ewing's sarcoma, neuroblastoma, and osteosarcoma tumors expressed high PLK1. We conclude that PLK1 could be a promising therapeutic target for the treatment of a wide range of pediatric solid tumors including rhabdomyosarcoma.

  5. High-Throughput Small Interfering RNA Screening Identifies Phosphatidylinositol 3-Kinase Class II Alpha as Important for Production of Human Cytomegalovirus Virions

    PubMed Central

    Polachek, William S.; Moshrif, Hanan F.; Franti, Michael; Coen, Donald M.; Sreenu, Vattipally B.

    2016-01-01

    ABSTRACT High-throughput small interfering RNA (siRNA) screening is a useful methodology to identify cellular factors required for virus replication. Here we utilized a high-throughput siRNA screen based on detection of a viral antigen by microscopy to interrogate cellular protein kinases and phosphatases for their importance during human cytomegalovirus (HCMV) replication and identified the class II phosphatidylinositol 3-kinase class II alpha (PI3K-C2A) as being involved in HCMV replication. Confirming this observation, infected cells treated with either pooled or individual siRNAs targeting PI3K-C2A mRNA produced approximately 10-fold less infectious virus than the controls. Western blotting and quantitative PCR analysis of infected cells treated with siRNAs indicated that depletion of PI3K-C2A slightly reduced the accumulation of late but not immediate early or early viral antigens and had no appreciable effect on viral DNA synthesis. Analysis of siRNA-treated cells by electron microscopy and Western blotting indicated that PI3K-C2A was not required for the production of viral capsids but did lead to increased numbers of enveloped capsids in the cytoplasm that had undergone secondary envelopment and a reduction in the amount of viral particles exiting the cell. Therefore, PI3K-C2A is a factor important for HCMV replication and has a role in the production of HCMV virions. IMPORTANCE There is limited information about the cellular factors required for human cytomegalovirus (HCMV) replication. Therefore, to identify proteins involved in HCMV replication, we developed a methodology to conduct a high-throughput siRNA screen of HCMV-infected cells. From our screening data, we focused our studies on the top hit from our screen, the lipid kinase phosphatidylinositol 3-kinase class II alpha (PI3K-C2A), as its role in HCMV replication was unknown. Interestingly, we found that PI3K-C2A is important for the production of HCMV virions and is involved in virion production

  6. A small interfering RNA targeting vascular endothelial growth factor efficiently inhibits growth of VX2 cells and VX2 tumor model of hepatocellular carcinoma in rabbit by transarterial embolization-mediated siRNA delivery

    PubMed Central

    Zou, Yu; Guo, Chuan-Gen; Yang, Zheng-Gang; Sun, Jun-Hui; Zhang, Min-Ming; Fu, Cai-Yun

    2016-01-01

    Introduction Hepatocellular carcinoma is currently the second leading cause of cancer-related deaths worldwide with an increasing incidence. Objective The objective of this study is to investigate the effect of vascular endothelial growth factor small interfering RNA (VEGF-siRNA) on rabbit VX2 carcinoma cell viability in vitro and the effect of transarterial embolization (TAE)-mediated VEGF-siRNA delivery on the growth of rabbit VX2 liver-transplanted model in vivo. Methods Quantitative reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blot technologies were used to detect the expression level of VEGF. TAE and computed tomography scan were used to deliver the VEGF-siRNA and detect the tumor volume in vivo, respectively. Microvessel density was detected by immunohistochemistry with CD34 antibody. A biochemical autoanalyzer was used to evaluate the hepatic and renal toxicity. Results The designed VEGF-siRNAs could effectively decrease the expression levels of VEGF mRNA and protein in vitro and in vivo. In vitro, the viability of rabbit VX2 carcinoma cells was reduced by 38.5%±7.3% (VEGF-siRNA no 1) and 30.0%±5.8% (VEGF-siRNA no 3) at 48 hours after transfection. Moreover, in rabbit VX2 liver-transplanted model, the growth ratios of tumors at 28 days after TAE-mediated siRNA delivery were 155.18%±19.42% in the control group, 79.67%±19.63% in the low-dose group, and 36.09%±15.73% in the high-dose group, with significant differences among these three groups. Microvessel density dropped to 34.22±4.01 and 22.63±4.07 in the low-dose group and high-dose group, respectively, compared with the control group (57.88±5.67), with significant differences among these three groups. Furthermore, inoculation of VX2 tumor into the liver itself at later stage induced significant increase in alanine aminotransferase and aspartate aminotransferase, indicating an obvious damage of liver functions, while treatment of VX2 tumor via TAE

  7. Therapeutic targeting of polo-like kinase 1 using RNA-interfering nanoparticles (iNOPs) for the treatment of non-small cell lung cancer.

    PubMed

    McCarroll, Joshua A; Dwarte, Tanya; Baigude, Huricha; Dang, Jason; Yang, Lu; Erlich, Rafael B; Kimpton, Kathleen; Teo, Joann; Sagnella, Sharon M; Akerfeldt, Mia C; Liu, Jie; Phillips, Phoebe A; Rana, Tariq M; Kavallaris, Maria

    2015-05-20

    Non-small cell lung cancer (NSCLC) remains the most common cause of cancer death worldwide due its resistance to chemotherapy and aggressive tumor growth. Polo-like kinase 1 (PLK1) is a serine-threonine protein kinase which is overexpressed in cancer cells, and plays a major role in regulating tumor growth. A number of PLK1 inhibitors are in clinical trial; however, poor tumor bioavailability and off-target effects limit their efficacy. Short-interfering-RNA (siRNA) holds promise as a class of therapeutics, which can selectively silence disease-causing genes. However, siRNA cannot enter cells without a delivery vehicle. Herein, we investigated whether RNAi-interfering nanoparticles could deliver siRNA to NSCLC cells and silence PLK1 expression in vitro and in vivo. iNOP-7 was non-toxic, and delivered siRNA with high efficiency to NSCLC cells. iNOP-7-PLK1 siRNA silenced PLK1 expression and reduced NSCLC growth in vitro. Notably, iNOP-7 delivered siRNA to orthotopic lung tumors in mice, and administration of iNOP-7-PLK1 siRNA reduced lung tumor burden. These novel data show that iNOP-7 can deliver siRNA against PLK1 to NSCLC cells, and decrease cell proliferation both in vitro and in vivo. iNOP-7-PLK1 siRNA may provide a novel therapeutic strategy for the treatment of NSCLC as well as other cancers which aberrantly express this gene.

  8. Caenorhabditis elegans RIG-I Homolog Mediates Antiviral RNA Interference Downstream of Dicer-Dependent Biogenesis of Viral Small Interfering RNAs

    PubMed Central

    Coffman, Stephanie R.; Lu, Jinfeng; Guo, Xunyang; Zhong, Jing; Broitman-Maduro, Gina; Li, Wan-Xiang; Lu, Rui; Maduro, Morris

    2017-01-01

    ABSTRACT Dicer enzymes process virus-specific double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) to initiate specific antiviral defense by related RNA interference (RNAi) pathways in plants, insects, nematodes, and mammals. Antiviral RNAi in Caenorhabditis elegans requires Dicer-related helicase 1 (DRH-1), not found in plants and insects but highly homologous to mammalian retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), intracellular viral RNA sensors that trigger innate immunity against RNA virus infection. However, it remains unclear if DRH-1 acts analogously to initiate antiviral RNAi in C. elegans. Here, we performed a forward genetic screen to characterize antiviral RNAi in C. elegans. Using a mapping-by-sequencing strategy, we uncovered four loss-of-function alleles of drh-1, three of which caused mutations in the helicase and C-terminal domains conserved in RLRs. Deep sequencing of small RNAs revealed an abundant population of Dicer-dependent virus-derived small interfering RNAs (vsiRNAs) in drh-1 single and double mutant animals after infection with Orsay virus, a positive-strand RNA virus. These findings provide further genetic evidence for the antiviral function of DRH-1 and illustrate that DRH-1 is not essential for the sensing and Dicer-mediated processing of the viral dsRNA replicative intermediates. Interestingly, vsiRNAs produced by drh-1 mutants were mapped overwhelmingly to the terminal regions of the viral genomic RNAs, in contrast to random distribution of vsiRNA hot spots when DRH-1 is functional. As RIG-I translocates on long dsRNA and DRH-1 exists in a complex with Dicer, we propose that DRH-1 facilitates the biogenesis of vsiRNAs in nematodes by catalyzing translocation of the Dicer complex on the viral long dsRNA precursors. PMID:28325765

  9. Insights into the therapeutic potential of hypoxia-inducible factor-1α small interfering RNA in malignant melanoma delivered via folate-decorated cationic liposomes

    PubMed Central

    Chen, Zhongjian; Zhang, Tianpeng; Wu, Baojian; Zhang, Xingwang

    2016-01-01

    Malignant melanoma (MM) represents the most dangerous form of skin cancer, and its incidence is expected to rise in the coming time. However, therapy for MM is limited by low topical drug concentration and multidrug resistance. This article aimed to develop folate-decorated cationic liposomes (fc-LPs) for hypoxia-inducible factor-1α (HIF-1α) small interfering (siRNA) delivery, and to evaluate the potential of such siRNA/liposome complexes in MM therapy. HIF-1α siRNA-loaded fc-LPs (siRNA-fc-LPs) were prepared by a film hydration method followed by siRNA incubation. Folate decoration of liposomes was achieved by incorporation of folate/oleic acid-diacylated oligochitosans. The resulting siRNA-fc-LPs were 95.3 nm in size with a ζ potential of 2.41 mV. The liposomal vectors exhibited excellent loading capacity and protective effect toward siRNA. The in vitro cell transfection efficiency was almost parallel to the commercially available Lipofectamine™ 2000. Moreover, the anti-melanoma activity of HIF-1α siRNA was significantly enhanced through fc-LPs. Western blot analysis and apoptosis test demonstrated that siRNA-fc-LPs substantially reduced the production of HIF-1α-associated protein and induced the apoptosis of hypoxia-tolerant melanoma cells. Our designed liposomal vectors might be applicable as siRNA delivery vehicle to systemically or topically treat MM. PMID:27042054

  10. Small interfering RNA knockdown of mini-TyrRS and mini-TrpRS effects angiogenesis in human umbilical vein endothelial cells in hypoxic culture.

    PubMed

    Zeng, Rui; Chen, Yu-Cheng; Zeng, Zhi; Liu, Rui; Qiang, Ou; Jiang, Xiao-Fei; Liu, Xiao-Xia; Li, Xian; Wang, Hao-Yu

    2008-03-01

    Aim We studied the role of mini-TyrRS and mini-TrpRS in angiogenesis by using small interfering RNA-mediated mini-TyrRS/mini-TrpRS knockout in hypoxic culture of human umbilical vein endothelial cells. Methods SiRNA was used as the main method to inhibited the gene function. Silencing efficiency was assayed by real-time reverse transcription-polymerase chain reaction and western blotting. The angiogenic activity in vitro was evaluated by transwell migration assay and Matrigel-induced capillary tube formation in hypoxic culture. Cell proliferation was determined by crystal violet staining. Results The results showed that levels of the mini-TyrRS/mini-TrpRS gene and protein in mock transfection group and negative control group were higher, but noticeably decreased in experimental group. However, no significant difference was detected between mock transfection group and negative control group, but there was a statistically significant difference compared with experimental group. For mini-TyrRS-siRNA group, the cell migration, tube formation and the rate of cell proliferation were respectively inhibited by (47.4, 56.3, 65.4, 73.7%), (60.5, 69.1, 75.9, 83.6%) and (40.4, 56.2, 61.2, 68.0%). For mini-TrpRS-siRNA, were respectively increased by (18.0, 33.8, 45.1, 56.4%), (18.3, 31.2, 40.3, 45.7%) and (8.4, 26.4, 38.2, 46.6%). Conclusion These results indicated that angiogenesis is either stimulated by mini-TyrRS or inhibited by mini-TrpRS in matrigel models in hypoxic culture, raising the possibility that mini-TyrRS stimulates a common downstream signaling event. Thus, naturally occurring fragments of two proteins involved in translation, TyrRS and TrpRS, have opposing activity on endothelial cell angiogenesis in the matrigel assays. The opposing activities of the two tRNA synthetases suggest tight regulation of the balance between pro- and anti-angiogenic stimuli.

  11. Deep Sequencing of Virus-Derived Small Interfering RNAs and RNA from Viral Particles Shows Highly Similar Mutational Landscapes of a Plant Virus Population

    PubMed Central

    Rupar, Matevž; Gutierrez-Aguirre, Ion; Curk, Tomaž; Kreuze, Jan F.

    2015-01-01

    ABSTRACT RNA viruses exist within a host as a population of mutant sequences, often referred to as quasispecies. Within a host, sequences of RNA viruses constitute several distinct but interconnected pools, such as RNA packed in viral particles, double-stranded RNA, and virus-derived small interfering RNAs. We aimed to test if the same representation of within-host viral population structure could be obtained by sequencing different viral sequence pools. Using ultradeep Illumina sequencing, the diversity of two coexisting Potato virus Y sequence pools present within a plant was investigated: RNA isolated from viral particles and virus-derived small interfering RNAs (the derivatives of a plant RNA silencing mechanism). The mutational landscape of the within-host virus population was highly similar between both pools, with no notable hotspots across the viral genome. Notably, all of the single-nucleotide polymorphisms with a frequency of higher than 1.6% were found in both pools. Some unique single-nucleotide polymorphisms (SNPs) with very low frequencies were found in each of the pools, with more of them occurring in the small RNA (sRNA) pool, possibly arising through genetic drift in localized virus populations within a plant and the errors introduced during the amplification of silencing signal. Sequencing of the viral particle pool enhanced the efficiency of consensus viral genome sequence reconstruction. Nonhomologous recombinations were commonly detected in the viral particle pool, with a hot spot in the 3′ untranslated and coat protein regions of the genome. We stress that they present an important but often overlooked aspect of virus population diversity. IMPORTANCE This study is the most comprehensive whole-genome characterization of a within-plant virus population to date and the first study comparing diversity of different pools of viral sequences within a host. We show that both virus-derived small RNAs and RNA from viral particles could be used for

  12. Suppression of wingless-type MMTV integration site family, member 1 expression by small interfering RNA inhibits U251 glioma cell growth in vitro

    PubMed Central

    DONG, LUN; DUAN, XIAO-CHUN; HAN, CHONG-XU; ZHANG, HENGZHU; WU, YONGKANG

    2015-01-01

    A Wingless-type MMTV integration site family, member 1 (Wnt-1) RNA interference expression vector was constructed during the present study, which was used to transfect the glioma U251 cell line and investigate its effect on glioma. Two 21-base oligonucleotides complementary to the coding sequence that was flanking the loop sequence were designed to form a DNA hairpin template for the target small interfering RNA (siRNA). The siRNA templates were cloned into the siRNA expression vector, pGPU6/green fluorescent protein (GFP)/Neo and the sequence was confirmed by DNA sequencing. The pGPU6/GFP/Neo-short hairpin RNA (shRNA)-Wnt-1 vector was subsequently transfected into U251 cells, and reverse transcription polymerase chain reaction and western blot analysis were used to evaluate the Wnt-1 gene silencing effect on U251 cell growth by MTT assay and flow cytometry. The Wnt-1 protein expression was significantly reduced following transfection with the recombinant plasmid, as determined by western blot analysis of the transfected U251 cells. This transfection exhibited a significantly higher death rate, as shown by MTT. Thus, the present study demonstrated that the pGPU6/GFP/Neo-shRNA-Wnt-1 vector inhibited Wnt-1 protein expression. However, further investigations regarding the Wnt signaling pathway in glioma pathogenesis are required. PMID:25435937

  13. Suppression of metastasis of human pancreatic cancer cells to the liver by small interfering RNA-mediated targeting of the midkine gene

    PubMed Central

    YU, LI; FAN, YU; CHEN, BAODING; HU, YUE; GAO, YINA; WEI, DA

    2013-01-01

    The present study aimed to ascertain whether suppression of midkine (MK) expression in pancreatic cancer cells inhibits metastasis to the liver. Human pancreatic cancer AsPC-1 cells were transfected with small interfering RNA (siRNA) targeting MK. siRNA against MK was observed to reduce the expression of MK mRNA and protein in a concentration- and time-dependent manner, and to decrease the number of migrating and tissue-penetrating cells in a concentration-dependent manner (P<0.005). Extracellular vascular endothelial growth factor (VEGF) concentrations were markedly reduced for the siRNA-transfected cells compared with those that were non-siRNA-transfected. The liver transmission rate and tumor nodule number in the animals harboring the siRNA-transfected cells were lower compared with those in the animals harboring the non-siRNA-transfected cells (P<0.005). These data indicate that metastasis of pancreatic cancer cells to the liver requires the expression of MK. The downregulation of VEGF expression is essential to the mechanism whereby suppression of MK expression constrains the metastasis of pancreatic cancer cells to the liver. PMID:24179520

  14. Topical Anti-Nuclear Factor-Kappa B Small Interfering RNA with Functional Peptides Containing Sericin-Based Hydrogel for Atopic Dermatitis

    PubMed Central

    Kanazawa, Takanori; Shizawa, Yuki; Takeuchi, Mayu; Tamano, Kuniko; Ibaraki, Hisako; Seta, Yasuo; Takashima, Yuuki; Okada, Hiroaki

    2015-01-01

    The small interfering RNA (siRNA) is suggested to offer a novel means of treating atopic dermatitis (AD) because it allows the specific silencing of genes related to AD pathogenesis. In our previous study, we found that siRNA targeted against RelA, an important nuclear factor-kappa B (NF-κB) subdomain, with functional peptides, showed therapeutic effects in a mouse model of AD. In the present study, to develop a topical skin application against AD, we prepared a hydrogel containing anti-RelA siRNA and functional peptides and determined the intradermal permeation and the anti-AD effects in an AD mouse model. We selected the silk protein, sericin (SC), which is a versatile biocompatible biomaterial to prepare hydrogel as an aqueous gel base. We found that the siRNA was more widely delivered to the site of application in AD-induced ear skin of mice after topical application via the hydrogel containing functional peptides than via the preparation without functional peptides. In addition, the ear thickness and clinical skin severity of the AD-induced mice treated with hydrogel containing anti-RelA siRNA with functional peptides improved more than that of mice treated with the preparation formulated with negative siRNA. PMID:26371030

  15. PACT- and RIG-I-Dependent Activation of Type I Interferon Production by a Defective Interfering RNA Derived from Measles Virus Vaccine

    PubMed Central

    Ho, Ting-Hin; Kew, Chun; Lui, Pak-Yin; Chan, Chi-Ping; Satoh, Takashi; Akira, Shizuo

    2015-01-01

    ABSTRACT The live attenuated measles virus vaccine is highly immunostimulatory. Identification and characterization of its components that activate the innate immune response might provide new strategies and agents for the rational design and development of chemically defined adjuvants. In this study, we report on the activation of type I interferon (IFN) production by a defective interfering (DI) RNA isolated from the Hu-191 vaccine strain of measles virus. We found that the Hu-191 virus induced IFN-β much more potently than the Edmonston strain. In the search for IFN-inducing species in Hu-191, we identified a DI RNA specifically expressed by this strain. This DI RNA, which was of the copy-back type, was predicted to fold into a hairpin structure with a long double-stranded stem region of 206 bp, and it potently induced the expression of IFN-β. Its IFN-β-inducing activity was further enhanced when both cytoplasmic RNA sensor RIG-I and its partner, PACT, were overexpressed. On the contrary, this activity was abrogated in cells deficient in PACT or RIG-I. The DI RNA was found to be associated with PACT in infected cells. In addition, both the 5′-di/triphosphate end and the double-stranded stem region on the DI RNA were essential for its activation of PACT and RIG-I. Taken together, our findings support a model in which a viral DI RNA is sensed by PACT and RIG-I to initiate an innate antiviral response. Our work might also provide a foundation for identifying physiological PACT ligands and developing novel adjuvants or antivirals. IMPORTANCE The live attenuated measles virus vaccine is one of the most successful human vaccines and has largely contained the devastating impact of a highly contagious virus. Identifying the components in this vaccine that stimulate the host immune response and understanding their mechanism of action might help to design and develop better adjuvants, vaccines, antivirals, and immunotherapeutic agents. We identified and characterized

  16. Cytoskeleton-Dependent Transport as a Potential Target for Interfering with Post-transcriptional HuR mRNA Regulons

    PubMed Central

    Eberhardt, Wolfgang; Badawi, Amel; Biyanee, Abhiruchi; Pfeilschifter, Josef

    2016-01-01

    The ubiquitous mRNA binding protein human antigen R (HuR), a member of the embryonal lethal abnormal vision protein family has a critical impact on the post-transcriptional control of AU-rich element bearing mRNA regulons implied in inflammation, senescence, and carcinogenesis. HuR in addition to mRNA stability can affect many other aspects of mRNA processing including splicing, polyadenylation, translation, modulation of miRNA repression, and intracellular mRNA trafficking. Since many of the identified HuR mRNA targets (“HuR mRNA regulons”) encode tumor-related proteins, HuR is not only discussed as an useful biomarker but also as promising therapeutic target for treatment of various human cancers. HuR which is most abundantly localized in the nucleus is translocated to the cytoplasm which is fundamental for most of the described HuR functions on target mRNAs. Accordingly, an elevation in cytoplasmic HuR was found in many tumors and correlated with a high grade of malignancy and a poor prognosis of patients. Therefore, direct interference with the intracellular trafficking of HuR offers an attractive approach to intervene with pathologically deregulated HuR functions. Data from several laboratories implicate that the integrity of the cytoskeleton is critical for HuR-mediated intracellular mRNA localization and translation. This review will particularly focus on drugs which have proven a direct inhibitory effect on HuR translocation. Based on the results from those studies, we will also discuss on the principle value of targeting cytoskeleton-dependent transport of HuR by natural or synthetic inhibitors as a potential therapeutic avenue for interfering with dysregulated post-transcriptional HuR mRNA regulons and related tumor cell functions. In spite of that, interfering with cytoplasmic HuR transport could highlight a so far underestimated action of microtubule inhibitors clinically used for cancer chemotherapy. PMID:27582706

  17. Silencing tissue inhibitors of metalloproteinases (TIMPs) with short interfering RNA reveals a role for TIMP-1 in hepatic stellate cell proliferation

    SciTech Connect

    Fowell, Andrew J.; Collins, Jane E.; Duncombe, Dale R.; Pickering, Judith A.; Rosenberg, William M.C.; Benyon, R. Christopher

    2011-04-08

    Research highlights: {yields} Myofibroblastic, activated hepatic stellate cells (HSC) play a pivotal role in the development of liver fibrosis. {yields} We used short interfering RNA (siRNA) to investigate the effects of autocrine TIMP-1 and -2 on HSC proliferation. {yields} Specific silencing of TIMP-1, but not TIMP-2, significantly reduces HSC proliferation and is associated with reduced Akt phosphorylation. {yields} TIMP-1 is localised in part to the HSC nucleus. {yields} TIMP-1 might promote liver fibrosis by means other than its previously described anti-apoptotic effect on HSC. -- Abstract: Myofibroblastic, activated hepatic stellate cells (HSC) play a pivotal role in the development of liver fibrosis through the secretion of fibrillar collagens and the tissue inhibitors of metalloproteinase (TIMP)-1 and -2. TIMPs are believed to promote hepatic fibrosis by inhibiting both matrix degradation and apoptosis of HSC. In other cell types, there is evidence that TIMP-1 has effects on proliferation, however the role of TIMPs in the regulation of HSC proliferation remains unexplored. Therefore, we have used short interfering RNA (siRNA) to investigate the effects of autocrine TIMP-1 and -2 on HSC proliferation. TIMP-1 and -2 siRNA were highly effective, producing peak target protein knockdown compared to negative control siRNA of 92% and 63%, respectively. Specific silencing of TIMP-1, using siRNA, significantly reduced HSC proliferation. TIMP-1 was localised in part to the HSC nucleus and TIMP-1 siRNA resulted in loss of both cytoplasmic and nuclear TIMP-1. Attenuated proliferation was associated with reduced Akt phosphorylation and was partially rescued by addition of recombinant TIMP-1. We have revealed a novel autocrine mitogenic effect of TIMP-1 on HSC, which may involve Akt-dependent and specific nuclear mechanisms of action. We suggest that TIMP-1 might promote liver fibrosis by means other than its previously described anti-apoptotic effect on HSC. Moreover

  18. In vivo study of Dicer-2-mediated immune response of the small interfering RNA pathway upon systemic infections of virulent and avirulent viruses in Bombus terrestris.

    PubMed

    Niu, Jinzhi; Smagghe, Guy; De Coninck, Dieter I M; Van Nieuwerburgh, Filip; Deforce, Dieter; Meeus, Ivan

    2016-03-01

    Recent studies suggest a potent role of the small interfering RNA (siRNA) pathway in the control of bee viruses and its usefulness to tackle these viral diseases. However, the involvement of the siRNA pathway in the defense against different bee viruses is still poorly understood. Therefore, in this report, we comprehensively analyzed the response of the siRNA pathway in bumblebees of Bombus terrestris to systemic infections of the virulent Israeli acute paralysis virus (IAPV) and the avirulent slow bee paralysis virus (SBPV). Our results showed that IAPV and SBPV infections induced the expression of Dicer-2. IAPV infections also triggered the production of predominantly 22 nt-long virus-derived siRNAs (vsiRNAs). Intriguingly, these 22 nt-long vsiRNAs showed a high proportion of antigenomic IAPV sequences. Conversely, these predominantly 22 nt-long vsiRNAs of SBPV were not detected in SBPV infected bees. Furthermore, an "RNAi-of-RNAi" experiment on Dicer-2 did not result in altered genome copy numbers of IAPV (n = 17-18) and also not of SBPV (n = 11-12). Based on these results, we can speculate about the importance of the siRNA pathway in bumblebees for the antiviral response. During infection of IAPV, this pathway is probably recruited but it might be insufficient to control viral infection in our experimental setup. The host can control SBPV infection, but aside from the induction of Dicer-2 expression, no further evidence of the antiviral activity of the siRNA pathway was observed. This report may also enhance the current understanding of the siRNA pathway in the innate immunity of non-model insects upon different viral infections.

  19. Caenorhabditis elegans RIG-I Homolog Mediates Antiviral RNA Interference Downstream of Dicer-Dependent Biogenesis of Viral Small Interfering RNAs.

    PubMed

    Coffman, Stephanie R; Lu, Jinfeng; Guo, Xunyang; Zhong, Jing; Jiang, Hongshan; Broitman-Maduro, Gina; Li, Wan-Xiang; Lu, Rui; Maduro, Morris; Ding, Shou-Wei

    2017-03-21

    Dicer enzymes process virus-specific double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) to initiate specific antiviral defense by related RNA interference (RNAi) pathways in plants, insects, nematodes, and mammals. Antiviral RNAi in Caenorhabditis elegans requires Dicer-related helicase 1 (DRH-1), not found in plants and insects but highly homologous to mammalian retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), intracellular viral RNA sensors that trigger innate immunity against RNA virus infection. However, it remains unclear if DRH-1 acts analogously to initiate antiviral RNAi in C. elegans Here, we performed a forward genetic screen to characterize antiviral RNAi in C. elegans Using a mapping-by-sequencing strategy, we uncovered four loss-of-function alleles of drh-1, three of which caused mutations in the helicase and C-terminal domains conserved in RLRs. Deep sequencing of small RNAs revealed an abundant population of Dicer-dependent virus-derived small interfering RNAs (vsiRNAs) in drh-1 single and double mutant animals after infection with Orsay virus, a positive-strand RNA virus. These findings provide further genetic evidence for the antiviral function of DRH-1 and illustrate that DRH-1 is not essential for the sensing and Dicer-mediated processing of the viral dsRNA replicative intermediates. Interestingly, vsiRNAs produced by drh-1 mutants were mapped overwhelmingly to the terminal regions of the viral genomic RNAs, in contrast to random distribution of vsiRNA hot spots when DRH-1 is functional. As RIG-I translocates on long dsRNA and DRH-1 exists in a complex with Dicer, we propose that DRH-1 facilitates the biogenesis of vsiRNAs in nematodes by catalyzing translocation of the Dicer complex on the viral long dsRNA precursors.IMPORTANCE The helicase and C-terminal domains of mammalian RLRs sense intracellular viral RNAs to initiate the interferon-regulated innate immunity against RNA virus infection. Both of the domains from

  20. A 28-day oral toxicity evaluation of small interfering RNAs and a long double-stranded RNA targeting vacuolar ATPase in mice.

    PubMed

    Petrick, Jay S; Moore, William M; Heydens, William F; Koch, Michael S; Sherman, James H; Lemke, Shawna L

    2015-02-01

    New biotechnology-derived crop traits have been developed utilizing the natural process of RNA interference (RNAi). However, plant-produced double stranded RNAs (dsRNAs) are not known to present a hazard to mammals because numerous biological barriers limit uptake and potential for activity. To evaluate this experimentally, dsRNA sequences matching the mouse vATPase gene (an established target for control of corn rootworms) were evaluated in a 28-day toxicity study with mice. Test groups were orally gavaged with escalating doses of either a pool of four 21-mer vATPase small interfering RNAs (siRNAs) or a 218-base pair vATPase dsRNA. There were no treatment-related effects on body weight, food consumption, clinical observations, clinical chemistry, hematology, gross pathology, or histopathology endpoints. The highest dose levels tested were considered to be the no observed adverse effect levels (NOAELs) for the 21-mer siRNAs (48 mg/kg/day) and the 218 bp dsRNA (64 mg/kg/day). As an additional exploratory endpoint, vATPase gene expression, was evaluated in selected gastrointestinal tract and systemic tissues. The results of this assay did not indicate treatment-related suppression of vATPase. The results of this study indicate that orally ingested dsRNAs, even those targeting a gene in the test species, do not produce adverse health effects in mammals.

  1. A Novel Repeat-Associated Small Interfering RNA-Mediated Silencing Pathway Downregulates Complementary Sense gypsy Transcripts in Somatic Cells of the Drosophila Ovary▿

    PubMed Central

    Pélisson, Alain; Sarot, Emeline; Payen-Groschêne, Geneviève; Bucheton, Alain

    2007-01-01

    Replication of the gypsy endogenous retrovirus involves contamination of the female germ line by adjacent somatic tissues. This is prevented by flam, an as-yet-uncloned heterochromatic pericentromeric locus, at the level of transcript accumulation in these somatic ovarian tissues. We tested the effect of a presumptive RNA silencing mechanism on the accumulation of RNAs produced by constructs containing various gypsy sequences and report that the efficiency of silencing is indeed correlated with the amount of complementary RNAs, 25 to 30 nucleotides in length, in the ovary. For instance, while these RNAs were found to display a three- to fivefold excess of the antisense strands, only the transcripts that contain the complementary sense gypsy sequences could be repressed, indicating that they are targeted at the RNA, not DNA, level. Their size and asymmetry in strand polarity are typical of the novel repeat-associated small interfering RNA (rasiRNA)-mediated pathway, recently suspected to prevent the deleterious expression of selfish DNA specifically in the germ line. Unlike microRNAs (but like rasiRNAs and, surprisingly, siRNAs as well), gypsy rasiRNAs are modified at the 3′ end. The rasiRNA-associated protein Piwi (but not Aub) is required for gypsy silencing, whereas Dicer-2 (which makes siRNAs) is not. In contrast, piwi, aub, and flam do not appear to affect somatic siRNA-mediated silencing. The amount of gypsy rasiRNAs is genetically determined by the flam locus in a provirus copy number-independent manner and is triggered in the somatic tissues by some pericentromeric provirus(es), which are thereby able to protect the germ line from retroviral invasion. PMID:17135323

  2. Small interfering RNA targeting ILK inhibits metastasis in human tongue cancer cells through repression of epithelial-to-mesenchymal transition

    SciTech Connect

    Xing, Yu; Qi, Jin; Deng, Shixiong; Wang, Cheng; Zhang, Luyu; Chen, Junxia

    2013-08-01

    Integrin-linked kinase (ILK) is a multifunctional serine/threonine kinase. Accumulating evidences suggest that ILK are involved in cell–matrix interactions, cell proliferation, invasion, migration, angiogenesis and Epithelial–mesenchymal transition (EMT). However, the underlying mechanisms remain largely unknown. EMT has been postulated as a prerequisite for metastasis. The reports have demonstrated that EMT was implicated in metastasis of oral squamous cell carcinomas. Therefore, here we further postulate that ILK might participate in EMT of tongue cancer. We showed that ILK siRNA inhibited EMT with low N-cadherin, Vimentin, Snail, Slug and Twist as well as high E-cadherin expression in vivo and in vitro. We found that knockdown of ILK inhibited cell proliferation, migration and invasion as well as changed cell morphology. We also demonstrated that ILK siRNA inhibited phosphorylation of downstream signaling targets Akt and GSK3β as well as reduced expression of MMP2 and MMP9. Furthermore, we found that the tongue tumor with high metastasis capability showed higher ILK, Vimentin, Snail, Slug and Twist as well as lower E-cadherin expression in clinical specimens. Finally, ILK siRNA led to the suppression for tumorigenesis and metastasis in vivo. Our findings suggest that ILK could be a novel diagnostic and therapeutic target for tongue cancer. Highlights: • ILK siRNA influences cell morphology, cell cycle, migration and invasion. • ILK siRNA affects the expression of proteins associated with EMT. • ILK expression is related to EMT in clinical human tongue tumors. • ILK siRNA inhibits metastasis of the tongue cancer cells through suppressing EMT.

  3. Small Interfering RNA Targeting Mitochondrial Calcium Uniporter Improves Cardiomyocyte Cell Viability in Hypoxia/Reoxygenation Injury by Reducing Calcium Overload

    PubMed Central

    Oropeza-Almazán, Yuriana; Vázquez-Garza, Eduardo; Chapoy-Villanueva, Héctor; Torre-Amione, Guillermo

    2017-01-01

    Intracellular Ca2+ mishandling is an underlying mechanism in hypoxia/reoxygenation (H/R) injury that results in mitochondrial dysfunction and cardiomyocytes death. These events are mediated by mitochondrial Ca2+ (mCa2+) overload that is facilitated by the mitochondrial calcium uniporter (MCU) channel. Along this line, we evaluated the effect of siRNA-targeting MCU in cardiomyocytes subjected to H/R injury. First, cardiomyocytes treated with siRNA demonstrated a reduction of MCU expression by 67%, which resulted in significant decrease in mitochondrial Ca2+ transport. siRNA treated cardiomyocytes showed decreased mitochondrial permeability pore opening and oxidative stress trigger by Ca2+ overload. Furthermore, after H/R injury MCU silencing decreased necrosis and apoptosis levels by 30% and 50%, respectively, and resulted in reduction in caspases 3/7, 9, and 8 activity. Our findings are consistent with previous conclusions that demonstrate that MCU activity is partly responsible for cellular injury induced by H/R and support the concept of utilizing siRNA-targeting MCU as a potential therapeutic strategy. PMID:28337252

  4. Respiratory syncytial virus infection in Fischer 344 rats is attenuated by short interfering RNA against the RSV-NS1 gene

    PubMed Central

    Kong, Xiaoyuan; Zhang, Weidong; Lockey, Richard F; Auais, Alexander; Piedimonte, Giovanni; Mohapatra, Shyam S

    2007-01-01

    Background Respiratory syncytial virus (RSV) causes severe bronchiolitis and is a risk factor for asthma. Since there is no commercially available vaccine against RSV, a short interfering RNA against the RSV-NS1gene (siNS1) was developed and its potential for decreasing RSV infection and infection-associated inflammation in rats was tested. Methods Plasmids encoding siNS1 or an unrelated siRNA were complexed with a chitosan nanoparticle delivery agent and administered intranasally. Control animals received a plasmid for a non-specific siRNA. After expression of the plasmid in lung cells for 24 hours, the rats were intranasally infected with RSV. Results Prophylaxis with siNS1 significantly reduced lung RSV titers and airway hyperreactivity to methacholine challenge compared to the control group. Lung sections from siNS1-treated rats showed a sizable reduction in goblet cell hyperplasia and in lung infiltration by inflammatory cells, both characteristics of asthma. Also, bronchoalveolar lavage samples from siNS1-treated animals had fewer eosinophils. Treatment of rats with siNS1 prior to RSV exposure was effective in reducing virus titers in the lung and in preventing the inflammation and airway hyperresponsiveness associated with the infection that has been linked to development of asthma. Conclusion The use of siNS1 prophylaxis may be an effective method for preventing RSV bronchiolitis and potentially reducing the later development of asthma associated with severe respiratory infections. PMID:17270047

  5. Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap

    PubMed Central

    Ayalew, Lisanework E.; Patel, Amrutlal K.; Gaba, Amit; Islam, Azharul; Tikoo, Suresh K.

    2016-01-01

    Earlier, targeting of DDX3 by few viral proteins has defined its role in mRNA transport and induction of interferon production. This study was conducted to investigate the function of bovine adenovirus (BAdV)-3 pVIII during virus infection. Here, we provided evidence regarding involvement of DDX3 in cap dependent cellular mRNA translation and demonstrated that targeting of DDX3 by adenovirus protein VIII interfered with cap-dependent mRNA translation function of DDX3 in virus infected cells. Adenovirus late protein pVIII interacted with DDX3 in transfected and BAdV-3 infected cells. pVIII inhibited capped mRNA translation in vitro and in vivo by limiting the amount of DDX3 and eIF3. Diminished amount of DDX3 and eIFs including eIF3, eIF4E, eIF4G, and PABP were present in cap binding complex in BAdV-3 infected or pVIII transfected cells with no trace of pVIII in cap binding complex. The total amount of eIFs appeared similar in uninfected or infected cells as BAdV-3 did not appear to degrade eIFs. The co-immunoprecipitation experiments indicated the absence of direct interaction between pVIII and eIF3, eIF4E, or PABP. These data indicate that interaction of pVIII with DDX3 interferes with the binding of eIF3, eIF4E and PABP to the 5′ Cap. We conclude that DDX3 promotes cap-dependent cellular mRNA translation and BAdV-3 pVIII inhibits translation of capped cellular mRNA possibly by interfering with the recruitment of eIFs to the capped cellular mRNA. PMID:28082972

  6. Silencing expression of the catalytic subunit of DNA-dependent protein kinase by small interfering RNA sensitizes human cells for radiation-induced chromosome damage, cell killing, and mutation

    NASA Technical Reports Server (NTRS)

    Peng, Yuanlin; Zhang, Qinming; Nagasawa, Hatsumi; Okayasu, Ryuichi; Liber, Howard L.; Bedford, Joel S.

    2002-01-01

    Targeted gene silencing in mammalian cells by RNA interference (RNAi) using small interfering RNAs (siRNAs) was recently described by Elbashir et al. (S. M. Elbashir et al., Nature (Lond.), 411: 494-498, 2001). We have used this methodology in several human cell strains to reduce expression of the Prkdc (DNA-PKcs) gene coding for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) that is involved in the nonhomologous end joining of DNA double-strand breaks. We have also demonstrated a radiosensitization for several phenotypic endpoints of radiation damage. In low-passage normal human fibroblasts, siRNA knock-down of DNA-PKcs resulted in a reduced capacity for restitution of radiation-induced interphase chromosome breaks as measured by premature chromosome condensation, an increased yield of acentric chromosome fragments at the first postirradiation mitosis, and an increased radiosensitivity for cell killing. For three strains of related human lymphoblasts, DNA-PKcs-targeted siRNA transfection resulted in little or no increase in radiosensitivity with respect to cell killing, a 1.5-fold decrease in induced mutant yield in TK6- and p53-null NH32 cells, but about a 2-fold increase in induced mutant yield in p53-mutant WTK1 cells at both the hypoxanthine quanine phosphoribosyl transferase (hprt) and the thymidine kinase loci.

  7. Regulation of protein degradation by insulin-degrading enzyme: analysis by small interfering RNA-mediated gene silencing.

    PubMed

    Fawcett, Janet; Permana, Paska A; Levy, Jennifer L; Duckworth, William C

    2007-12-01

    Proteins are vital to the overall structure of cells and to the function of cells in the form of enzymes. Thus the control of protein metabolism is among the most important aspects of cellular metabolism. Insulin's major effect on protein metabolism in the adult animal is inhibition of protein degradation. This is via inhibition of proteasome activity via an interaction with insulin-degrading enzyme (IDE). IDE is responsible for the majority of cellular insulin degradation. We hypothesized that a reduction in IDE would reduce insulin degradation and insulin's ability to inhibit protein degradation. HepG2 cells were transfected with siRNA against human IDE and insulin degradation and protein degradation measured. Both IDE mRNA and protein were reduced by >50% in the IDE siRNA transfected cells. Insulin degradation was reduced by approximately 50%. Cells were labeled with [3H]-leucine to investigate protein degradation. Short-lived protein degradation was unchanged in the cells with reduced IDE expression. Long-lived and very-long-lived protein degradation was reduced in the cells with reduced IDE expression (14.0+/-0.16 vs. 12.5+/-0.07%/4h (long-lived), 9.6+/-2.2% vs. 7.3+/-0.2%/3h (very-long-lived), control vs. IDE transfected, respectively, P<0.005). The inhibition of protein degradation by insulin was reduced 37-76% by a decreased expression of IDE in HepG2 cells. This shows that IDE is involved in cellular insulin metabolism and provides further evidence that insulin inhibits protein degradation via an interaction with IDE.

  8. Prevention of airway inflammation with topical cream containing imiquimod and small interfering RNA for natriuretic peptide receptor

    PubMed Central

    Wang, Xiaoqin; Xu, Weidong; Mohapatra, Subhra; Kong, Xiaoyuan; Li, Xu; Lockey, Richard F; Mohapatra, Shyam S

    2008-01-01

    Background Asthma is a complex disease, characterized by reversible airway obstruction, hyperresponsiveness and chronic inflammation. Principle pharmacologic treatments for asthma include bronchodilating beta2-agonists and anti-inflammatory glucocorticosteroids; but these agents do not target the main cause of the disease, the generation of pathogenic Th2 cells. We previously reported reduction in allergic inflammation in mice deficient in the ANP receptor NPRA. Here we determined whether siRNA for natriuretic peptide receptor A (siNPRA) protected against asthma when administered transdermally. Methods Imiquimod cream mixed with chitosan nanoparticles containing either siRNA green indicator (siGLO) or siNPRA was applied to the skin of mice. Delivery of siGLO was confirmed by fluorescence microscopy. The anti-inflammatory activity of transdermal siNPRA was tested in OVA-sensitized mice by measuring airway hyperresponsiveness, eosinophilia, lung histopathology and pro-inflammatory cytokines. Results SiGLO appearing in the lung proved the feasibility of transdermal delivery. In a mouse asthma model, BALB/c mice treated with imiquimod cream containing siNPRA chitosan nanoparticles showed significantly reduced airway hyperresponsiveness, eosinophilia, lung histopathology and pro-inflammatory cytokines IL-4 and IL-5 in lung homogenates compared to controls. Conclusion These results demonstrate that topical cream containing imiquimod and siNPRA nanoparticles exerts an anti-inflammatory effect and may provide a new and simple therapy for asthma. PMID:18279512

  9. Silencing tumor necrosis factor-alpha in vitro from small interfering RNA-decorated titanium nanotube array can facilitate osteogenic differentiation of mesenchymal stem cells

    PubMed Central

    Wang, Zhenlin; Hu, Zhiqiang; Zhang, Dawei; Zhuo, Mengchuan; Cheng, Jiwei; Xu, Xingping; Xing, Yongming; Fan, Jie

    2016-01-01

    Titanium implants are known for their bone bonding ability. However, the osseointegration may be severely disturbed in the inflammation environment. In order to enhance osseointegration of the implant in an inflamed environment, the small interfering RNA (siRNA) targeting tumor necrosis factor alpha (TNF-α) was used to functionalize titanium surface for gene silencing. The chitosan–tripolyphosphate–hyaluronate complexes were used to formulate nanoparticles (NPs) with siRNA, which were adsorbed directly by the anodized titanium surface. The surface characterization was analyzed by scanning electron microscope, atomic force microscopy, as well as contact angle measurement. The fluorescence microscope was used to monitor the degradation of the layer. The coculture system was established with mesenchymal stem cells (MSCs) grown directly on functionalized titanium surface and RAW264.7 cells (preactivated by lipopolysaccharide) grown upside in a transwell chamber. The transfection and knockdown efficiency of TNF-α in RAW264.7 cells were determined by fluorescence microscope, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay. The cytoskeleton and osteogenic differentiation of MSCs were also analyzed. Regular vertical aligned nanotubes (~100 nm diameter and ~300 nm length) were generated after anodization of polished titanium. After loading with NPs, the nanotubes were filled and covered by a layer of amorphous particles. The surface topography changed and wettability decreased after covering with NPs. As expected, a burst degradation of the film was observed, which could provide sufficient NPs in the released supernatant and result in transfection and knockdown effects in RAW264.7 cells. The cytoskeleton arrangement of MSCs was elongated and the osteogenic differentiation was also significantly improved on NPs loading surface. In conclusion, the siRNA decorated titanium implant could simultaneously suppress inflammation and improve

  10. Small interfering RNA-mediated silencing of nicotinamide phosphoribosyltransferase (NAMPT) and lysosomal trafficking regulator (LYST) induce growth inhibition and apoptosis in human multiple myeloma cells: A preliminary study.

    PubMed

    Bong, Ivyna Pau Ni; Ng, Ching Ching; Fakiruddin, Shaik Kamal; Lim, Moon Nian; Zakaria, Zubaidah

    2016-11-10

    Multiple myeloma (MM) is a malignancy of B lymphocytes or plasma cells. Our array-based comparative genomic hybridization findings revealed chromosomal gains at 7q22.3 and 1q42.3, where nicotinamide (NAM) phosphoribosyltransferase (NAMPT) and lysosomal trafficking regulator (LYST) genes are localized, respectively. This led us to further study the functions of these genes in myeloma cells. NAMPT is a key enzyme involved in nicotinamide adenine dinucleotide salvage pathway, and it is frequently overexpressed in human cancers. In contrast, little is known about the function of LYST in cancer. The expression of LYST is shown to affect lysosomal size, granule size, and autophagy in human cells. In this study, the effects of small interfering RNA (siRNA)-mediated silencing of NAMPT and LYST on cell proliferation and apoptosis were evaluated in RPMI 8226 myeloma cells. Transfection efficiencies were determined by quantitative real time reverse transcriptase PCR. Cell proliferation was determined using MTT assay, while apoptosis was analyzed with flow cytometry using Annexin V-fluorescein isothiocyanate/propidium iodide assay. The NAMPT protein expression in siRNA-treated cells was estimated by enzyme-linked immunosorbent assay. Our results showed that NAMPT and LYST were successfully knockdown by siRNA transfection (p < 0.05). NAMPT or LYST gene silencing significantly inhibited cell proliferation and induced apoptosis in RPMI 8226 cells (p < 0.05). Silencing of NAMPT gene also decreased NAMPT protein levels (p < 0.01). Our study demonstrated that NAMPT and LYST play pivotal roles in the molecular pathogenesis of MM. This is the first report describing the possible functions of LYST in myelomagenesis and its potential role as a therapeutic target in MM.

  11. Small interfering RNA-mediated silencing of nicotinamide phosphoribosyltransferase (NAMPT) and lysosomal trafficking regulator (LYST) induce growth inhibition and apoptosis in human multiple myeloma cells: A preliminary study

    PubMed Central

    Bong, Ivyna Pau Ni; Ng, Ching Ching; Fakiruddin, Shaik Kamal; Lim, Moon Nian; Zakaria, Zubaidah

    2016-01-01

    Multiple myeloma (MM) is a malignancy of B lymphocytes or plasma cells. Our array-based comparative genomic hybridization findings revealed chromosomal gains at 7q22.3 and 1q42.3, where nicotinamide (NAM) phosphoribosyltransferase (NAMPT) and lysosomal trafficking regulator (LYST) genes are localized, respectively. This led us to further study the fprotein expression in unctions of these genes in myeloma cells. NAMPT is a key enzyme involved in nicotinamide adenine dinucleotide salvage pathway, and it is frequently overexpressed in human cancers. In contrast, little is known about the function of LYST in cancer. The expression of LYST is shown to affect lysosomal size, granule size, and autophagy in human cells. In this study, the effects of small interfering RNA (siRNA)-mediated silencing of NAMPT and LYST on cell proliferation and apoptosis were evaluated in RPMI 8226 myeloma cells. Transfection efficiencies were determined by quantitative real time reverse transcriptase PCR. Cell proliferation was determined using MTT assay, while apoptosis was analyzed with flow cytometry using Annexin V-fluorescein isothiocyanate/propidium iodide assay. The NAMPT protein expression in siRNA-treated cells was estimated by enzyme-linked immunosorbent assay. Our results showed that NAMPT and LYST were successfully knockdown by siRNA transfection (p < 0.05). NAMPT or LYST gene silencing significantly inhibited cell proliferation and induced apoptosis in RPMI 8226 cells (p < 0.05). Silencing of NAMPT gene also decreased NAMPT protein levels (p < 0.01). Our study demonstrated that NAMPT and LYST play pivotal roles in the molecular pathogenesis of MM. This is the first report describing the possible functions of LYST in myelomagenesis and its potential role as a therapeutic target in MM. PMID:27754828

  12. Silencing tumor necrosis factor-alpha in vitro from small interfering RNA-decorated titanium nanotube array can facilitate osteogenic differentiation of mesenchymal stem cells.

    PubMed

    Wang, Zhenlin; Hu, Zhiqiang; Zhang, Dawei; Zhuo, Mengchuan; Cheng, Jiwei; Xu, Xingping; Xing, Yongming; Fan, Jie

    2016-01-01

    Titanium implants are known for their bone bonding ability. However, the osseointegration may be severely disturbed in the inflammation environment. In order to enhance osseointegration of the implant in an inflamed environment, the small interfering RNA (siRNA) targeting tumor necrosis factor alpha (TNF-α) was used to functionalize titanium surface for gene silencing. The chitosan-tripolyphosphate-hyaluronate complexes were used to formulate nanoparticles (NPs) with siRNA, which were adsorbed directly by the anodized titanium surface. The surface characterization was analyzed by scanning electron microscope, atomic force microscopy, as well as contact angle measurement. The fluorescence microscope was used to monitor the degradation of the layer. The coculture system was established with mesenchymal stem cells (MSCs) grown directly on functionalized titanium surface and RAW264.7 cells (preactivated by lipopolysaccharide) grown upside in a transwell chamber. The transfection and knockdown efficiency of TNF-α in RAW264.7 cells were determined by fluorescence microscope, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay. The cytoskeleton and osteogenic differentiation of MSCs were also analyzed. Regular vertical aligned nanotubes (~100 nm diameter and ~300 nm length) were generated after anodization of polished titanium. After loading with NPs, the nanotubes were filled and covered by a layer of amorphous particles. The surface topography changed and wettability decreased after covering with NPs. As expected, a burst degradation of the film was observed, which could provide sufficient NPs in the released supernatant and result in transfection and knockdown effects in RAW264.7 cells. The cytoskeleton arrangement of MSCs was elongated and the osteogenic differentiation was also significantly improved on NPs loading surface. In conclusion, the siRNA decorated titanium implant could simultaneously suppress inflammation and improve

  13. Cytoplasmic viral RNA-dependent RNA polymerase disrupts the intracellular splicing machinery by entering the nucleus and interfering with Prp8.

    PubMed

    Liu, Yen-Chin; Kuo, Rei-Lin; Lin, Jing-Yi; Huang, Peng-Nien; Huang, Yi; Liu, Hsuan; Arnold, Jamine J; Chen, Shu-Jen; Wang, Robert Yung-Liang; Cameron, Craig E; Shih, Shin-Ru

    2014-06-01

    The primary role of cytoplasmic viral RNA-dependent RNA polymerase (RdRp) is viral genome replication in the cellular cytoplasm. However, picornaviral RdRp denoted 3D polymerase (3D(pol)) also enters the host nucleus, where its function remains unclear. In this study, we describe a novel mechanism of viral attack in which 3D(pol) enters the nucleus through the nuclear localization signal (NLS) and targets the pre-mRNA processing factor 8 (Prp8) to block pre-mRNA splicing and mRNA synthesis. The fingers domain of 3D(pol) associates with the C-terminal region of Prp8, which contains the Jab1/MPN domain, and interferes in the second catalytic step, resulting in the accumulation of the lariat form of the splicing intermediate. Endogenous pre-mRNAs trapped by the Prp8-3D(pol) complex in enterovirus-infected cells were identified and classed into groups associated with cell growth, proliferation, and differentiation. Our results suggest that picornaviral RdRp disrupts pre-mRNA splicing processes, that differs from viral protease shutting off cellular transcription and translation which contributes to the pathogenesis of viral infection.

  14. Cytoplasmic Viral RNA-Dependent RNA Polymerase Disrupts the Intracellular Splicing Machinery by Entering the Nucleus and Interfering with Prp8

    PubMed Central

    Liu, Yen-Chin; Kuo, Rei-Lin; Lin, Jing-Yi; Huang, Peng-Nien; Huang, Yi; Liu, Hsuan; Arnold, Jamine J.; Chen, Shu-Jen; Wang, Robert Yung-Liang; Cameron, Craig E.; Shih, Shin-Ru

    2014-01-01

    The primary role of cytoplasmic viral RNA-dependent RNA polymerase (RdRp) is viral genome replication in the cellular cytoplasm. However, picornaviral RdRp denoted 3D polymerase (3Dpol) also enters the host nucleus, where its function remains unclear. In this study, we describe a novel mechanism of viral attack in which 3Dpol enters the nucleus through the nuclear localization signal (NLS) and targets the pre-mRNA processing factor 8 (Prp8) to block pre-mRNA splicing and mRNA synthesis. The fingers domain of 3Dpol associates with the C-terminal region of Prp8, which contains the Jab1/MPN domain, and interferes in the second catalytic step, resulting in the accumulation of the lariat form of the splicing intermediate. Endogenous pre-mRNAs trapped by the Prp8-3Dpol complex in enterovirus-infected cells were identified and classed into groups associated with cell growth, proliferation, and differentiation. Our results suggest that picornaviral RdRp disrupts pre-mRNA splicing processes, that differs from viral protease shutting off cellular transcription and translation which contributes to the pathogenesis of viral infection. PMID:24968230

  15. Effects of silencing S100A8 and S100A9 with small interfering RNA on the migration of CNE1 nasopharyngeal carcinoma cells.

    PubMed

    Yan, Lin-Lin; Huang, Yuan-Jiao; Yi, Xiang; Yan, Xue-Min; Cai, Yan; He, Qin; Han, Zi-Jian

    2015-06-01

    The calcium-binding S100 proteins are involved in functions such as cell growth, differentiation, migration, adhesion and signal transduction. S100A8 and S100A9 are highly expressed in a variety of tumor cells, and are implicated in tumor development and progression. However, the role of S100A8 and S100A9 in nasopharyngeal carcinoma (NPC) cell migration is unclear. The present study investigated the effect of S100A8 and S100A9 on migration using a NPC cell line, CNE1. The CNE1 cells were transfected with S100A8 or S100A9 small interfering RNA (siRNA). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect S100A8 and S100A9 gene expression. Following the downregulation of S100A8 or S100A9, the effects on cell migration were determined using wound-healing assays. The expression of matrix metalloproteinase-7 (MMP7), a member of the MMP family that is associated with tumor cell invasion and migration, was also detected by RT-qPCR. S100A8 and S100A9 siRNAs effectively suppressed S100A8 and S100A9 gene expression, and substantially inhibited the migration of the CNE1 cells. In addition, MMP7 expression was reduced to varying extents in S100A8 and S100A9 siRNA-treated cells compared with controls. Thus, S100A8 and S100A9 promoted the migration of CNE1 NPC cells.

  16. A single dose of EGLN1 siRNA yields increased erythropoiesis in nonhuman primates.

    PubMed

    Abrams, Marc T; Koser, Martin; Burchard, Julja; Strapps, Walter; Mehmet, Huseyin; Gindy, Marian; Zaller, Dennis; Sepp-Lorenzino, Laura; Stickens, Dominique

    2014-12-01

    Decreased production of erythropoietin (EPO) causes anemia in patients with chronic kidney disease, and recombinant human EPO is used to treat renal failure associated anemia. The liver, the main EPO-producing organ in utero, maintains the capacity to produce EPO in the adult but in insufficient quantities to restore hemoglobin levels to normal in patients with impaired renal function. Inhibition of prolyl-4-hydroxylase domain (PHD) proteins is known to cause an increase in EPO production through its effects on hypoxia inducible factor. Here, we utilized small interfering RNA (siRNA) targeting EGLN1, the gene encoding the PHD2 protein, to investigate the phenotypic consequences in nonhuman primates. A single, well-tolerated intravenous dose of an optimized EGLN1 siRNA encapsulated in a lipid nanoparticle formulation caused robust mRNA silencing in the liver, leading to increases in serum EPO and hemoglobin. The siRNA-induced erythropoiesis was dose-dependent and was sustained for at least 2 months. These data point to the potential for an RNA interference-based, liver-targeted therapeutic approach for the treatment of anemia.

  17. Combined integrin phosphoproteomic analyses and small interfering RNA--based functional screening identify key regulators for cancer cell adhesion and migration.

    PubMed

    Chen, Yanling; Lu, Bingwen; Yang, Qingkai; Fearns, Colleen; Yates, John R; Lee, Jiing-Dwan

    2009-04-15

    Integrins interact with extracellular matrix (ECM) and deliver intracellular signaling for cell proliferation, survival, and motility. During tumor metastasis, integrin-mediated cell adhesion to and migration on the ECM proteins are required for cancer cell survival and adaptation to the new microenvironment. Using stable isotope labeling by amino acids in cell culture-mass spectrometry, we profiled the phosphoproteomic changes induced by the interactions of cell integrins with type I collagen, the most common ECM substratum. Integrin-ECM interactions modulate phosphorylation of 517 serine, threonine, or tyrosine residues in 513 peptides, corresponding to 357 proteins. Among these proteins, 33 key signaling mediators with kinase or phosphatase activity were subjected to small interfering RNA-based functional screening. Three integrin-regulated kinases, DBF4, PAK2, and GRK6, were identified for their critical role in cell adhesion and migration possibly through their regulation of actin cytoskeleton arrangement. Altogether, we not only depict an integrin-modulated phosphorylation network during cell-ECM protein interactions but also reveal novel regulators for cell adhesion and migration.

  18. Shock-induced poration, cholesterol flip-flop and small interfering RNA transfection in a phospholipid membrane: Multimillion atom, microsecond molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Choubey, Amit

    performing a 15 mus all-atom MD simulation of a DPPC-CHOL bilayer. We find that the CHOL flip-flop rates are on the sub microsecond timescale. These results are verified by performing various independent parallel replica (PR) simulations. Our PR simulations provide significant boost in sampling of the flip-flop events. We observe that the CHOL flip-flop can induce membrane order, regulate membrane-bending energy, and facilitate membrane relaxation. The rapid flip-flop rates reported here have important implications for the role of CHOL in mechanical properties of cell membranes, formation of domains, and maintaining CHOL concentration asymmetry in plasma membrane. Our PR approach can reach submillisecond time scales and bridge the gap between MD simulations and Nuclear Magnetic Resonance (NMR) experiments on CHOL flip-flop dynamics in membranes. The last project deals with transfection barriers encountered by a bare small interfering RNA (siRNA) in a phospholipid bilayer. SiRNA molecules play a pivotal role in therapeutic applications. A key limitation to the widespread implementation of siRNA-based therapeutics is the difficulty of delivering siRNA-based drugs to cells. We have examined structural and mechanical barriers to siRNA passage across a phospholipid bilayer using all-atom MD simulations. We find that the electrostatic interaction between the anionic siRNA and head groups of phospholipid molecules induces a phase transformation from the liquid crystalline to ripple phase. Steered MD simulations reveal that the siRNA transfection through the ripple phase requires a force of ˜ 1.5 nN.

  19. Short-interfering RNA-mediated silencing of proliferating cell nuclear antigen inhibit proliferation and induce apoptosis in HeLa cells.

    PubMed

    Hao, H; Xin, T; Nancai, Y; Yanxia, W; Qian, L; Wei, M; Yandong, Y; Hanju, H

    2008-01-01

    Proliferating cell nuclear antigen (PCNA) is an important protein for DNA polymerase delta in the nucleus, and shown to have a fundamental role in cellular proliferation. It is overexpressed to support cell growth in cervical carcinoma. To study its role in stress response, we design and use short hairpin RNA (shRNA) to inhibit PCNA expression in HeLa cells and validate its effect on cell proliferation. In this study, three PCNA-shRNA expression vectors are constructed and introduced into HeLa cells, and the cell cycle is analyzed by flow cytometry. Apoptotic cell is detected by single cell gel electrophoresis assay (comet assay), and caspase cleavage is studied also. Expression of PCNA is assessed by real-time reverse transcription-polymerase chain reaction and Western blot analysis. Upon transient transfection with plasmid encoding shRNA, it is found that expression of PCNA decreased in shRNA-transfected cells, downregulation of PCNA inhibit cell growth and induce apoptosis in HeLa cells. PCNA downregulation also increase cell population in the G0-G1 phase. In conclusion, our findings demonstrate that shRNA can inhibit the DNA replication and induce apoptosis in HeLa cells effectively and, therefore, could be used as a new potential anticancer tool for therapy of human cervical carcinoma.

  20. A genome-wide small interfering RNA (siRNA) screen reveals nuclear factor-κB (NF-κB)-independent regulators of NOD2-induced interleukin-8 (IL-8) secretion.

    PubMed

    Warner, Neil; Burberry, Aaron; Pliakas, Maria; McDonald, Christine; Núñez, Gabriel

    2014-10-10

    NOD2 encodes an intracellular multidomain pattern recognition receptor that is the strongest known genetic risk factor in the pathogenesis of Crohn disease (CD), a chronic relapsing inflammatory disorder of the intestinal tract. NOD2 functions as a sensor for bacterial cell wall components and activates proinflammatory and antimicrobial signaling pathways. Here, using a genome-wide small interfering RNA (siRNA) screen, we identify numerous genes that regulate secretion of the proinflammatory cytokine IL-8 in response to NOD2 activation. Moreover, many of the identified IL-8 regulators are linked by protein-protein interactions, revealing subnetworks of highly connected IL-8 regulators implicated in processes such as vesicle formation, mRNA stability, and protein ubiquitination and trafficking. A TNFα counterscreen to induce IL-8 secretion in an NOD2-independent manner reveals that the majority of the identified regulators affect IL-8 secretion irrespective of the initiating stimuli. Using immortalized macrophages, we validate the ubiquitin protease, USP8, and the endosomal sorting protein, VPS28, as negative regulators of NOD2-induced cytokine secretion. Interestingly, several genes that affect NOD2-induced IL-8 secretion are present in loci associated with CD risk by genome-wide association studies, supporting a role for the NOD2/IL-8 pathway, and not just NOD2, in the pathogenesis of CD. Overall, this screen provides a valuable resource in the advancement of our understanding of the genes that regulate the secretion of IL-8.

  1. Silk worm Bm1 SINE RNA increases following cellular insults.

    PubMed

    Kimura, R H; Choudary, P V; Schmid, C W

    1999-08-15

    The effect of cell stresses upon the expression of the Bm1 short interspersed element (SINE) family in cultured silk worm cells is examined. Primer extension analysis shows that Bm1 repeats are transcribed by RNA polymerase III (Pol III) into cytoplasmic RNAs. Five consecutive T residues, which would normally terminate Pol III transcription, occur within the Bm1 consensus and are included within cDNA sequences representing these transcripts. In analogy to mammalian SINEs, the level of the Bm1 transcripts increases in response to either heat shock, inhibiting protein synthesis by cycloheximide or viral infection. The lifetime of Bm1 RNA increases following cell insults so that post-transcriptional events partially account for stress induced increases in its abundance. In the case of heat shock, the increase in Bm1 RNA follows the transient increase in hsp70 mRNA indicating that this response is temporally regulated to occur later in heat shock recovery. These results support the proposal that SINE RNAs serve a role in the cell stress response that predates the divergence of insects and mammals implying that SINEs are essentially a class of cell stress genes.

  2. PUNCTATE VASCULAR EXPRESSION1 Is a Novel Maize Gene Required for Leaf Pattern Formation That Functions Downstream of the Trans-Acting Small Interfering RNA Pathway1[C][W][OA

    PubMed Central

    Zhang, Xiaolan; Douglas, Ryan N.; Strable, Josh; Lee, Michelle; Buckner, Brent; Janick-Buckner, Diane; Schnable, Patrick S.; Timmermans, Marja C.P.; Scanlon, Michael J.

    2012-01-01

    The maize (Zea mays) gene RAGGED SEEDLING2-R (RGD2-R) encodes an ARGONAUTE7-like protein required for the biogenesis of trans-acting small interfering RNA, which regulates the accumulation of AUXIN RESPONSE FACTOR3A transcripts in shoots. Although dorsiventral polarity is established in the narrow and cylindrical leaves of rgd2-R mutant plants, swapping of adaxial/abaxial epidermal identity occurs and suggests a model wherein RGD2 is required to coordinate dorsiventral and mediolateral patterning in maize leaves. Laser microdissection-microarray analyses of the rgd2-R mutant shoot apical meristem identified a novel gene, PUNCTATE VASCULAR EXPRESSION1 (PVE1), that is down-regulated in rgd2-R mutant apices. Transcripts of PVE1 provide an early molecular marker for vascular morphogenesis. Reverse genetic analyses suggest that PVE1 functions during vascular development and in mediolateral and dorsiventral patterning of maize leaves. Molecular genetic analyses of PVE1 and of rgd2-R;pve1-M2 double mutants suggest a model wherein PVE1 functions downstream of RGD2 in a pathway that intersects and interacts with the trans-acting small interfering RNA pathway. PMID:22669891

  3. Genetic modulation of RNA metabolism in Drosophilia. I. Increased rate of ribosomal RNA synthesis.

    PubMed

    Clark, S H; Strausbaugh, L D; Kiefer, B I

    1977-08-01

    It has been suggested that a particular Y chromosome which is rDNA-deficient (YbbSuVar-5) may be associated with an increased utilization of rDNA template in adult testes (Shermoen and Kiefer 1975). To extend the observations on this chromosome, experiments were designed to determine if the chromosome has an effect on rRNA synthesis in bobbed adults and on classic bobbed phenotypes (shortened and thinner scutellar bristles and delayed development). Specific activity measurements were made on rRNA extracted from adult males of the genotypes car bb/YbbSuVar-5, which are rDNA-deficient to the same extent, and from Samarkand+ isogenic (Sam+ iso), which is a wild-type stock. The resulting data demonstrated that the presence of the YbbSuVar-5 chromosome increases the rate of ribosomal RNA synthesis in adult flies. In addition, it was found that the presence of this particular Y chromosome restores wild-type bristle phenotype and development time. Appropriate genetic crosses indicate that the observed effects (increased rRNA synthesis, restoration of wild-type phenotype) are a function of this particular Y chromosome, and are not due to autosomal factors. The results of these experiments suggest that the rate of rRNA accumulation is under genetic control.

  4. Polo-like kinase-1 as a novel target in neoplastic mast cells: demonstration of growth-inhibitory effects of small interfering RNA and the Polo-like kinase-1 targeting drug BI 2536

    PubMed Central

    Peter, Barbara; Gleixner, Karoline V.; Cerny-Reiterer, Sabine; Herrmann, Harald; Winter, Viviane; Hadzijusufovic, Emir; Ferenc, Veronika; Schuch, Karina; Mirkina, Irina; Horny, Hans-Peter; Pickl, Winfried F.; Müllauer, Leonhard; Willmann, Michael; Valent, Peter

    2011-01-01

    Background In advanced systemic mastocytosis the response of neoplastic mast cells to conventional drugs is poor and the prognosis is bad. Current research is, therefore, attempting to identify novel drug targets in neoplastic mast cells. Polo-like kinase-1 is a serine/threonine kinase that plays an essential role in mitosis and has recently been introduced as a new target in myeloid leukemias and solid tumors. Design and Methods In the present study, we analyzed the expression and function of Polo-like kinase-1 in neoplastic mast cells in systemic mastocytosis. Results As determined by immunostaining, primary neoplastic mast cells as well as the human mast cell leukemia cell line HMC-1 displayed phosphorylated Polo-like kinase-1. In addition, neoplastic mast cells expressed Polo-like kinase-1 mRNA. Polo-like kinase-1-specific small interfering RNA induced apoptosis in neoplastic mast cells, whereas no effect was seen with a control small interfering RNA. BI 2536, a drug targeting Polo-like kinase-1, was found to inhibit proliferation in HMC-1 cells in a dose-dependent manner. BI 2536 also inhibited the growth of primary neoplastic mast cells and cells of the canine mastocytoma cell line C2. The growth-inhibitory effects of BI 2536 on neoplastic mast cells were found to be associated with mitotic arrest and subsequent apoptosis. Finally, BI 2536 was found to synergize with the KIT-targeting kinase inhibitor midostaurin (PKC412) in inhibiting the growth of neoplastic mast cells. In control experiments, BI 2536 did not induce apoptosis in normal cultured mast cells. Conclusions Collectively, our data show that Polo-like kinase-1 is a potential therapeutic target in neoplastic mast cells. Targeting Polo-like kinase-1 may be an attractive pharmacological concept in the management of advanced systemic mastocytosis. PMID:21242189

  5. C3a Increases VEGF and Decreases PEDF mRNA Levels in Human Retinal Pigment Epithelial Cells.

    PubMed

    Long, Qin; Cao, Xiaoguang; Bian, Ailing; Li, Ying

    2016-01-01

    Complement activation, specifically complement 3 (C3) activation and C3a generation, contributes to an imbalance between angiogenic stimulation by vascular endothelial growth factor (VEGF) and angiogenic inhibition by pigment epithelial derived factor (PEDF), leading to pathological angiogenesis. This study aimed to investigate the effects of C3a and small interfering RNA (siRNA) targeting C3 on the levels of VEGF and PEDF mRNAs in human retinal pigment epithelial (RPE) cells. ARPE-19 cells were cultured in the presence of exogenous C3a at 0.1 μM and 0.3 μM C3a for 24, 48, and 72 hours. 0.1 pmol/μL duplexes of siRNA targeting C3 were applied for C3a inhibition by transfecting ARPE-19 cells for 48 hours. RT-PCR was performed to examine the level of VEGF and PEDF mRNA. A random siRNA duplex was set for control siRNA. Results demonstrated that exogenous C3a significantly upregulated VEGF and downregulated PEDF mRNA levels in cultured ARPE-19 cells, and siRNA targeting C3 transfection reversed the above changes, significantly reducing VEGF and enhancing PEDF mRNAs level in ARPE-19 cells compared to the control. The present data provided evidence that reducing C3 activation can decreases VEGF and increase PEDF mRNA level in RPE and may serve as a potential therapy in pathological angiogenesis.

  6. C3a Increases VEGF and Decreases PEDF mRNA Levels in Human Retinal Pigment Epithelial Cells

    PubMed Central

    Long, Qin; Cao, Xiaoguang; Bian, Ailing

    2016-01-01

    Complement activation, specifically complement 3 (C3) activation and C3a generation, contributes to an imbalance between angiogenic stimulation by vascular endothelial growth factor (VEGF) and angiogenic inhibition by pigment epithelial derived factor (PEDF), leading to pathological angiogenesis. This study aimed to investigate the effects of C3a and small interfering RNA (siRNA) targeting C3 on the levels of VEGF and PEDF mRNAs in human retinal pigment epithelial (RPE) cells. ARPE-19 cells were cultured in the presence of exogenous C3a at 0.1 μM and 0.3 μM C3a for 24, 48, and 72 hours. 0.1 pmol/μL duplexes of siRNA targeting C3 were applied for C3a inhibition by transfecting ARPE-19 cells for 48 hours. RT-PCR was performed to examine the level of VEGF and PEDF mRNA. A random siRNA duplex was set for control siRNA. Results demonstrated that exogenous C3a significantly upregulated VEGF and downregulated PEDF mRNA levels in cultured ARPE-19 cells, and siRNA targeting C3 transfection reversed the above changes, significantly reducing VEGF and enhancing PEDF mRNAs level in ARPE-19 cells compared to the control. The present data provided evidence that reducing C3 activation can decreases VEGF and increase PEDF mRNA level in RPE and may serve as a potential therapy in pathological angiogenesis. PMID:27747237

  7. Lettuce chlorosis virus P23 Suppresses RNA Silencing and Induces Local Necrosis with Increased Severity at Raised Temperatures.

    PubMed

    Kubota, Kenji; Ng, James C K

    2016-06-01

    RNA silencing functions as an antivirus defense strategy in plants, one that plant viruses counter by producing viral suppressors of RNA silencing (VSRs). VSRs have been identified in three members of the genus Crinivirus but they do not all share identical suppression mechanisms. Here, we used Agrobacterium co-infiltration assays to investigate the suppressor activity of proteins encoded by Lettuce chlorosis virus (LCV). Of 7 LCV proteins (1b, P23, HSP70 homolog, P60, CP, CPm, and P27) tested for the suppression of silencing of green fluorescent protein (GFP) expression in wild-type Nicotiana benthamiana plants, only P23 suppressed the onset of local silencing. Small-interfering (si)RNA accumulation was reduced in leaves co-infiltrated with P23, suggesting that P23 inhibited the accumulation or enhanced the degradation of siRNA. P23 also inhibited the cell-to-cell and systemic movement of RNA silencing in GFP-expressing transgenic N. benthamiana plants. Expression of P23 via agroinfiltration of N. benthamiana leaves induced local necrosis that increased in severity at elevated temperatures, a novelty given that a direct temperature effect on necrosis severity has not been reported for the other crinivirus VSRs. These results further affirm the sophistication of crinivirus VSRs in mediating the evasion of host's antiviral defenses and in symptom modulation.

  8. Analysis of the small interfering RNA profiles of randomly inserted pTRM-TRI6 Fusarium graminearum mutants and their DON related phenotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Deoxynivalenol (DON) production by Fusarium graminearum requires activation of the trichothecene pathway in which TRI5 catalyzes the first step of trichothecene synthesis and TRI6 is a transcription factor activates the pathway. RNA interference (RNAi) has emerged as a useful fungal genetics tool f...

  9. Postexposure Application of Fas Receptor Small-Interfering RNA to Suppress Sulfur Mustard-Induced Apoptosis in Human Airway Epithelial Cells: Implication for a Therapeutic Approach

    DTIC Science & Technology

    2013-01-01

    pathogenic mechanisms, e.g., DNA damage, are initiated without much delay but the tissue injury is not seen until after a lag period of 12 to 24 hours...measured at 450 nm using a ThermoMax microplate reader (MDS Analytical Technologies, Sunnyvale, CA). The recombi - nant human FasL/TNFSF6 produced in CHO...alkylating form that reacts readily with cellular functional molecules (e.g., DNA , RNA, proteins, etc.) to initiate its toxic mecha- nisms. At body temperature

  10. Deposition of insoluble elastin by pulmonary fibroblasts from patients with COPD is increased by treatment with versican siRNA

    PubMed Central

    Wu, Lian; Zhang, Jing; Qu, Jie Ming; Bai, Chun-xue; Merrilees, Mervyn J

    2017-01-01

    A reduced content of alveolar elastic fibers is a key feature of COPD lung. Despite continued elastogenic potential by alveolar fibroblasts in the lung affected by COPD, repair of elastic fibers does not take place, which is due to increased levels of the chondroitin sulfate proteoglycan versican that inhibits the assembly of tropoelastin into fibers. In this study, primary pulmonary fibroblast cell lines from COPD and non-COPD patients were treated with a small interfering RNA (siRNA) against versican to determine if knockdown of versican could restore the deposition of insoluble elastin. Versican siRNA treatment reduced versican expression and secretion by pulmonary fibroblasts from both COPD and non-COPD patients (P<0.01) and significantly increased deposition of insoluble elastin in the COPD cell cultures (P<0.05). The treatment, however, did not significantly affect production of soluble elastin (tropoelastin) in either the COPD or non-COPD cell cultures, supporting a role for versican in inhibiting assembly but not synthesis of tropoelastin. These results suggest that removal or knockdown of versican may be a possible therapeutic strategy for increasing deposition of insoluble elastin and stimulating repair of elastic fibers in COPD lung. PMID:28138236

  11. Measles Virus Defective Interfering RNAs Are Generated Frequently and Early in the Absence of C Protein and Can Be Destabilized by Adenosine Deaminase Acting on RNA-1-Like Hypermutations

    PubMed Central

    Pfaller, Christian K.; Mastorakos, George M.; Matchett, William E.; Ma, Xiao; Samuel, Charles E.

    2015-01-01

    ABSTRACT Defective interfering RNAs (DI-RNAs) of the viral genome can form during infections of negative-strand RNA viruses and outgrow full-length viral genomes, thereby modulating the severity and duration of infection. Here we document the frequent de novo generation of copy-back DI-RNAs from independent rescue events both for a vaccine measles virus (vac2) and for a wild-type measles virus (IC323) as early as passage 1 after virus rescue. Moreover, vaccine and wild-type C-protein-deficient (C-protein-knockout [CKO]) measles viruses generated about 10 times more DI-RNAs than parental virus, suggesting that C enhances the processivity of the viral polymerase. We obtained the nucleotide sequences of 65 individual DI-RNAs, identified breakpoints and reinitiation sites, and predicted their structural features. Several DI-RNAs possessed clusters of A-to-G or U-to-C transitions. Sequences flanking these mutation sites were characteristic of those favored by adenosine deaminase acting on RNA-1 (ADAR1), which catalyzes in double-stranded RNA the C-6 deamination of adenosine to produce inosine, which is recognized as guanosine, a process known as A-to-I RNA editing. In individual DI-RNAs the transitions were of the same type and occurred on both sides of the breakpoint. These patterns of mutations suggest that ADAR1 edits unencapsidated DI-RNAs that form double-strand RNA structures. Encapsidated DI-RNAs were incorporated into virus particles, which reduced the infectivity of virus stocks. The CKO phenotype was dominant: DI-RNAs derived from vac2 with a CKO suppressed the replication of vac2, as shown by coinfections of interferon-incompetent lymphatic cells with viruses expressing different fluorescent reporter proteins. In contrast, coinfection with a C-protein-expressing virus did not counteract the suppressive phenotype of DI-RNAs. IMPORTANCE Recombinant measles viruses (MVs) are in clinical trials as cancer therapeutics and as vectored vaccines for HIV-AIDS and

  12. Induction of CML28-specific cytotoxic T cell responses using co-transfected dendritic cells with CML28 DNA vaccine and SOCS1 small interfering RNA expression vector

    SciTech Connect

    Zhou Hongsheng; Zhang Donghua . E-mail: hanson2008@gmail.com; Wang Yaya; Dai Ming; Zhang Lu; Liu Wenli; Liu Dan; Tan Huo; Huang Zhenqian

    2006-08-18

    CML28 is an attractive target for antigen-specific immunotherapy. SOCS1 represents an inhibitory control mechanism for DC antigen presentation and the magnitude of adaptive immunity. In this study, we evaluated the potential for inducing CML28-specific cytotoxic T lymphocytes (CTL) responses by dendritic cells (DCs)-based vaccination. We constructed a CML28 DNA vaccine and a SOCS1 siRNA vector and then cotransfect monocyte-derived DCs. Flow cytometry analysis showed gene silencing of SOCS1 resulted in higher expressions of costimulative moleculars in DCs. Mixed lymphocyte reaction (MLR) indicated downregulation of SOCS1 stronger capability to stimulate proliferation of responder cell in DCs. The CTL assay revealed transfected DCs effectively induced autologous CML28-specific CTL responses and the lytic activities induced by SOCS1-silenced DCs were significantly higher compared with those induced by SOCS1-expressing DCs. These results in our study indicates gene silencing of SOCS1 remarkably enhanced the cytotoxicity efficiency of CML28 DNA vaccine in DCs.

  13. Globally increased ultraconserved noncoding RNA expression in pancreatic adenocarcinoma

    PubMed Central

    Lee, Eun Joo; Gusev, Yuriy; Allard, David; Sutaria, Dhruvitkumar S.; Badawi, Mohamed; Elgamal, Ola A.; Lerner, Megan R.; Brackett, Daniel J.; Calin, George A.; Schmittgen, Thomas D.

    2016-01-01

    Transcribed ultraconserved regions (T-UCRs) are a class of non-coding RNAs with 100% sequence conservation among human, rat and mouse genomes. T-UCRs are differentially expressed in several cancers, however their expression in pancreatic adenocarcinoma (PDAC) has not been studied. We used a qPCR array to profile all 481 T-UCRs in pancreatic cancer specimens, pancreatic cancer cell lines, during experimental pancreatic desmoplasia and in the pancreases of P48Cre/wt; KrasLSL-G12D/wt mice. Fourteen, 57 and 29% of the detectable T-UCRs were differentially expressed in the cell lines, human tumors and transgenic mouse pancreases, respectively. The vast majority of the differentially expressed T-UCRs had increased expression in the cancer. T-UCRs were monitored using an in vitro model of the desmoplastic reaction. Twenty-five % of the expressed T-UCRs were increased in the HPDE cells cultured on PANC-1 cellular matrix. UC.190, UC.233 and UC.270 were increased in all three human data sets. siRNA knockdown of each of these three T-UCRs reduced the proliferation of MIA PaCa-2 cells up to 60%. The expression pattern among many T-UCRs in the human and mouse pancreases closely correlated with one another, suggesting that groups of T-UCRs are co-activated in PDAC. Successful knockout of the transcription factor EGR1 in PANC-1 cells caused a reduction in the expression of a subset of T-UCRs suggesting that EGR1 may control T-UCR expression in PDAC. We report a global increase in expression of T-UCRs in both human and mouse PDAC. Commonalties in their expression pattern suggest a similar mechanism of transcriptional upregulation for T-UCRs in PDAC. PMID:27363020

  14. Acid-Sensitive Sheddable PEGylated PLGA Nanoparticles Increase the Delivery of TNF-α siRNA in Chronic Inflammation Sites

    PubMed Central

    Aldayel, Abdulaziz M; Naguib, Youssef W; O'Mary, Hannah L; Li, Xu; Niu, Mengmeng; Ruwona, Tinashe B; Cui, Zhengrong

    2016-01-01

    There has been growing interest in utilizing small interfering RNA (siRNA) specific to pro-inflammatory cytokines, such as tumor necrosis factor-α ( TNF-α), in chronic inflammation therapy. However, delivery systems that can increase the distribution of the siRNA in chronic inflammation sites after intravenous administration are needed. Herein we report that innovative functionalization of the surface of siRNA-incorporated poly (lactic-co-glycolic) acid (PLGA) nanoparticles significantly increases the delivery of the siRNA in the chronic inflammation sites in a mouse model. The TNF-α siRNA incorporated PLGA nanoparticles were prepared by the standard double emulsion method, but using stearoyl-hydrazone-polyethylene glycol 2000, a unique acid-sensitive surface active agent, as the emulsifying agent, which renders (i) the nanoparticles PEGylated and (ii) the PEGylation sheddable in low pH environment such as that in chronic inflammation sites. In a mouse model of lipopolysaccharide-induced chronic inflammation, the acid-sensitive sheddable PEGylated PLGA nanoparticles showed significantly higher accumulation or distribution in chronic inflammation sites than PLGA nanoparticles prepared with an acid-insensitive emulsifying agent (i.e., stearoyl-amide-polyethylene glycol 2000) and significantly increased the distribution of the TNF-α siRNA incorporated into the nanoparticles in inflamed mouse foot. PMID:27434685

  15. MORF9 increases the RNA-binding activity of PLS-type pentatricopeptide repeat protein in plastid RNA editing.

    PubMed

    Yan, Junjie; Zhang, Qunxia; Guan, Zeyuan; Wang, Qiang; Li, Li; Ruan, Fengying; Lin, Rongcheng; Zou, Tingting; Yin, Ping

    2017-04-10

    RNA editing is a post-transcriptional process that modifies the genetic information on RNA molecules. In flowering plants, RNA editing usually alters cytidine to uridine in plastids and mitochondria. The PLS-type pentatricopeptide repeat (PPR) protein and the multiple organellar RNA editing factor (MORF, also known as RNA editing factor interacting protein (RIP)) are two types of key trans-acting factors involved in this process. However, how they cooperate with one another remains unclear. Here, we have characterized the interactions between a designer PLS-type PPR protein (PLS)3PPR and MORF9, and found that RNA-binding activity of (PLS)3PPR is drastically increased on MORF9 binding. We also determined the crystal structures of (PLS)3PPR, MORF9 and the (PLS)3PPR-MORF9 complex. MORF9 binding induces significant compressed conformational changes of (PLS)3PPR, revealing the molecular mechanisms by which MORF9-bound (PLS)3PPR has increased RNA-binding activity. Similarly, increased RNA-binding activity is observed for the natural PLS-type PPR protein, LPA66, in the presence of MORF9. These findings significantly expand our understanding of MORF function in plant organellar RNA editing.

  16. Blocking miRNA Biogenesis in Adult Forebrain Neurons Enhances Seizure Susceptibility, Fear Memory, and Food Intake by Increasing Neuronal Responsiveness.

    PubMed

    Fiorenza, Anna; Lopez-Atalaya, Jose P; Rovira, Victor; Scandaglia, Marilyn; Geijo-Barrientos, Emilio; Barco, Angel

    2016-04-01

    The RNase Dicer is essential for the maturation of most microRNAs, a molecular system that plays an essential role in fine-tuning gene expression. To gain molecular insight into the role of Dicer and the microRNA system in brain function, we conducted 2 complementary RNA-seq screens in the hippocampus of inducible forebrain-restricted Dicer1 mutants aimed at identifying the microRNAs primarily affected by Dicer loss and their targets, respectively. Functional genomics analyses predicted the main biological processes and phenotypes associated with impaired microRNA maturation, including categories related to microRNA biology, signal transduction, seizures, and synaptic transmission and plasticity. Consistent with these predictions, we found that, soon after recombination, Dicer-deficient mice exhibited an exaggerated seizure response, enhanced induction of immediate early genes in response to different stimuli, stronger and more stable fear memory, hyperphagia, and increased excitability of CA1 pyramidal neurons. In the long term, we also observed slow and progressive excitotoxic neurodegeneration. Overall, our results indicate that interfering with microRNA biogenesis causes an increase in neuronal responsiveness and disrupts homeostatic mechanisms that protect the neuron against overactivation, which may explain both the initial and late phenotypes associated with the loss of Dicer in excitatory neurons.

  17. Low-Fidelity Polymerases of Alphaviruses Recombine at Higher Rates To Overproduce Defective Interfering Particles

    PubMed Central

    Poirier, Enzo Z.; Mounce, Bryan C.; Rozen-Gagnon, Kathryn; Hooikaas, Peter Jan; Stapleford, Kenneth A.; Moratorio, Gonzalo

    2015-01-01

    ABSTRACT Low-fidelity RNA-dependent RNA polymerases for many RNA virus mutators have been shown to confer attenuated phenotypes, presumably due to increased mutation rates. Additionally, for many RNA viruses, replication to high titers results in the production of defective interfering particles (DIs) that also attenuate infection. We hypothesized that fidelity, recombination, and DI production are tightly linked. We show that a Sindbis virus mutator replicating at a high multiplicity of infection manifests an earlier and greater accumulation of DIs than its wild-type counterpart. The isolated DIs interfere with the replication of full-length virus in a dose-dependent manner. Importantly, the ability of the mutator virus to overproduce DIs could be linked to an increased recombination frequency. These data confirm that RNA-dependent RNA polymerase fidelity and recombination are inversely correlated for this mutator. Our findings suggest that defective interference resulting from higher recombination rates may be more detrimental to RNA virus mutators than the increase in mutational burden. IMPORTANCE Replication, adaptation, and evolution of RNA viruses rely in large part on their low-fidelity RNA-dependent RNA polymerase. Viruses artificially modified in their polymerases to decrease fidelity (mutator viruses) are attenuated in vivo, demonstrating the important role of fidelity in viral fitness. However, attenuation was attributed solely to the modification of the viral mutation rate and the accumulation of detrimental point mutations. In this work, we described an additional phenotype of mutator viruses: an increased recombination rate leading to defective interfering particle (DI) overproduction. Because DIs are known for their inhibitory effect on viral replication, our work suggests that fidelity variants may be attenuated in vivo via several mechanisms. This has important implications in the development of fidelity variants as live attenuated vaccine strains

  18. Anti-DNA:RNA antibodies and silicon photonic microring resonators: increased sensitivity for multiplexed microRNA detection.

    PubMed

    Qavi, Abraham J; Kindt, Jared T; Gleeson, Martin A; Bailey, Ryan C

    2011-08-01

    In this paper, we present a method for the sensitive detection of microRNAs (miRNAs) utilizing an antibody that specifically recognizes DNA:RNA heteroduplexes and a silicon photonic microring resonator array transduction platform. Microring resonator arrays are covalently functionalized with DNA capture probes that are complementary to solution phase miRNA targets. Following hybridization on the sensor, the anti-DNA:RNA antibody is introduced and binds selectively to the heteroduplexes, giving a larger signal than the original miRNA hybridization due to the increased mass of the antibody, as compared to the 22-mer oligoribonucleotide. Furthermore, the secondary recognition step is performed in neat buffer solution and at relatively higher antibody concentrations, facilitating the detection of miRNAs of interest. The intrinsic sensitivity of the microring resonator platform coupled with the amplification provided by the anti-DNA:RNA antibodies allows for the detection of microRNAs at concentrations as low as 10 pM (350 amol). The simplicity and sequence generality of this amplification method position it as a promising tool for high-throughput, multiplexed miRNA analysis as well as a range of other RNA based detection applications.

  19. Denervation of rat adrenal glands markedly increases preproenkephalin mRNA.

    PubMed Central

    Kilpatrick, D L; Howells, R D; Fleminger, G; Udenfriend, S

    1984-01-01

    The effect of denervation on the expression of rat adrenal proenkephalin has been examined. Following splanchnicectomy there was a several-fold increase in the steady-state levels of preproenkephalin mRNA, which became maximal after 24-48 hr (greater than 10-fold). These results indicate that the previously observed increase in rat adrenal enkephalin-containing peptides following denervation occurs entirely by a pretranslational mechanism. The increase in preproenkephalin mRNA was accompanied by a 50-75% decrease in rat adrenal poly(A)+ RNA. Neural input thus exerts a profound trophic influence on proenkephalin gene expression and RNA metabolism in rat adrenals. Images PMID:6594691

  20. Denervation of rat adrenal glands markedly increases preproenkephalin mRNA.

    PubMed

    Kilpatrick, D L; Howells, R D; Fleminger, G; Udenfriend, S

    1984-11-01

    The effect of denervation on the expression of rat adrenal proenkephalin has been examined. Following splanchnicectomy there was a several-fold increase in the steady-state levels of preproenkephalin mRNA, which became maximal after 24-48 hr (greater than 10-fold). These results indicate that the previously observed increase in rat adrenal enkephalin-containing peptides following denervation occurs entirely by a pretranslational mechanism. The increase in preproenkephalin mRNA was accompanied by a 50-75% decrease in rat adrenal poly(A)+ RNA. Neural input thus exerts a profound trophic influence on proenkephalin gene expression and RNA metabolism in rat adrenals.

  1. Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

    PubMed

    Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

    1995-02-10

    Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain.

  2. Exercise and adrenaline increase PGC-1α mRNA expression in rat adipose tissue

    PubMed Central

    Sutherland, Lindsey N; Bomhof, Marc R; Capozzi, Lauren C; Basaraba, Susan A U; Wright, David C

    2009-01-01

    The purpose of the present investigation was to explore the effects of exercise and adrenaline on the mRNA expression of PGC-1α, a master regulator of mitochondrial biogenesis, in rat abdominal adipose tissue. We hypothesized that (1) exercise training would increase PGC-1α mRNA expression in association with increases in mitochondrial marker enzymes, (2) adrenaline would increase PGC-1α mRNA expression and (3) the effect of exercise on PGC-1α mRNA expression in white adipose tissue would be attenuated by a β-blocker. Two hours of daily swim training for 4 weeks led to increases in mitochondrial marker proteins and PGC-1α mRNA expression in epididymal and retroperitoneal fat depots. Additionally, a single 2 h bout of exercise led to increases in PGC-1α mRNA expression immediately following exercise cessation. Adrenaline treatment of adipose tissue organ cultures led to dose-dependent increases in PGC-1α mRNA expression. A supra-physiological concentration of adrenaline increased PGC-1α mRNA expression in epididymal but not retroperitoneal adipose tissue. β-Blockade attenuated the effects of an acute bout of exercise on PGC-1α mRNA expression in epididymal but not retroperitoneal fat pads. In summary, this is the first investigation to demonstrate that exercise training, an acute bout of exercise and adrenaline all increase PGC-1α mRNA expression in rat white adipose tissue. Furthermore it would appear that increases in circulating catecholamine levels may be one potential mechanism mediating exercise induced increases in PGC-1α mRNA expression in rat abdominal adipose tissue. PMID:19221126

  3. RNA Sequencing Identifies New RNase III Cleavage Sites in Escherichia coli and Reveals Increased Regulation of mRNA.

    PubMed

    Gordon, Gina C; Cameron, Jeffrey C; Pfleger, Brian F

    2017-03-28

    Ribonucleases facilitate rapid turnover of RNA, providing cells with another mechanism to adjust transcript and protein levels in response to environmental conditions. While many examples have been documented, a comprehensive list of RNase targets is not available. To address this knowledge gap, we compared levels of RNA sequencing coverage of Escherichia coli and a corresponding RNase III mutant to expand the list of known RNase III targets. RNase III is a widespread endoribonuclease that binds and cleaves double-stranded RNA in many critical transcripts. RNase III cleavage at novel sites found in aceEF, proP, tnaC, dctA, pheM, sdhC, yhhQ, glpT, aceK, and gluQ accelerated RNA decay, consistent with previously described targets wherein RNase III cleavage initiates rapid degradation of secondary messages by other RNases. In contrast, cleavage at three novel sites in the ahpF, pflB, and yajQ transcripts led to stabilized secondary transcripts. Two other novel sites in hisL and pheM overlapped with transcriptional attenuators that likely serve to ensure turnover of these highly structured RNAs. Many of the new RNase III target sites are located on transcripts encoding metabolic enzymes. For instance, two novel RNase III sites are located within transcripts encoding enzymes near a key metabolic node connecting glycolysis and the tricarboxylic acid (TCA) cycle. Pyruvate dehydrogenase activity was increased in an rnc deletion mutant compared to the wild-type (WT) strain in early stationary phase, confirming the novel link between RNA turnover and regulation of pathway activity. Identification of these novel sites suggests that mRNA turnover may be an underappreciated mode of regulating metabolism.IMPORTANCE The concerted action and overlapping functions of endoribonucleases, exoribonucleases, and RNA processing enzymes complicate the study of global RNA turnover and recycling of specific transcripts. More information about RNase specificity and activity is needed to make

  4. Repeated stress increases catalytic TrkB mRNA in rat hippocampus.

    PubMed

    Nibuya, M; Takahashi, M; Russell, D S; Duman, R S

    1999-05-28

    Northern blot analysis was utilized to distinguish between catalytic and truncated TrkB mRNA on the basis of transcript size. Repeated (10 days), but not acute, immobilization stress significantly increased levels of catalytic TrkB mRNA, but did not influence expression of truncated TrkB transcripts in rat hippocampus. Exposure to another paradigm, a combination of different, unpredictable stressors, also increased levels of catalytic, but not truncated, TrkB mRNA. In situ hybridization analysis demonstrated that chronic stress up-regulated TrkB mRNA in CA1 and CA3 pyramidal and dentate gyrus granule cells layers of hippocampus. As previously reported, both acute and chronic immobilization stress decreased expression of BDNF mRNA, suggesting that up-regulation of catalytic TrkB mRNA may be a compensatory adaptation to repeated stress.

  5. Single-base mutations at position 2661 of Escherichia coli 23S rRNA increase efficiency of translational proofreading.

    PubMed Central

    Melançon, P; Tapprich, W E; Brakier-Gingras, L

    1992-01-01

    Two single-base substitutions were constructed in the 2660 loop of Escherichia coli 23S rRNA (G2661-->C or U) and were introduced into the rrnB operon cloned in plasmid pKK3535. Ribosomes were isolated from bacteria transformed with the mutated plasmids and assayed in vitro in a poly(U)-directed system for their response to the misreading effect of streptomycin, neomycin, and gentamicin, three aminoglycoside antibiotics known to impair the proofreading control of translational accuracy. Both mutations decreased the stimulation of misreading by these drugs, but neither interfered with their binding to the ribosome. The response of the mutant ribosomes to these drugs suggests that the 2660 loop, which belongs to the elongation factor Tu binding site, is involved in the proofreading step of the accuracy control. In vivo, both mutations reduced read-through of nonsense codons and frameshifting, which can also be related to the increased efficiency in proofreading control which they confer to ribosomes. PMID:1281147

  6. Atherogenic diet increases cholesteryl ester transfer protein messenger RNA levels in rabbit liver.

    PubMed

    Quinet, E M; Agellon, L B; Kroon, P A; Marcel, Y L; Lee, Y C; Whitlock, M E; Tall, A R

    1990-02-01

    Cholesteryl ester transfer activity is increased in plasma of cholesterol-fed rabbits. To investigate the mechanisms leading to changes in activity, we measured cholesteryl ester transfer protein (CETP) mass by RIA and CETP mRNA abundance by Northern and slot blot analysis using a human CETP cDNA probe in control (n = 8) and cholesterol-fed rabbits (n = 10). Cholesterol feeding (chow plus 0.5% cholesterol, 10% corn oil) for 30 d increased CETP mass in plasma 3.2-fold in the cholesterol-fed rabbits (12.45 +/- 0.82 micrograms/ml) compared with controls (3.86 +/- 0.38 micrograms/ml). In the hypercholesterolemic rabbit, liver CETP mRNA levels were increased 2.8 times control mRNA levels. Actin, apo E, lecithin-cholesterol acyltransferase, and albumin mRNA abundances were unchanged. In contrast to the widespread tissue distribution in humans, CETP mRNA was not detected in extrahepatic tissues of either control or cholesterol-fed animals. Using a sensitive RNase protection assay, the increase in liver CETP mRNA was detectable within 3 d of beginning the high cholesterol diet. Thus, in response to the atherogenic diet there is an early increase in liver CETP mRNA, probably causing increased CETP synthesis and secretion, and increased plasma CETP. The results indicate that the CETP gene may be regulated by diet-induced changes in lipid metabolism.

  7. RNA-Based Methods Increase the Detection of Fecal Bacteria and Fecal Identifiers in Environmental Waters

    EPA Science Inventory

    We evaluated the use of qPCR RNA-based methods in the detection of fecal bacteria in environmental waters. We showed that RNA methods can increase the detection of fecal bacteria in multiple water matrices. The data suggest that this is a viable alternative for the detection of a...

  8. Adaptive antenna arrays for weak interfering signals

    NASA Technical Reports Server (NTRS)

    Gupta, I. J.

    1985-01-01

    The interference protection provided by adaptive antenna arrays to an Earth station or satellite receive antenna system is studied. The case where the interference is caused by the transmission from adjacent satellites or Earth stations whose signals inadverently enter the receiving system and interfere with the communication link is considered. Thus, the interfering signals are very weak. To increase the interference suppression, one can either decrease the thermal noise in the feedback loops or increase the gain of the auxiliary antennas in the interfering signal direction. Both methods are examined. It is shown that one may have to reduce the noise correlation to impractically low values and if directive auxiliary antennas are used, the auxiliary antenna size may have to be too large. One can, however, combine the two methods to achieve the specified interference suppression with reasonable requirements of noise decorrelation and auxiliary antenna size. Effects of the errors in the steering vector on the adaptive array performance are studied.

  9. Post-transcriptional regulation tends to attenuate the mRNA noise and to increase the mRNA gain

    NASA Astrophysics Data System (ADS)

    Shi, Changhong; Wang, Shuqiang; Zhou, Tianshou; Jiang, Yiguo

    2015-10-01

    Post-transcriptional regulation is ubiquitous in prokaryotic and eukaryotic cells, but how it impacts gene expression remains to be fully explored. Here, we analyze a simple gene model in which we assume that mRNAs are produced in a constitutive manner but are regulated post-transcriptionally by a decapping enzyme that switches between the active state and the inactive state. We derive the analytical mRNA distribution governed by a chemical master equation, which can be well used to analyze the mechanism of how post-transcription regulation influences the mRNA expression level including the mRNA noise. We demonstrate that the mean mRNA level in the stochastic case is always higher than that in the deterministic case due to the stochastic effect of the enzyme, but the size of the increased part depends mainly on the switching rates between two enzyme states. More interesting is that we find that in contrast to transcriptional regulation, post-transcriptional regulation tends to attenuate noise in mRNA. Our results provide insight into the role of post-transcriptional regulation in controlling the transcriptional noise.

  10. RNA Sequencing Identifies New RNase III Cleavage Sites in Escherichia coli and Reveals Increased Regulation of mRNA

    PubMed Central

    Gordon, Gina C.; Cameron, Jeffrey C.

    2017-01-01

    ABSTRACT Ribonucleases facilitate rapid turnover of RNA, providing cells with another mechanism to adjust transcript and protein levels in response to environmental conditions. While many examples have been documented, a comprehensive list of RNase targets is not available. To address this knowledge gap, we compared levels of RNA sequencing coverage of Escherichia coli and a corresponding RNase III mutant to expand the list of known RNase III targets. RNase III is a widespread endoribonuclease that binds and cleaves double-stranded RNA in many critical transcripts. RNase III cleavage at novel sites found in aceEF, proP, tnaC, dctA, pheM, sdhC, yhhQ, glpT, aceK, and gluQ accelerated RNA decay, consistent with previously described targets wherein RNase III cleavage initiates rapid degradation of secondary messages by other RNases. In contrast, cleavage at three novel sites in the ahpF, pflB, and yajQ transcripts led to stabilized secondary transcripts. Two other novel sites in hisL and pheM overlapped with transcriptional attenuators that likely serve to ensure turnover of these highly structured RNAs. Many of the new RNase III target sites are located on transcripts encoding metabolic enzymes. For instance, two novel RNase III sites are located within transcripts encoding enzymes near a key metabolic node connecting glycolysis and the tricarboxylic acid (TCA) cycle. Pyruvate dehydrogenase activity was increased in an rnc deletion mutant compared to the wild-type (WT) strain in early stationary phase, confirming the novel link between RNA turnover and regulation of pathway activity. Identification of these novel sites suggests that mRNA turnover may be an underappreciated mode of regulating metabolism. PMID:28351917

  11. Phosphorylation of DGCR8 increases its intracellular stability and induces a progrowth miRNA profile.

    PubMed

    Herbert, Kristina M; Pimienta, Genaro; DeGregorio, Suzanne J; Alexandrov, Andrei; Steitz, Joan A

    2013-11-27

    During miRNA biogenesis, the microprocessor complex (MC), which is composed minimally of Drosha, an RNase III enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary miRNA (pri-miRNA) in order to release the pre-miRNA stem-loop structure. Using phosphoproteomics, we mapped 23 phosphorylation sites on full-length human DGCR8 expressed in insect or mammalian cells. DGCR8 can be phosphorylated by mitogenic ERK/MAPK, indicating that DGCR8 phosphorylation may respond to and integrate extracellular cues. The expression of phosphomimetic DGCR8 or inhibition of phosphatases increased the cellular levels of DGCR8 and Drosha proteins. Increased levels of phosphomimetic DGCR8 were not due to higher mRNA levels, altered DGCR8 localization, or DGCR8's ability to self-associate, but rather to an increase in protein stability. MCs incorporating phosphomutant or phosphomimetic DGCR8 were not altered in specific processing activity. However, HeLa cells expressing phosphomimetic DGCR8 exhibited a progrowth miRNA expression profile and increased proliferation and scratch closure rates relative to cells expressing phosphomutant DGCR8.

  12. Phosphorylation of DGCR8 Increases Its Intracellular Stability and Induces a Progrowth miRNA Profile

    PubMed Central

    Herbert, Kristina M.; Pimienta, Genaro; DeGregorio, Suzanne J.; Alexandrov, Andrei; Steitz, Joan A.

    2014-01-01

    SUMMARY During miRNA biogenesis, the microprocessor complex (MC), which is composed minimally of Drosha, an RNase III enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary miRNA (pri-miRNA) in order to release the pre-miRNA stem-loop structure. Using phosphoproteomics, we mapped 23 phosphorylation sites on full-length human DGCR8 expressed in insect or mammalian cells. DGCR8 can be phosphorylated by mitogenic ERK/MAPK, indicating that DGCR8 phosphorylation may respond to and integrate extracellular cues. The expression of phosphomimetic DGCR8 or inhibition of phosphatases increased the cellular levels of DGCR8 and Drosha proteins. Increased levels of phosphomimetic DGCR8 were not due to higher mRNA levels, altered DGCR8 localization, or DGCR8’s ability to self-associate, but rather to an increase in protein stability. MCs incorporating phosphomutant or phosphomimetic DGCR8 were not altered in specific processing activity. However, HeLa cells expressing phosphomimetic DGCR8 exhibited a progrowth miRNA expression profile and increased proliferation and scratch closure rates relative to cells expressing phosphomutant DGCR8. PMID:24239349

  13. Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts.

    PubMed

    Ferizi, Mehrije; Aneja, Manish K; Balmayor, Elizabeth R; Badieyan, Zohreh Sadat; Mykhaylyk, Olga; Rudolph, Carsten; Plank, Christian

    2016-12-15

    Modified nucleotide chemistries that increase the half-life (T1/2) of transfected recombinant mRNA and the use of non-native 5'- and 3'-untranslated region (UTR) sequences that enhance protein translation are advancing the prospects of transcript therapy. To this end, a set of UTR sequences that are present in mRNAs with long cellular T1/2 were synthesized and cloned as five different recombinant sequence set combinations as upstream 5'-UTR and/or downstream 3'-UTR regions flanking a reporter gene. Initial screening in two different cell systems in vitro revealed that cytochrome b-245 alpha chain (CYBA) combinations performed the best among all other UTR combinations and were characterized in detail. The presence or absence of CYBA UTRs had no impact on the mRNA stability of transfected mRNAs, but appeared to enhance the productivity of transfected transcripts based on the measurement of mRNA and protein levels in cells. When CYBA UTRs were fused to human bone morphogenetic protein 2 (hBMP2) coding sequence, the recombinant mRNA transcripts upon transfection produced higher levels of protein as compared to control transcripts. Moreover, transfection of human adipose mesenchymal stem cells with recombinant hBMP2-CYBA UTR transcripts induced bone differentiation demonstrating the osteogenic and therapeutic potential for transcript therapy based on hybrid UTR designs.

  14. Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts

    PubMed Central

    Ferizi, Mehrije; Aneja, Manish K.; Balmayor, Elizabeth R.; Badieyan, Zohreh Sadat; Mykhaylyk, Olga; Rudolph, Carsten; Plank, Christian

    2016-01-01

    Modified nucleotide chemistries that increase the half-life (T1/2) of transfected recombinant mRNA and the use of non-native 5′- and 3′-untranslated region (UTR) sequences that enhance protein translation are advancing the prospects of transcript therapy. To this end, a set of UTR sequences that are present in mRNAs with long cellular T1/2 were synthesized and cloned as five different recombinant sequence set combinations as upstream 5′-UTR and/or downstream 3′-UTR regions flanking a reporter gene. Initial screening in two different cell systems in vitro revealed that cytochrome b-245 alpha chain (CYBA) combinations performed the best among all other UTR combinations and were characterized in detail. The presence or absence of CYBA UTRs had no impact on the mRNA stability of transfected mRNAs, but appeared to enhance the productivity of transfected transcripts based on the measurement of mRNA and protein levels in cells. When CYBA UTRs were fused to human bone morphogenetic protein 2 (hBMP2) coding sequence, the recombinant mRNA transcripts upon transfection produced higher levels of protein as compared to control transcripts. Moreover, transfection of human adipose mesenchymal stem cells with recombinant hBMP2-CYBA UTR transcripts induced bone differentiation demonstrating the osteogenic and therapeutic potential for transcript therapy based on hybrid UTR designs. PMID:27974853

  15. Exercise increases hexokinase II mRNA, but not activity in obesity and type 2 diabetes.

    PubMed

    Cusi, K J; Pratipanawatr, T; Koval, J; Printz, R; Ardehali, H; Granner, D K; Defronzo, R A; Mandarino, L J

    2001-05-01

    Glucose phosphorylation, catalyzed by hexokinase, is the first committed step in glucose uptake in skeletal muscle. Hexokinase II (HKII) is the isoform that is present in muscle and is regulated by insulin and muscle contraction. Glucose phosphorylation and HKII expression are both reduced in obese and type 2 diabetic subjects. A single bout of exercise increases HKII mRNA and activity in muscle from healthy subjects. The present study was performed to determine if a moderate exercise increases HKII mRNA expression and activity in patients with type 2 diabetes. Muscle biopsies were performed before and 3 hours after a single bout of cycle ergometer exercise in obese and type 2 diabetic patients. HKII mRNA and activity and glycogen synthase activity were determined in the muscle biopsies. Exercise increased HKII mRNA in obese and diabetic subjects by 1.67 +/- 0.34 and 1.87 +/- 0.26-fold, respectively (P <.05 for both). Exercise did not significantly increase HKI mRNA. When HKII mRNA increases were compared with the 2.26 +/- 0.36-fold increase in HKII mRNA previously reported for healthy lean subjects, no statistically significant differences were found. In contrast to the increase in HKII activity observed after exercise by lean healthy controls, exercise did not increase HKII activity in obese nondiabetic or diabetic subjects. Exercise increased glycogen synthase activity (GS(0.1) and GS(FV)) significantly in both obese nondiabetic and type 2 diabetic patients. The present results indicate that there is a posttranscriptional defect in the response of HKII expression to exercise in obese and type 2 diabetic subjects. This defect may contribute to reduced HKII activity and glucose uptake in these patients.

  16. RNA containing pyrrolocytidine base analogs: increased binding affinity and fluorescence that responds to hybridization.

    PubMed

    Wahba, Alexander S; Damha, Masad J; Hudson, Robert H E

    2008-01-01

    6-Phenylpyrrolocytidine and 6-methoxymethylene-pyrrolocytidine are base-modified nucleosides with remarkable fluorescence properties. These analogs produce increased binding affinity to both RNA and DNA targets when incorporated into oligoribonucleotides. The fluorescence observed for the single-stranded oligomers is quenched upon duplex formation with either RNA or DNA targets. The fluorescence response depends on the nature of the 6-substituent and the sequence position of the modified nucleoside.

  17. Prefrontal microRNA-221 Mediates Environmental Enrichment-Induced Increase of Locomotor Sensitivity to Nicotine

    PubMed Central

    Gomez, Adrian M.; Altomare, Diego; Sun, Wei-Lun; Midde, Narasimha M.; Ji, Hao; Shtutman, Michael; Turner, Jill R.; Creek, Kim E.

    2016-01-01

    Background: Environmental enrichment alters susceptibility in developing drug addiction. We have demonstrated that rats raised in an enriched condition are more sensitive than rats raised in an impoverished condition to nicotine-induced locomotor activity, and this is associated with alterations of phosphorylated extracellular signal-regulated kinase 1/2 within the prefrontal cortex. This study determined the impact of microRNA-221 in the prefrontal cortex on phosphorylated extracellular signal-regulated kinase 1/2 and the enriched environment-dependent behavioral changes in response to nicotine. Methods: A microRNA array was conducted to profile microRNA expression in the prefrontal cortex of enriched condition and impoverished condition rats in response to repeated nicotine (0.35mg/kg, s.c.) administration. microRNA-221 in the prefrontal cortex, nucleus accumbens, and striatum was further verified by quantitative real-time PCR. Lentiviral-mediated overexpression of microRNA-221 in PC12 cells and the medial prefrontal cortex was performed to determine the effects of microRNA-221 on nicotine-mediated phosphorylated extracellular signal-regulated kinase 1/2, phosphorylated cAMP-response element-binding protein, and locomotor activity. Results: microRNA-221 was profoundly upregulated in the prefrontal cortex but not in nucleus accumbens and striatum of enriched condition rats relative to impoverished condition rats following repeated administration of nicotine. Overexpression of lentiviral-microRNA-221 attenuated nicotine-induced increase in phosphorylated extracellular signal-regulated kinase 1/2 in PC12 cells. Lentiviral-microRNA-221 overexpression in the medial prefrontal cortex further increased locomotor activity in impoverished condition but not in enriched condition rats in response to repeated nicotine administration. Accordingly, lentiviral-microRNA-221 attenuated nicotine-induced increases in phosphorylated extracellular signal-regulated kinase 1/2 and

  18. Proteasome inhibition increases DNA and RNA oxidation in astrocyte and neuron cultures.

    PubMed

    Ding, Qunxing; Dimayuga, Edgardo; Markesbery, William R; Keller, Jeffrey N

    2004-12-01

    Increased levels of nucleic acid oxidation have been described as part of normal brain aging and have been demonstrated to occur in multiple neurological disorders. The basis for increased nucleic acid oxidation in each of these conditions is presently unknown. Proteasome inhibition occurs in a host of neurodegenerative conditions and likely contributes to increased levels of oxidative damage and neurotoxicity. In the present study we demonstrate for the first time the ability of proteasome inhibition to increase the level of nucleic acid oxidation in primary neuron and astrocyte cultures. Administration of proteasome inhibitors (MG262, MG115) at concentrations that do not induce neuron death in the first 24 h of treatment, dramatically increase the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 8-hydroxyguanosine (8OHG) immunoreactivity in both cell types. Neurons underwent larger increases in nucleic acid oxidation compared to astrocyte cultures. While both DNA and RNA oxidation were observed following proteasome inhibition, RNA appeared to undergo a greater degree of oxidation than DNA. Both 18S and 28S ribosomal RNA were dramatically decreased following proteasome inhibition. Interestingly, an accumulation of unprocessed and/or cross-linked RNA species was observed following proteasome inhibition. Taken together, these data indicate the ability of proteasome inhibition to increase the levels of nucleic acid oxidation in both neurons and astrocytes, and suggest that proteasome inhibition may have deleterious effects on transcription and translation in both neurons and glia.

  19. Infusion of Gabrα6 siRNA into the trigeminal ganglia increased the myogenic orofacial nociceptive response of ovariectomized rats treated with 17β-estradiol.

    PubMed

    Kramer, P R; Bellinger, L L

    2014-10-10

    High levels of 17β-estradiol (E2) have been found to reduce inflammatory temporomandibular joint (TMJ) pain. A search for genes effected by a high concentration of estradiol showed an increase in GABAA receptor subunit alpha 6 (Gabrα6) in the trigeminal ganglia (TG). Blockade of Gabrα6 expression in the TG increases masseter muscle nociception in male rats, but the relationship between estradiol's effect on nociception and Gabrα6 expression remains unclear in females. To address this knowledge gap we hypothesized that reducing Gabrα6 expression in the TG will increase the orofacial nociceptive response of ovariectomized female rats treated with estradiol. To administer hormone osmotic pumps were placed in rats that dispensed a low diestrus plasma concentration of 17β-estradiol, in addition, 17β-estradiol was injected to produce a high proestrus plasma concentration of estradiol. A ligature was then placed around the masseter tendon to induce a nociceptive response; a model for TMJ muscle pain. Gabrα6 small interfering RNA (siRNA) was later infused into the TG and the nociceptive response was measured using von Frey filaments and a meal duration assay. GABAA receptor expression was measured in the TG and trigeminal nucleus caudalis and upper cervical region (Vc-C1). Ligature significantly increased the nociceptive response but a high proestrus concentration of 17β-estradiol attenuated this response. Gabrα6 siRNA infusion decreased Gabrα6 expression in the TG and Vc-C1 but increased the nociceptive response after 17β-estradiol treatment. The results suggest estradiol decreased the orofacial nociceptive response, in part, by causing an increase in Gabrα6 expression.

  20. Engineering small interfering RNAs by strategic chemical modification.

    PubMed

    Bramsen, Jesper B; Kjems, Jørgen

    2013-01-01

    Synthetic small interfering RNAs (siRNAs) have revolutionized functional genomics in mammalian cell cultures due to their reliability, efficiency, and ease of use. This success, however, has not fully translated into siRNA applications in vivo and in siRNA therapeutics where initial optimism has been dampened by a lack of efficient delivery strategies and reports of siRNA off-target effects and immunogenicity. Encouragingly, most aspects of siRNA behavior can be addressed by careful engineering of siRNAs incorporating beneficial chemical modifications into discrete nucleotide positions during siRNA synthesis. Here, we review the literature (Subheadings 1 -3) and provide a quick guide (Subheading 4) to how the performance of siRNA can be improved by chemical modification to suit specific applications in vitro and in vivo.

  1. Overexpression of Dicer as a Result of Reduced let-7 microRNA Levels Contributes to Increased Cell Proliferation of Oral Cancer Cells

    PubMed Central

    Jakymiw, Andrew; Patel, Rushi S.; Deming, Natasha; Bhattacharyya, Indraneel; Shah, Priya; Lamont, Richard J.; Stewart, Carol M.; Cohen, Donald M.; Chan, Edward K.L.

    2010-01-01

    Recent reports have demonstrated that Dicer, an RNase III endonuclease required for microRNA (miRNA) maturation, is aberrantly expressed in different types of cancer. Furthermore, Dicer has been reported to be regulated by the let-7 family of miRNA genes. We hypothesize that Dicer is aberrantly expressed in oral cancer cells due to altered expressions of let-7, and that Dicer contributes to the development and progression of the disease. Western blot examination of Dicer protein levels in four head and neck squamous cell carcinoma (HNSCC) cell lines, including two oral cancer cell lines, demonstrated that Dicer had between 4 to 24 fold higher expression levels when compared to normal human primary gingival epithelial cells. Furthermore, five of six oral cancer tissues analyzed by indirect immunofluorescence had increased Dicer protein expression, compared to normal gingival epithelial tissue. The Dicer mRNA levels were not found to correlate well with protein expression in the HNSCC cell lines, suggesting that Dicer protein expression was post-transcriptionally regulated. Analysis of let-7a and let-7b levels in HNSCC cell lines by real-time PCR demonstrated that let-7b, but not let-7a, was significantly reduced in the HNSCC cell lines compared to control cells. Lastly, transfection of oral cancer cells with chemically synthesized let-7b and small interfering RNAs targeting Dicer significantly inhibited cell proliferation up to 83% and >100%, respectively, as early as three days post-transfection. Together, these data demonstrate that elevated expression levels of Dicer in oral cancer cells correlate with down-regulation of let-7b and increased cell proliferation. PMID:20232482

  2. Increased Serine-Arginine (SR) Protein Phosphorylation Changes Pre-mRNA Splicing in Hypoxia*

    PubMed Central

    Jakubauskiene, Egle; Vilys, Laurynas; Makino, Yuichi; Poellinger, Lorenz; Kanopka, Arvydas

    2015-01-01

    The removal of introns from mRNA precursors (pre-mRNAs) is an essential step in eukaryotic gene expression. The splicing machinery heavily contributes to biological complexity and especially to the ability of cells to adapt to altered cellular conditions. Inhibitory PAS domain protein (IPAS), a dominant negative regulator of hypoxia-inducible gene expression, is generated from hypoxia inducible transcription factor-3α (HIF-3α) pre-mRNA by an alternative splicing mechanism. Inactivation of the IPAS transcript in mice leads to the neo-vascularization of the cornea, suggesting that IPAS is an important regulator of anti-angiogenesis in this tissue. For the first time we demonstrate that serine-arginine (SR) proteins are involved in oxygen tension-dependent changes in pre-mRNA splicing. SR proteins isolated from hypoxic cells differentially interact with RNA (compared with proteins isolated from cells cultured under normoxic conditions). They possess the differential ability to activate hypoxia-dependent splice sites, and they are more phosphorylated than those isolated from normoxic HeLa cells. We also show that expression of SR protein kinases (CLK1, SRPK1, SRPK2) in hypoxic cells is elevated at mRNA and protein levels. Increased expression of CLK1 kinase is regulated by HIFs. Reduction of CLK1 cellular expression levels reduces hypoxia-dependent full-length carbonic anhydrase IX (CAIX) mRNA and CAIX protein formation and changes hypoxia-dependent cysteine-rich angiogenic inducer 61 (Cyr61) mRNA isoform formation profiles. PMID:26023237

  3. Prolactin increases hepatic Na+/taurocholate co-transport activity and messenger RNA post partum.

    PubMed Central

    Ganguly, T C; Liu, Y; Hyde, J F; Hagenbuch, B; Meier, P J; Vore, M

    1994-01-01

    We have shown that Na+/taurocholate co-transport activity is decreased in pregnancy, but rebounds post partum relative to non-pregnant controls, and that activity can be increased by treatment with ovine prolactin [Ganguly, Hyde and Vore (1993) J. Pharmacol. Exp. Ther. 267, 82-87]. To determine the basis for these effects, Na+/taurocholate co-transport was determined in purified basolateral liver plasma-membrane (bLPM) vesicles and compared with steady-state mRNA levels encoding the Na+/taurocholate-co-transporting polypeptide (Ntcp) in non-pregnant controls, pregnant rats (19-20 days pregnant), rats post partum (48 h post partum) and rats post partum treated with bromocriptine to inhibit prolactin secretion. Na+/taurocholate co-transport activity (nmol/5 s per mg of protein) in bLPM was decreased from 10.4 +/- 1.8 in non-pregnant controls to 7.9 +/- 0.6 in bLPM in pregnant rats, but rebounded to 17.5 +/- 1.3 post partum; treatment of rats post partum with bromocriptine to inhibit prolactin secretion decreased activity to 14.1 +/- 0.9. Northern and slot-blot analyses revealed similar changes in mRNA for Ntcp, so that a positive correlation was observed between Na+/taurocholate co-transport activity and Ntcp mRNA. Furthermore, treatment of ovariectomized rats with ovine prolactin increased Ntcp mRNA 10-fold compared with solvent-treated controls, consistent with the 2-fold increase in Vmax, for Na+/taurocholate co-transport in isolated hepatocytes. These data are the first to demonstrate endogenous physiological regulation by prolactin of Ntcp mRNA in parallel with Na+/taurocholate co-transport activity. Images Figure 2 PMID:7945260

  4. Increased N6-methyladenosine in Human Sperm RNA as a Risk Factor for Asthenozoospermia.

    PubMed

    Yang, Ying; Huang, Wei; Huang, Jing-Tao; Shen, Fan; Xiong, Jun; Yuan, Er-Feng; Qin, Shan-shan; Zhang, Ming; Feng, Yu-Qi; Yuan, Bi-Feng; Liu, Song-Mei

    2016-04-13

    Male infertility is a worldwide medical problem. Asthenozoospermia is a common cause of infertility. Epigenetic modifications of DNA and histones have been shown to influence human infertility, but no research has explored whether N(6)-methyladenosine (m(6)A) level in RNA is associated with asthenozoospermia. Here, we collected a total of 52 semen samples, including 20 asthenozoospermia patients and 32 healthy controls. An LC-ESI-MS/MS method was used to detect m(6)A contents in sperm RNA, and real-time PCR was performed to determine the mRNA expression of demethylase (FTO, ALKBH5), methyltransferase (METTL3, METTL14, WTAP) and an m(6)A-selective-binding protein (YTHDF2). We found that m(6)A content (p = 0.033) and the mRNA expression of METTL3 (p = 0.016) and METTL14 (p = 0.025) in asthenozoospermia patients were significantly higher than those of controls. Increased m(6)A content was a risk factor for asthenozoospermia (odds ratio (OR) 3.229, 95% confidence interval (CI) 1.178 - 8.853, p = 0.023). Moreover, m(6)A content was correlated with the expression of METTL3 (r = 0.303, p = 0.032) and with sperm motility (progressive motility: r = -0.288, p = 0.038; non-progressive motility: r = -0.293, p = 0.037; immotility: r = 0.387, p = 0.005). Our data suggest that increased m(6)A content is a risk factor for asthenozoospermia and affects sperm motility. Methyltransferases, particularly METTL3, play key roles in increasing m(6)A contents in sperm RNA.

  5. Increased N6-methyladenosine in Human Sperm RNA as a Risk Factor for Asthenozoospermia

    PubMed Central

    Yang, Ying; Huang, Wei; Huang, Jing-Tao; Shen, Fan; Xiong, Jun; Yuan, Er-Feng; Qin, Shan-shan; Zhang, Ming; Feng, Yu-Qi; Yuan, Bi-Feng; Liu, Song-Mei

    2016-01-01

    Male infertility is a worldwide medical problem. Asthenozoospermia is a common cause of infertility. Epigenetic modifications of DNA and histones have been shown to influence human infertility, but no research has explored whether N6-methyladenosine (m6A) level in RNA is associated with asthenozoospermia. Here, we collected a total of 52 semen samples, including 20 asthenozoospermia patients and 32 healthy controls. An LC-ESI-MS/MS method was used to detect m6A contents in sperm RNA, and real-time PCR was performed to determine the mRNA expression of demethylase (FTO, ALKBH5), methyltransferase (METTL3, METTL14, WTAP) and an m6A-selective-binding protein (YTHDF2). We found that m6A content (p = 0.033) and the mRNA expression of METTL3 (p = 0.016) and METTL14 (p = 0.025) in asthenozoospermia patients were significantly higher than those of controls. Increased m6A content was a risk factor for asthenozoospermia (odds ratio (OR) 3.229, 95% confidence interval (CI) 1.178 – 8.853, p = 0.023). Moreover, m6A content was correlated with the expression of METTL3 (r = 0.303, p = 0.032) and with sperm motility (progressive motility: r = −0.288, p = 0.038; non-progressive motility: r = −0.293, p = 0.037; immotility: r = 0.387, p = 0.005). Our data suggest that increased m6A content is a risk factor for asthenozoospermia and affects sperm motility. Methyltransferases, particularly METTL3, play key roles in increasing m6A contents in sperm RNA. PMID:27072590

  6. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  7. Increased levels of rat liver RNA polymerase I(A) and I(B) following the administration of triiodothyronine.

    PubMed

    Zoncheddu, A; Accomando, R; Pertica, M; Carlini, A; Orunesu, M

    1981-06-15

    The levels of the transcribing RNA polymerase I(B) in the nucleus and of the non-transcribing RNA polymerase I(A) in the cytoplasm are both approximately doubled 24 h after a single i.p. injection of triiodothyronine into thyroidectomized rats. This suggests that the triiodothyronine-induced stimulation of ribosomal RNA synthesis is associated with an increase in the total RNA polymerase I content of rat liver cells.

  8. Feeding and insulin increase leptin translation. Importance of the leptin mRNA untranslated regions.

    PubMed

    Lee, Mi-Jeong; Yang, Rong-Ze; Gong, Da-Wei; Fried, Susan K

    2007-01-05

    The post-transcriptional mechanisms by which feeding and insulin increase leptin production are poorly understood. Starvation of 6-7-week-old rats for 14 h decreased leptin mRNA level by only 22% but decreased plasma levels, adipose tissue leptin content, and release by over 75%. The decreased leptin with starvation was explained by >85% decrease in relative rates of leptin biosynthesis measured by metabolic labeling and immunoprecipitation. In vitro insulin treatment of adipose tissue from fed or starved rats for 2 h increased relative rates of leptin biosynthesis by 2-3-fold, and the effect was blocked by inhibition of phosphatidylinositol 3-kinase or mammalian target of rapamycin. Consistent with the hypothesis that feeding/insulin increases leptin translation, more leptin mRNA was associated with polysomes in adipose tissue of fed than starved rats, and in vitro incubation of adipose tissue of starved rats with insulin shifted leptin mRNA into polysomes. To assess the mechanisms regulating leptin translation, chimeric human leptin untranslated region (UTR) reporter constructs were transiently transfected into differentiated 3T3-L1 adipocytes. The 5'-UTR of leptin mRNA increased luciferase reporter activity 2-3-fold, whereas the full-length 3'-UTR (nucleotides 1-2804) was inhibitory (-65%). Sequences between nucleotides 462 and 1130 of the leptin 3'-UTR conferred most of the inhibitory effect. Insulin stimulated the expression of constructs that included both the full-length 5'-UTR and the inhibitory 3'-UTR, and the effect was blocked by inhibition of phosphatidylinositol 3-kinase or mammalian target of rapamycin. Our data suggest that insulin derepresses leptin translation by a mechanism that requires both the 5'-UTR and the 3'-UTR and may contribute to the increase in leptin production with feeding.

  9. Increased level of H19 long noncoding RNA promotes invasion, angiogenesis, and stemness of glioblastoma cells.

    PubMed

    Jiang, Xiaochun; Yan, Yukui; Hu, Minghua; Chen, Xiande; Wang, Yaxian; Dai, Yi; Wu, Degang; Wang, Yongsheng; Zhuang, Zhixiang; Xia, Hongping

    2016-01-01

    OBJECT Increased levels of H19 long noncoding RNA (lncRNA) have been observed in many cancers, suggesting that overexpression of H19 may be important in the development of carcinogenesis. However, the role of H19 in human glioblastoma is still unclear. The object of this study was to examine the level of H19 in glioblastoma samples and investigate the role of H19 in glioblastoma carcinogenesis. METHODS Glioblastoma and nontumor brain tissue specimens were obtained from tissue obtained during tumor resection in 30 patients with glioblastoma. The level of H19 lncRNA was detected by real-time quantitative reverse transcription polymerase chain reaction. The role of H19 in invasion, angiogenesis, and stemness of glioblastoma cells was then investigated using commercially produced cell lines (U87 and U373). The effects of H19 overexpression on glioblastoma cell invasion and angiogenesis were detected by in vitro Matrigel invasion and endothelial tube formation assay. The effects of H19 on glioblastoma cell stemness and tumorigenicity were investigated by neurosphere formation and an in vivo murine xenograft model. RESULTS The authors found that H19 is significantly overexpressed in glioblastoma tissues, and the level of expression was associated with patient survival. In the subsequent investigations, the authors found that overexpression of H19 promotes glioblastoma cell invasion and angiogenesis in vitro. Interestingly, H19 was also significantly overexpressed in CD133(+) glioblastoma cells, and overexpression of H19 was associated with increased neurosphere formation of glioblastoma cells. Finally, stable overexpression of H19 was associated with increased tumor growth in the murine xenograft model. CONCLUSIONS The results of this study suggest that increased expression of H19 lncRNA promotes invasion, angiogenesis, stemness, and tumorigenicity of glioblastoma cells. Taken together, these findings indicate that H19 plays an important role in tumorigenicity and

  10. Hypotensive but not normotensive haemorrhage increases tryptophan hydroxylase-2 mRNA in caudal midline medulla.

    PubMed

    Brown, Heidi J; Henderson, Luke A; Keay, Kevin A

    2006-05-08

    Severe blood loss triggers shock, a precipitous hypotension and bradycardia. The integrity of (i) neurons in the vasodepressor region of the caudal midline medulla and (ii) central 5-HT neurotransmission are critical for the expression of haemorrhagic shock. This study investigated whether progressive blood loss triggers altered synthesis of 5-HT in the vasodepressor region of the caudal midline medulla by measuring changes in relative expression levels of tryptophan hydroxylase 2 (TpH 2) mRNA, the rate-limiting enzyme in the synthesis of neuronal 5-HT. Hypotensive but not normotensive haemorrhage triggered a significant increase in TpH 2 mRNA in the vasodepressor region of the caudal midline medulla, identifying an important role for 5-HT-containing caudal midline medullary neurons in haemorrhagic shock.

  11. Acute exercise induces biphasic increase in respiratory mRNA in skeletal muscle

    SciTech Connect

    Ikeda, Shin-ichi; Kizaki, Takako; Haga, Shukoh; Ohno, Hideki; Takemasa, Tohru

    2008-04-04

    Peroxisome proliferator-activated receptor {gamma} coactivator-1{alpha} (PGC-1{alpha}) promotes the expression of oxidative enzymes in skeletal muscle. We hypothesized that activation of the p38 MAPK (mitogen-activated protein kinase) in response to exercise was associated with exercise-induced PGC-1{alpha} and respiratory enzymes expression and aimed to demonstrate this under the physiological level. We subjected mice to a single bout of treadmill running and found that the exercise induced a biphasic increase in the expression of respiratory enzymes mRNA. The second phase of the increase was accompanied by an increase in PGC-1{alpha} protein, but the other was not. Administration of SB203580 (SB), an inhibitor of p38 MAPK, suppressed the increase in PGC-1{alpha} expression and respiratory enzymes mRNA in both phases. These data suggest that p38 MAPK is associated with the exercise-induced expression of PGC-1{alpha} and biphasic increase in respiratory enzyme mRNAs in mouse skeletal muscle under physiological conditions.

  12. Oct-2 forms a complex with Oct-1 on the iNOS promoter and represses transcription by interfering with recruitment of RNA PolII by Oct-1

    PubMed Central

    Bentrari, Fatima; Chantôme, Aurelie; Knights, Andrew; Jeannin, Jean-François; Pance, Alena

    2015-01-01

    Oct-1 (POU2f1) and Oct-2 (POU2f2) are members of the POU family of transcription factors. They recognize the same DNA sequence but fulfil distinct functions: Oct-1 is ubiquitous and regulates a variety of genes while Oct-2 is restricted to B-cells and neurones. Here we examine the interplay and regulatory mechanisms of these factors to control the inducible nitric oxide synthase (iNOS, NOS2). Using two breast cancer cell lines as a comparative model, we found that MCF-7 express iNOS upon cytokine stimulation while MDA-MB-231 do not. Oct-1 is present in both cell lines but MDA-MB-231also express high levels of Oct-2. Manipulation of Oct-2 expression in these cell lines demonstrates that it is directly responsible for the repression of iNOS in MDA-MB-231. In MCF-7 cells Oct-1 binds the iNOS promoter, recruits RNA PolII and triggers initiation of transcription. In MDA-MB-231 cells, both Oct-1 and Oct-2 bind the iNOS promoter, forming a higher-order complex which fails to recruit RNA PolII, and as a consequence iNOS transcription does not proceed. Unravelling the mechanisms of transcription factor activity is paramount to the understanding of gene expression patterns that determine cell behaviour. PMID:26271992

  13. Expression of the RNA-binding protein TIAR is increased in neurons after ischemic cerebral injury.

    PubMed

    Jin, K; Li, W; Nagayama, T; He, X; Sinor, A D; Chang, J; Mao, X; Graham, S H; Simon, R P; Greenberg, D A

    2000-03-15

    T-cell restricted intracellular antigen-related protein (TIAR) is an RNA recognition motif-type RNA-binding protein that has been implicated in the apoptotic death of T-lymphocytes and retinal pigment epithelial cells. Western blots prepared with a monoclonal antibody against TIAR showed expression in normal rat hippocampus, and induction by 15 min of global cerebral ischemia. This increased expression was evident at 8 hr after ischemia and maximal at 24 hr, whereas expression at 72 hr was reduced below basal levels. Expression of TIAR protein was also increased in parietal cortex 6 and 24 hr after 90 min of focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion, as well as in cultured cortical neurons and astroglia after exposure to hypoxia in vitro. Immunocytochemistry showed that increased expression of TIAR occurred mainly in the CA1 sector of hippocampus 24 hr after global ischemia, and in cortical and striatal neurons 24 hr after 20 or 90 min of focal ischemia. Double-labeling studies showed that TIAR protein expression was co-localized with DNA damage in neuronal cells. The findings suggest that TIAR may be involved in neuronal cell death after cerebral ischemic injury.

  14. Interfering ribonucleic acids that suppress expression of multiple unrelated genes

    PubMed Central

    Passioura, Toby; Gozar, Mary M; Goodchild, Amber; King, Andrew; Arndt, Greg M; Poidinger, Michael; Birkett, Donald J; Rivory, Laurent P

    2009-01-01

    Background Short interfering RNAs (siRNAs) have become the research tool of choice for gene suppression, with human clinical trials ongoing. The emphasis so far in siRNA therapeutics has been the design of one siRNA with complete complementarity to the intended target. However, there is a need for multi-targeting interfering RNA in diseases in which multiple gene products are of importance. We have investigated the possibility of using a single short synthetic duplex RNA to suppress the expression of VEGF-A and ICAM-1; genes implicated in the progression of ocular neovascular diseases such as diabetic retinopathy. Results Duplex RNA were designed to have incomplete complementarity with the 3'UTR sequences of both target genes. One such duplex, CODEMIR-1, was found to suppress VEGF and ICAM-1 by 90 and 60%, respectively in ARPE-19 cells at a transfected concentration of 40 ng/mL. Use of a cyan fusion reporter with target sites constructed in its 3'UTR demonstrated that the repression of VEGF and ICAM-1 by CODEMIR-1 was indeed due to interaction with the target sequence. An exhaustive analysis of sequence variants of CODEMIR-1 demonstrated a clear positive correlation between activity against VEGF (but not ICAM-1) and the length of the contiguous complementary region (from the 5' end of the guide strand). Various strategies, including the use of inosine bases at the sites of divergence of the target sequences were investigated. Conclusion Our work demonstrates the possibility of designing multitargeting dsRNA to suppress more than one disease-altering gene. This warrants further investigation as a possible therapeutic approach. PMID:19531249

  15. Spinal muscular atrophy phenotype is ameliorated in human motor neurons by SMN increase via different novel RNA therapeutic approaches.

    PubMed

    Nizzardo, Monica; Simone, Chiara; Dametti, Sara; Salani, Sabrina; Ulzi, Gianna; Pagliarani, Serena; Rizzo, Federica; Frattini, Emanuele; Pagani, Franco; Bresolin, Nereo; Comi, Giacomo; Corti, Stefania

    2015-06-30

    Spinal muscular atrophy (SMA) is a primary genetic cause of infant mortality due to mutations in the Survival Motor Neuron (SMN) 1 gene. No cure is available. Antisense oligonucleotides (ASOs) aimed at increasing SMN levels from the paralogous SMN2 gene represent a possible therapeutic strategy. Here, we tested in SMA human induced pluripotent stem cells (iPSCs) and iPSC-differentiated motor neurons, three different RNA approaches based on morpholino antisense targeting of the ISSN-1, exon-specific U1 small nuclear RNA (ExSpeU1), and Transcription Activator-Like Effector-Transcription Factor (TALE-TF). All strategies act modulating SMN2 RNA: ASO affects exon 7 splicing, TALE-TF increase SMN2 RNA acting on the promoter, while ExSpeU1 improves pre-mRNA processing. These approaches induced up-regulation of full-length SMN mRNA and differentially affected the Delta-7 isoform: ASO reduced this isoform, while ExSpeU1 and TALE-TF increased it. All approaches upregulate the SMN protein and significantly improve the in vitro SMA motor neurons survival. Thus, these findings demonstrate that therapeutic tools that act on SMN2 RNA are able to rescue the SMA disease phenotype. Our data confirm the feasibility of SMA iPSCs as in vitro disease models and we propose novel RNA approaches as potential therapeutic strategies for treating SMA and other genetic neurological disorders.

  16. Delivery of Small Interfering RNAs to Cells via Exosomes.

    PubMed

    Wahlgren, Jessica; Statello, Luisa; Skogberg, Gabriel; Telemo, Esbjörn; Valadi, Hadi

    2016-01-01

    Exosomes are small membrane bound vesicles between 30 and 100 nm in diameter of endocytic origin that are secreted into the extracellular environment by many different cell types. Exosomes play a role in intercellular communication by transferring proteins, lipids, and RNAs to recipient cells.Exosomes from human cells could be used as vectors to provide cells with therapeutic RNAs. Here we describe how exogenous small interfering RNAs may successfully be introduced into various kinds of human exosomes using electroporation and subsequently delivered to recipient cells. Methods used to confirm the presence of siRNA inside exosomes and cells are presented, such as flow cytometry, confocal microscopy, and Northern blot.

  17. Increased expression and localization of the RNA-binding protein HuD and GAP-43 mRNA to cytoplasmic granules in DRG neurons during nerve regeneration.

    PubMed

    Anderson, K D; Merhege, M A; Morin, M; Bolognani, F; Perrone-Bizzozero, N I

    2003-09-01

    The neuronal-specific RNA-binding protein, HuD, binds to a U-rich regulatory element of the 3' untranslated region (3' UTR) of the GAP-43 mRNA and delays the onset of its degradation. We have recently shown that overexpression of HuD in embryonic rat cortical cells accelerated the time course of normal neurite outgrowth and resulted in a twofold increase in GAP-43 mRNA levels. Given this evidence, we sought to investigate the involvement of HuD during nerve regeneration. It is known that HuD protein and GAP-43 mRNA are expressed in the dorsal root ganglia (DRG) of adult rat and that GAP-43 is upregulated in DRG neurons during regeneration. In this study, we examined the expression patterns and levels of HuD and GAP-43 mRNA in DRG neurons following sciatic nerve injury using a combination of in situ hybridization, immunocytochemistry, and quantitative RT-PCR. GAP-43 and HuD expression increased in the ipsilateral DRG during the first 3 weeks of regeneration, with peak values seen at 7 days postcrush. At this time point, the levels of HuD and GAP-43 mRNAs in the ipsilateral DRG increased by twofold and sixfold, respectively, relative to the contralateral DRG. Not only were the temporal patterns of expression of HuD protein and GAP-43 mRNA similar, but also they were found to colocalize in the cytoplasm of DRG neurons. Moreover, both molecules were distributed in cytoplasmic granules containing ribosomal RNA. In conclusion, our results suggest that HuD is involved in the upregulation of GAP-43 expression observed at early stages of peripheral nerve regeneration.

  18. Permissive effect of dexamethasone on the increase of proenkephalin mRNA induced by depolarization of chromaffin cells

    SciTech Connect

    Naranjo, J.R.; Mocchetti, I.; Schwartz, J.P.; Costa, E.

    1986-03-01

    In cultured bovine chromaffin cells, changes in the dynamic state of enkephalin stores elicited experimentally were studied by measuring cellular proenkephalin mRNA, as well as enkephalin precursors and authentic enkephalin content of cells and culture media. In parallel, tyrosine hydroxylase mRNA and catecholamine cell content were also determined. Low concentrations (0.5-100 pM) of dexamethasone increased the cell contents of proenkephalin mRNA and enkephalin-containing peptides. High concentrations of the hormone(1 ..mu..M) were required to increase the cell contents of tyrosine hydroxylase mRNA and catecholamines. Depolarization of the cells with 10 ..mu..M veratridine resulted in a depletion of enkephalin and catecholamine stores after 24 hr. The enkephalin, but not the catecholamine, content was restored by 48 hr. An increase in proenkephalin mRNA content might account for the recovery; this increase was curtailed by tetrodotoxin and enhanced by 10 pM dexamethasone. Tyrosine hydroxylase mRNA content was not significantly modified by depolarization, even in the presence of 1 ..mu..M dexamethasone. Aldosterone, progesterone, testosterone, or estradiol (1 ..mu..M) failed to change proenkephalin mRNA. Hence, dexamethasone appears to exert a specific permissive action on the stimulation of the proenkephalin gene elicited by depolarization. Though the catecholamines and enkephalins are localized in the same chromaffin granules and are coreleased by depolarization, the genes coding for the processes that are rate limiting in the production of these neuromodulators can be differentially regulated.

  19. Positive AMPA receptor modulation rapidly stimulates BDNF release and increases dendritic mRNA translation.

    PubMed

    Jourdi, Hussam; Hsu, Yu-Tien; Zhou, Miou; Qin, Qingyu; Bi, Xiaoning; Baudry, Michel

    2009-07-08

    Brain-derived neurotrophic factor (BDNF) stimulates local dendritic mRNA translation and is involved in formation and consolidation of memory. 2H,3H,6aH-pyrrolidino[2'',1''-3',2']1,3-oxazino[6',5'-5,4]-benzo[e]1,4-dioxan-10-one (CX614), one of the best-studied positive AMPA receptor modulators (also known as ampakines), increases BDNF mRNA and protein and facilitates long-term potentiation (LTP) induction. Several other ampakines also improve performance in various behavioral and learning tasks. Since local dendritic protein synthesis has been implicated in LTP stabilization and in memory consolidation, this study investigated whether CX614 could influence synaptic plasticity by upregulating dendritic protein translation. CX614 treatment of primary neuronal cultures and acute hippocampal slices rapidly activated the translation machinery and increased local dendritic protein synthesis. CX614-induced activation of translation was blocked by K252a [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester], CNQX, APV, and TTX, and was inhibited in the presence of an extracellular BDNF scavenger, TrkB-Fc. The acute effect of CX614 on translation was mediated by increased BDNF release as demonstrated with a BDNF scavenging assay using TrkB-Fc during CX614 treatment of cultured primary neurons and was blocked by nifedipine, ryanodine, and lack of extracellular Ca(2+) in acute hippocampal slices. Finally, CX614, like BDNF, rapidly increased dendritic translation of an exogenous translation reporter. Together, our results demonstrate that positive modulation of AMPA receptors rapidly stimulates dendritic translation, an effect mediated by BDNF secretion and TrkB receptor activation. They also suggest that increased BDNF secretion and stimulation of local protein synthesis contribute to the effects of ampakines on synaptic plasticity.

  20. Alkane-modified short polyethyleneimine for siRNA delivery.

    PubMed

    Schroeder, Avi; Dahlman, James E; Sahay, Gaurav; Love, Kevin T; Jiang, Shan; Eltoukhy, Ahmed A; Levins, Christopher G; Wang, Yingxia; Anderson, Daniel G

    2012-06-10

    RNA interference (RNAi) is a highly specific gene-silencing mechanism triggered by small interfering RNA (siRNA). Effective intracellular delivery requires the development of potent siRNA carriers. Here, we describe the synthesis and screening of a series of siRNA delivery materials. Short polyethyleneimine (PEI, Mw 600) was selected as a cationic backbone to which lipid tails were conjugated at various levels of saturation. In solution these polymer-lipid hybrids self-assemble to form nanoparticles capable of complexing siRNA. The complexes silence genes specifically and with low cytotoxicity. The efficiency of gene knockdown increased as the number of lipid tails conjugated to the PEI backbone increased. This is explained by reducing the binding affinity between the siRNA strands to the complex, thereby enabling siRNA release after cellular internalization. These results highlight the importance of complexation strength when designing siRNA delivery materials.

  1. Short interfering RNA directed against the GOLPH3 gene enhances the effect of chemotherapy against oral squamous cell carcinoma by regulating Caspase3, Bcl2 and cytochrome-c expression

    PubMed Central

    Cui, Guo-Hui; Li, Wen-Xin; Lin, Zhi-Yong; Zhang, Wei-Qun; Hu, Zhao-Hui; Wang, Li

    2015-01-01

    Growing evidence reported that Golgi phosphoprotein 3 (GOLPH3) was involved in the progression of several human cancers. To determine whether knockout of GOLPH3 enhances the effect of Chemotherapy against cell growth of oral squamous cell carcinoma in vitro. OSCC cells were transfected with Golph3 plasmid, Golph3-RNAi and the relative control plasmids. Transfected Tca-8113 cells treated with cis-Dichlorodiamineplatinum (DDP; 0, 0.05, 0.25, 1.25, 6.25 and 31.25 ug/ml) or Paclitaxe (0, 2, 10, 50, 250 and 1250 nM) or Adriamycin (0, 0.25, 0.5, 1, 2 and 4 ug/ml) for 24 h, respectively, was determined using MTT assay. Apoptosis-related protein expression Cytochrome-C, Caspase3 and Bcl-2 was analyzed by RT-PCR and western blots. Result of MTT showed that Golph3-RNAi transfected Tca-8113 cells enhanced the effect of chemotherapy, and the effect was strengthened with the increasing concentration of drugs, and the Golph3 plasmid transfected Tca-8113 cells showed the opposite effect. RT-PCR and western blots assays revealed that expression of cytochrome-C and caspase3 were up-regulated, while Bcl-2 expression was down-regulated in Golph3-RNAi transfected Tca-8113 cells. Taken together, this study demonstrated that GOLPH3 had potent pro-tumor growth and decreased the effect of Chemotherapy, and its mechanism is primarily via cell anti-apoptosis, down-regulating the expression of cytochrome-C and caspase3, up-regulating Bcl-2 expression. PMID:26550222

  2. Lack of the Delta Subunit of RNA Polymerase Increases Virulence Related Traits of Streptococcus mutans

    PubMed Central

    Xue, Xiaoli; Sztajer, Helena; Buddruhs, Nora; Petersen, Jörn; Rohde, Manfred; Talay, Susanne R.; Wagner-Döbler, Irene

    2011-01-01

    The delta subunit of the RNA polymerase, RpoE, maintains the transcriptional specificity in Gram-positive bacteria. Lack of RpoE results in massive changes in the transcriptome of the human dental caries pathogen Streptococcus mutans. In this study, we analyzed traits of the ΔrpoE mutant which are important for biofilm formation and interaction with oral microorganisms and human cells and performed a global phenotypic analysis of its physiological functions. The ΔrpoE mutant showed higher self-aggregation compared to the wild type and coaggregated with other oral bacteria and Candida albicans. It formed a biofilm with a different matrix structure and an altered surface attachment. The amount of the cell surface antigens I/II SpaP and the glucosyltransferase GtfB was reduced. The ΔrpoE mutant displayed significantly stronger adhesion to human extracellular matrix components, especially to fibronectin, than the wild type. Its adhesion to human epithelial cells HEp-2 was reduced, probably due to the highly aggregated cell mass. The analysis of 1248 physiological traits using phenotype microarrays showed that the ΔrpoE mutant metabolized a wider spectrum of carbon sources than the wild type and had acquired resistance to antibiotics and inhibitory compounds with various modes of action. The reduced antigenicity, increased aggregation, adherence to fibronection, broader substrate spectrum and increased resistance to antibiotics of the ΔrpoE mutant reveal the physiological potential of S. mutans and show that some of its virulence related traits are increased. PMID:21625504

  3. Elevated CXCL1 increases hepatocellular carcinoma aggressiveness and is inhibited by miRNA-200a

    PubMed Central

    Gao, Jie; Gao, Peng-Ji; Ni, Yan-bing; Zhu, Ji-Ye

    2016-01-01

    In this study, we investigated the value of measurement of the chemokine CXCL1 in clinical management of hepatocellular carcinoma (HCC) and its possible role in the molecular pathogenesis of HCC. High CXCL1 expression predicted recurrence in HCC patients and promoted tumor progression in both in vivo and in vitro experimental systems. Overexpression of CXCL1 increased mitochondrial metabolism and activated the epithelial-to-mesenchymal transition (EMT). Using computational analysis we identified the microRNA miR-200a as a putative post-transcriptional regulator of CXCL1. We found that levels of miR-200a were inversely correlated with CXCL1 expression in HCC patient tissue samples by northern blot and qRT-PCR. Furthermore, CXCL1 was identified as a direct target which was bound and inhibited by miR- 200a. These findings provide new insights into the role of CXCL1 in HCC and its post-transcriptional regulation and suggest it may be a prognostic indicator for poor outcomes and a potential target for therapy. PMID:27542259

  4. Increased myogenic repressor Id mRNA and protein levels in hindlimb muscles of aged rats.

    PubMed

    Alway, Stephen E; Degens, Hans; Lowe, Dawn A; Krishnamurthy, Gururaj

    2002-02-01

    The objective of this study was to determine if levels of repressors to myogenic regulatory factors (MRFs) differ between muscles from young adult and aged animals. Total RNA from plantaris, gastrocnemius, and soleus muscles of Fischer 344 x Brown Norway rats aged 9 mo (young adult, n = 10) and 37 mo (aged, n = 10) was reverse transcribed and then amplified by PCR. To obtain a semiquantitative measure of the mRNA levels, PCR signals were normalized to cyclophilin or 18S signals from the corresponding reverse transcription product. Normalization to cyclophilin and 18S gave similar results. The mRNA levels of MyoD and myogenin were approximately 275-650% (P < 0.001) and approximately 500-1,100% (P < 0.001) greater, respectively, in muscles from aged compared with young adults. In contrast, the protein levels were lower in plantaris and gastrocnemius muscles and similar in the soleus muscle of aged vs. young adult rats. Id repressor mRNA levels were approximately 300-900% greater in fast and slow muscles of aged animals (P < or = 0.02), and Mist 1 mRNA was approximately 50% greater in the plantaris and gastrocnemius muscles (P < 0.01). The mRNA level of Twist mRNA was not significantly affected by aging. Id-1, Id-2, and Id-3 protein levels were approximately 17-740% greater (P < 0.05) in hindlimb muscles of aged rats compared with young adult rats. The elevated levels of Id mRNA and protein suggest that MRF repressors may play a role in gene regulation of fast and slow muscles in aged rats.

  5. Cell homeostasis in a Leishmania major mutant overexpressing the spliced leader RNA is maintained by an increased proteolytic activity.

    PubMed

    Toledo, Juliano S; Ferreira, Tiago R; Defina, Tânia P A; Dossin, Fernando de M; Beattie, Kenneth A; Lamont, Douglas J; Cloutier, Serge; Papadopoulou, Barbara; Schenkman, Sergio; Cruz, Angela K

    2010-10-01

    Although several stage-specific genes have been identified in Leishmania, the molecular mechanisms governing developmental gene regulation in this organism are still not well understood. We have previously reported an attenuation of virulence in Leishmania major and L. braziliensis carrying extra-copies of the spliced leader RNA gene. Here, we surveyed the major differences in proteome and transcript expression profiles between the spliced leader RNA overexpressor and control lines using two-dimensional gel electrophoresis and differential display reverse transcription PCR, respectively. Thirty-nine genes related to stress response, cytoskeleton, proteolysis, cell cycle control and proliferation, energy generation, gene transcription, RNA processing and post-transcriptional regulation have abnormal patterns of expression in the spliced leader RNA overexpressor line. The evaluation of proteolytic pathways in the mutant revealed a selective increase of cysteine protease activity and an exacerbated ubiquitin-labeled protein population. Polysome profile analysis and measurement of cellular protein aggregates showed that protein translation in the spliced leader RNA overexpressor line is increased when compared to the control line. We found that L. major promastigotes maintain homeostasis in culture when challenged with a metabolic imbalance generated by spliced leader RNA surplus through modulation of intracellular proteolysis. However, this might interfere with a fine-tuned gene expression control necessary for the amastigote multiplication in the mammalian host.

  6. Dexamethasone increases growth hormone (GH)-releasing hormone (GRH) receptor mRNA levels in cultured rat anterior pituitary cells.

    PubMed

    Tamaki, M; Sato, M; Matsubara, S; Wada, Y; Takahara, J

    1996-06-01

    To examine the effects of glucocorticoid (GC) on growth hormone (GH)-releasing hormone (GRH) receptor gene expression, a highly-sensitive and quantitative reverse-transcribed polymerase chain reaction (RT-PCR) method was used in this study. Rat anterior pituitary cells were isolated and cultured for 4 days. The cultured cells were treated with dexamethasone for 2, 6, and 24 h. GRH receptor mRNA levels were determined by competitive RT-PCR using a recombinant RNA as the competitor. Dexamethasone significantly increased GRH receptor mRNA levels at 5 nM after 6- and 24 h-incubations, and the maximal effect was found at 25 nM. The GC receptor-specific antagonist, RU 38486 completely eliminated the dexamethasone-induced enhancement of GRH receptor mRNA levels. Dexamethasone did not alter the mRNA levels of beta-actin and prolactin at 5 nM for 24 h, whereas GH mRNA levels were significantly increased by the same treatment. The GH response to GRH was significantly enhanced by the 24-h incubation with 5 nM dexamethasone. These findings suggest that GC stimulates GRH receptor gene expression through the ligand-activated GC receptors in the rat somatotrophs. The direct effects of GC on the GRH receptor gene could explain the enhancement of GRH-induced GH secretion.

  7. Hypocalcemia increases and hypercalcemia decreases the steady-state level of parathyroid hormone messenger RNA in the rat.

    PubMed Central

    Yamamoto, M; Igarashi, T; Muramatsu, M; Fukagawa, M; Motokura, T; Ogata, E

    1989-01-01

    To examine the effects of serum calcium concentrations on PTH biosynthesis, rats were made hyper- (serum total calcium, approximately 3.5 mM) or hypocalcemic (approximately 1.25 mM) and steady-state levels of PTH mRNA in parathyroid cells were measured by the primer extension method using a 32P-labeled synthetic oligomer. PTH mRNA levels increased about twofold in the rats made slightly hypocalcemic by infusion of calcium-free solution and decreased slightly in those made hypercalcemic by CaCl2 infusion (120-150 mumol/h) compared with the levels present in nonfasting control rats. Infusion of calcitonin (0.5 U/h) or EGTA (90 mumol/h) with calcium-free solution increased PTH mRNA levels further (two- to sevenfold) above the levels present in animals infused with calcium-free solution alone. These changes in PTH mRNA levels were observed after 48- but not 24-h infusion, and there was an inverse correlation between PTH mRNA levels and serum calcium concentrations. The results suggest that changes in serum calcium concentrations in the near physiological range regulate the biosynthesis of PTH by affecting steady-state levels of PTH mRNA when hypercalcemia or hypocalcemia continues for a relatively long period. Images PMID:2493484

  8. Reduced genetic distance and high replication levels increase the RNA recombination rate of hepatitis delta virus.

    PubMed

    Lin, Chia-Chi; Yang, Zhi-Wei; Iang, Shan-Bei; Chao, Mei

    2015-01-02

    Hepatitis delta virus (HDV) replication is carried out by host RNA polymerases. Since homologous inter-genotypic RNA recombination is known to occur in HDV, possibly via a replication-dependent process, we hypothesized that the degree of sequence homology and the replication level should be related to the recombination frequency in cells co-expressing two HDV sequences. To confirm this, we separately co-transfected cells with three different pairs of HDV genomic RNAs and analyzed the obtained recombinants by RT-PCR followed by restriction fragment length polymorphism and sequencing analyses. The sequence divergence between the clones ranged from 24% to less than 0.1%, and the difference in replication levels was as high as 100-fold. As expected, significant differences were observed in the recombination frequencies, which ranged from 0.5% to 47.5%. Furthermore, varying the relative amounts of parental RNA altered the dominant recombinant species produced, suggesting that template switching occurs frequently during the synthesis of genomic HDV RNA. Taken together, these data suggest that during the host RNA polymerase-driven RNA recombination of HDV, both inter- and intra-genotypic recombination events are important in shaping the genetic diversity of HDV.

  9. Interfering with Gendered Development: A Timely Intervention

    ERIC Educational Resources Information Center

    Blaise, Mindy

    2014-01-01

    Instead of relying on colonial and Western developmental logic to understand and research gender, this paper proposes interfering as a strategy toward generating gender knowledges that are more inclusive to other-than-Western concepts and contexts. This paper shows how post-developmental perspectives interfere with psychological and biological…

  10. Increased Circulating Exosomal miRNA-223 Is Associated with Acute Ischemic Stroke

    PubMed Central

    Chen, Yajing; Song, Yaying; Huang, Jun; Qu, Meijie; Zhang, Yu; Geng, Jieli; Zhang, Zhijun; Liu, Jianrong; Yang, Guo-Yuan

    2017-01-01

    Recent studies have demonstrated that exosomal microRNAs (miRNAs) are novel biomarkers and therapeutic targets for various diseases including vascular disease. However, specific exosomal miRNAs expression in stroke patients has not been reported yet. Here, we explored whether circulating exosomal miRNAs can serve as potential biomarkers for the diagnosis of acute ischemic stroke and discussed the potential for clinical application. Blood samples were collected from acute ischemic stroke patients within the first 72 h (n = 50). Circulating exosomes were exacted by Exoquick exosome isolation kit and characterized by transmission electron microscopy. Western blot was performed to assess the expression of exosomal protein makers. Exosomal miRNA-223 (miR-223) was detected by RT-PCR assay. The relationship between the expression levels of miR-223 and National Institutes of Health Stroke Scale (NIHSS) scores, brain infarct volume, and neurological outcomes were analyzed. Circulating exosomes were isolated and the size of vesicles ranged between 30 and 100 nm. The identification of exosomes was further confirmed by the detection of specific exosomal protein markers CD9, CD63, and Tsg101. Exosomal miR-223 in acute ischemic stroke patients was significantly upregulated compared to control group (p < 0.001). Exosomal miR-223 level was positively correlated with NIHSS scores (r = 0.31, p = 0.03). Exosomal miR-223 expression in stroke patients with poor outcomes was higher than those with good outcomes (p < 0.05). Increased exosomal miR-223 was associated with acute ischemic stroke occurrence, stroke severity, and short-term outcomes. Future studies with large sample are needed to assess the clinical application of exosomal miR-223 as a novel biomarker for ischemic stroke diagnosis. PMID:28289400

  11. The number of preproghrelin mRNA expressing cells is increased in mice with activity-based anorexia.

    PubMed

    François, Marie; Barde, Swapnali; Achamrah, Najate; Breton, Jonathan; do Rego, Jean-Claude; Coëffier, Moïse; Hökfelt, Tomas; Déchelotte, Pierre; Fetissov, Sergueï O

    2015-06-01

    Plasma levels of ghrelin, an orexigenic peptide, are increased during conditions of chronic starvation, such as in patients with anorexia nervosa. However, it is not known whether such increase can be related to the number of preproghrelin mRNA-expressing cells in the stomach, and if chronic starvation may activate a tentative central ghrelin production. In this work, in situ hybridization technique was used to analyze the presence and number of preproghrelin mRNA-expressing cells in the stomach and the hypothalamus of mice with activity-based anorexia (ABA) induced by the combination of running wheel activity with progressive, during 10 days, feeding-time restriction (FTR) and compared with sedentary FTR, ABA pair-fed (PF) and ad libitum-fed control mice. All food-restricted mice lost more than 20% of body weight. Body weight loss was similar in ABA and PF mice, but it was more pronounced than in FTR mice. Food intake was also lower in ABA than in FTR mice. Preproghrelin mRNA-expressing cells in the stomach were increased proportionally to the body weight loss in all food-restricted groups with the highest number in ABA mice. No preproghrelin mRNA-producing cells were detectable in the hypothalamus of either control or food-restricted mice. Thus, the increased number of gastric preproghrelin mRNA-producing cells during chronic starvation proportionally to the body weight loss and reduced food intake may underlie increased plasma ghrelin. Hyperactivity-induced anorexia appears to further increase the number of preproghrelin mRNA-producing cells in the stomach. No evidence was found for ghrelin expression in the hypothalamus, not even in any of the present experimental models.

  12. Effect of template size on accumulation of defective interfering RNAs in protoplasts.

    PubMed Central

    Zhang, C; Simon, A E

    1994-01-01

    A turnip protoplast system has been used to study the effects of template size and sequence on the replication and/or stability of a small defective interfering (DI) RNA associated with turnip crinkle virus. Our results indicated that as little as a single base difference in the size of the molecule in some regions, rather than the specific sequence, affected the level of DI RNA accumulating in protoplasts. Images PMID:7966644

  13. Increased glutamate decarboxylase mRNA levels in the striatum and pallidum of MPTP-treated primates.

    PubMed

    Soghomonian, J J; Pedneault, S; Audet, G; Parent, A

    1994-10-01

    The mRNA levels encoding for the enzyme glutamate decarboxylase (GAD67) were measured by computerized image analysis after in situ hybridization histochemistry and radioautography in the striatum and pallidum of normal squirrel monkeys (Saimiri sciureus), or after treatment with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). All MPTP-injected monkeys exhibited profound motor deficits including akinesia. The dopaminergic innervation, as visualized and quantified on x-ray films after 3H-mazindol binding on tissue sections, was uniformly lost throughout the striatum of MPTP-treated monkeys. Brain sections processed with a probe synthesized from a feline or human GAD67 cDNA exhibited intense radioautographic labeling throughout the striatum. When measured on x-ray films, the intensity of GAD67 mRNA labeling was increased in the striatum of MPTP-treated versus control monkeys. Increased labeling reached statistical significance in the dorsolateral sector of the rostral putamen and throughout the putamen and the caudate at the caudal, postcommissural, level. Analysis of emulsion radioautographs demonstrated that the increase in GAD67 mRNA labeling in MPTP-treated monkeys occurred in individual neurons of the striatum. In the external and internal segments of the pallidum, numerous neurons labeled with the GAD67 cRNA probe were visualized on emulsion radioautographs. The intensity of GAD67 mRNA labeling in single neurons of both pallidal segments was increased in MPTP-treated versus control monkeys. Construction of the histograms of frequency distribution of labeling indicated that this increase occurred in a majority of labeled neurons. The present study demonstrates that GAD67 mRNA levels are significantly altered in the striatum and pallidum of parkinsonian monkeys. The preferential increase of GAD67 mRNA labeling in the dorsolateral putamen, which receives afferents from the sensorimotor cortex, provides further evidence of the involvement of

  14. Increased expression of miRNA-146a in Alzheimer’s disease transgenic mouse models

    PubMed Central

    Li, Y.Y.; Cui, J.G.; Hill, J.M.; Bhattacharjee, S.; Zhao, Y.; Lukiw, W.J.

    2017-01-01

    A mouse and human brain-enriched micro-RNA-146a (miRNA-146a) is known to be important in modulating the innate immune response and inflammatory signaling in certain immunological and brain cell types. In this study we examined miRNA-146a levels in early-, moderate- and late-stage Alzheimer’s disease (AD) neocortex and hippocampus, in several human primary brain and retinal cell lines, and in 5 different transgenic mouse models of AD including Tg2576, TgCRND8, PSAPP, 3xTg-AD and 5xFAD. Inducible expression of miRNA-146a was found to be significantly up-regulated in a primary co-culture of human neuronal–glial (HNG) cells stressed using interleukin1-beta (IL-1β), and this up-regulation was quenched using specific NF-κB inhibitors including curcumin. Expression of miRNA-146a correlated with senile plaque density and synaptic pathology in Tg2576 and in 5xFAD transgenic mouse models used in the study of this common neurodegenerative disorder. PMID:20934487

  15. ROCK inhibition enhances microRNA function by promoting deadenylation of targeted mRNAs via increasing PAIP2 expression

    PubMed Central

    Yoshikawa, Takeshi; Wu, Jianfeng; Otsuka, Motoyuki; Kishikawa, Takahiro; Ohno, Motoko; Shibata, Chikako; Takata, Akemi; Han, Felicia; Kang, Young Jun; Chen, Chyi-Ying A.; Shyu, Ann-Bin; Han, Jiahuai; Koike, Kazuhiko

    2015-01-01

    The reduced expression levels and functional impairment of global miRNAs are related to various human diseases, including cancers. However, relatively little is known about how global miRNA function may be upregulated. Here, we report that global miRNA function can be enhanced by Rho-associated, coiled-coil-containing protein kinase (ROCK) inhibitors. The regulation of miRNA function by ROCK inhibitors is mediated, at least in part, by poly(A)-binding protein-interacting protein 2 (PAIP2), which enhances poly(A)-shortening of miRNA-targeted mRNAs and leads to global upregulation of miRNA function. In the presence of a ROCK inhibitor, PAIP2 expression is enhanced by the transcription factor hepatocyte nuclear factor 4 alpha (HNF4A) through increased ROCK1 nuclear localization and enhanced ROCK1 association with HNF4A. Our data reveal an unexpected role of ROCK1 as a cofactor of HNF4A in enhancing PAIP2 transcription. ROCK inhibitors may be useful for the various pathologies associated with the impairment of global miRNA function. PMID:26187994

  16. MiRNA expression profiling in human gliomas: upregulated miR-363 increases cell survival and proliferation.

    PubMed

    Conti, Alfredo; Romeo, Sara G; Cama, Annamaria; La Torre, Domenico; Barresi, Valeria; Pezzino, Gaetana; Tomasello, Chiara; Cardali, Salvatore; Angileri, Filippo F; Polito, Francesca; Ferlazzo, Guido; Di Giorgio, Rosamaria; Germanò, Antonino; Aguennouz, M'hammed

    2016-10-01

    The role of microRNAs (miRNAs) in glioma biology is increasingly recognized. To investigate the regulatory mechanisms governing the malignant signature of gliomas with different grades of malignancy, we analyzed miRNA expression profiles in human grade I-IV tumor samples and primary glioma cell cultures. Multiplex real-time PCR was used to profile miRNA expression in a set of World Health Organization (WHO) grade I (pilocytic astrocytoma), II (diffuse fibrillary astrocytoma), and IV (glioblastoma multiforme) astrocytic tumors and primary glioma cell cultures. Primary glioma cell cultures were used to evaluate the effect of transfection of specific miRNAs and miRNA inhibitors. miRNA microarray showed that a set of miRNAs was consistently upregulated in all glioma samples. miR-363 was upregulated in all tumor specimens and cell lines, and its expression correlated with tumor grading. The transfection of glioma cells with the specific inhibitor of miR-363 increased the expression level of tumor suppressor growth-associated protein 43 (GAP-43). Transfection of miR-363 induced cell survival, while inhibition of miR-363 significantly reduced glioma cell viability. Furthermore, miRNA-363 inhibition induced the downregulation of AKT, cyclin-D1, matrix metalloproteinase (MMP)-2, MMP-9, and Bcl-2 and upregulation of caspase 3. Together, these data suggest that the upregulation of miR-363 may play a role in malignant glioma signature.

  17. Effect of the increased stability of the penicillin amidase mRNA on the protein expression levels.

    PubMed

    Viegas, Sandra C; Schmidt, Dorothea; Kasche, Volker; Arraiano, Cecília M; Ignatova, Zoya

    2005-09-12

    Several factors at transcriptional, post-transcriptional or post-translational level determine the fate of a target protein and can severely restrict its yield. Here, we focus on the post-transcriptional regulation of the biosynthesis of the periplasmic protein, penicillin amidase (PA). The PA mRNA stability was determined under depleted RNase conditions in strains carrying single or multiple RNase deletions. Single deletion of the endonuclease RNase E yielded, as the highest, a fourfold stabilization of the PA mRNA. This effect, however, was reduced twice at post-translational level. The RNase II, generating secondary exonucleolytic cleavages in the mRNA, although not significantly influencing the PA mRNA decay, led also to an increase of the amount of mature PA. The non-proportional correlation between increased mRNA longevity and amount of active enzyme propose that the rational strategies for yield improvement must be based on a simultaneous tuning of more than one yield restricting factor.

  18. Exercise-mimicking treatment fails to increase Fndc5 mRNA & irisin secretion in primary human myotubes.

    PubMed

    Kurdiova, Timea; Balaz, Miroslav; Mayer, Alexander; Maderova, Denisa; Belan, Vitazoslav; Wolfrum, Christian; Ukropec, Jozef; Ukropcova, Barbara

    2014-06-01

    Irisin, myokine secreted by skeletal muscle, was suggested to mediate some of exercise health benefits via "browning" of white adipose tissue. However, mounting evidence contradicts the regulatory role of exercise for muscle irisin production/secretion in humans. Thus, we explored the direct effect of exercise-mimicking treatment on irisin in human primary muscle cells in vitro. Human primary muscle cell cultures were established from lean, obese prediabetic and type-2-diabetic individuals. Complex metabolic phenotyping included assessment of insulin sensitivity (euglycemic hyperinsulinemic clamp) and adiposity content&distribution (MRI&MRS). In vitro exercise-mimicking treatment (forskolin+ionomycin) was delivered in 1-h pulse/day during differentiation. Fndc5 mRNA (qRT-PCR) and secreted irisin (ELISA) were determined in cells and media. Exercise-mimicking treatment more than doubled Pgc1α mRNA in differentiated muscle cells. Nevertheless, Fndc5 mRNA was reduced by 18% and irisin in media by 20%. Moreover, Fncd5 mRNA was increased in myotubes derived from individuals with type-2-diabetes, independent on exercise-mimicking treatment. Fndc5 mRNA in cells was positively related to fasting glycemia (p=0.0001) and negatively to whole-body insulin sensitivity (p<0.05). Collectively, our data do not support the role of exercise-related signaling pathways in irisin regulation in human skeletal muscle and confirm our previous observations on increased Fndc5 expression in muscle cells from individuals with type-2-diabetes.

  19. Increased angiotensin-I converting enzyme gene expression in the failing human heart. Quantification by competitive RNA polymerase chain reaction.

    PubMed Central

    Studer, R; Reinecke, H; Müller, B; Holtz, J; Just, H; Drexler, H

    1994-01-01

    Local activation of the components of the renin angiotensin system in the heart is regarded as an important modulator of cardiac phenotype and function; however, little is known about their presence, regulation, and potential activation in the human heart. To investigate the gene expression of major angiotensin-II-forming enzymes in left ventricles of normal (n = 9) and failing human hearts (n = 20), we established a competitive RNA-polymerase chain reaction (PCR) for mRNA quantification of angiotensin-I converting enzyme (ACE) and human heart chymase. For each gene, competitor RNA targets with small internal deletions were used as internal standards to quantify the original number of transcripts and to control reverse transcription and PCR. In PCR, each target and the corresponding competitor were amplified by competing for the same primer oligonucleotides. The variability of ACE RNA-PCR was 11% indicating a high reproducibility of this method. In addition, ACE mRNA levels obtained by competitive RNA-PCR correlated favorably with traditional slot blot hybridization (r = 0.69, n = 10; P < 0.05). Compared with nonfailing hearts, the number of ACE transcripts referred to 100 ng of total RNA was increased threefold in patients with chronic heart failure (4.2 +/- 2.5 vs. 12.8 +/- 6 x 10(5); P < 0.0005). In contrast, no significant difference was found in chymase gene expression between normal and failing hearts. Thus, the expression of the cardiac ACE but not of human heart chymase is upregulated in failing human heart indicating an activation of the cardiac renin-angiotensin system in patients with advanced heart failure. Images PMID:8040271

  20. Lactobacillus acidophilus Increases the Anti-apoptotic Micro RNA-21 and Decreases the Pro-inflammatory Micro RNA-155 in the LPS-Treated Human Endothelial Cells.

    PubMed

    Kalani, Mehdi; Hodjati, Hossein; Sajedi Khanian, Mahdi; Doroudchi, Mehrnoosh

    2016-06-01

    Given the anti-inflammatory and protective role of probiotics in atherosclerosis and the regulatory role of micro RNA (miRNA) in endothelial cell (dys) functions, this study aimed to investigate the effect of Lactobacillus acidophilus (La) on cellular death and the expression of miRNA-21, 92a, 155, and 663 in human umbilical vein endothelial cell (HUVEC) induced by Escherichia coli lipopolysaccharide (Ec-LPS). LPS-treated and untreated HUVECs were cultured in the presence of different La conditions such as La-conditioned media (LaCM), La water extract (LaWE), La culture-filtered (LaFS) and unfiltered supernatants (LaUFS). After 24 h, apoptosis, necrosis and the levels of the mentioned miRNAs were measured using flow cytometry and real-time PCR methods, respectively. LaCM decreased apoptosis, necrosis and inflammatory miR-155 and conversely increased anti-apoptotic miR-21 in Ec-LPS-treated HUVECs. Association analysis revealed negative correlations between necrosis and the levels of miR-21, miR-92a, and miR-155. The beneficial effects of L. acidophilus on the ECs death and expression of atherosclerosis related miRNAs in these cells imply a new aspect of its regulation in cardiovascular diseases rather than previously described ones and suggest this probiotic bacterium as a candidate in the preventative therapy of atherosclerosis.

  1. Knock down of the myostatin gene by RNA interference increased body weight in chicken.

    PubMed

    Bhattacharya, T K; Shukla, R; Chatterjee, R N; Dushyanth, K

    2017-01-10

    Myostatin is a negative regulator of muscular growth in poultry and other animals. Of several approaches, knocking down the negative regulator is an important aspect to augment muscular growth in chicken. Knock down of myostatin gene has been performed by shRNA acting against the expression of gene in animals. Two methods of knock down of gene in chicken such as embryo manipulation and sperm mediated method have been performed. The hatching percentage in embryo manipulation and sperm mediated method of knock down was 58.0 and 41.5%, respectively. The shRNA in knock down chicken enhanced body weight at 6 weeks by 26.9%. The dressing percentage and serum biochemical parameters such as SGPT and alkaline phosphatase differed significantly (P<0.05) between knock down and control birds. It is concluded that knocking down the myostatin gene successfully augmented growth in chicken.

  2. In vivo co-localization of enzymes on RNA scaffolds increases metabolic production in a geometrically dependent manner.

    PubMed

    Sachdeva, Gairik; Garg, Abhishek; Godding, David; Way, Jeffrey C; Silver, Pamela A

    2014-08-01

    Co-localization of biochemical processes plays a key role in the directional control of metabolic fluxes toward specific products in cells. Here, we employ in vivo scaffolds made of RNA that can bind engineered proteins fused to specific RNA binding domains. This allows proteins to be co-localized on RNA scaffolds inside living Escherichia coli. We assembled a library of eight aptamers and corresponding RNA binding domains fused to partial fragments of fluorescent proteins. New scaffold designs could co-localize split green fluorescent protein fragments to produce activity as measured by cell-based fluorescence. The scaffolds consisted of either single bivalent RNAs or RNAs designed to polymerize in one or two dimensions. The new scaffolds were used to increase metabolic output from a two-enzyme pentadecane production pathway that contains a fatty aldehyde intermediate, as well as three and four enzymes in the succinate production pathway. Pentadecane synthesis depended on the geometry of enzymes on the scaffold, as determined through systematic reorientation of the acyl-ACP reductase fusion by rotation via addition of base pairs to its cognate RNA aptamer. Together, these data suggest that intra-cellular scaffolding of enzymatic reactions may enhance the direct channeling of a variety of substrates.

  3. Aryl hydrocarbon induction of rat cytochrome P-450d results from increased precursor RNA processing.

    PubMed Central

    Silver, G; Krauter, K S

    1990-01-01

    We have previously demonstrated that cytochrome P-450d mRNA accumulation is induced at a posttranscriptional level by 3-methylcholanthrene (MCA) in primary cultures of rat hepatocytes grown in serum-free hormonally defined medium. Using dactinomycin chase experiments in this culture system, we found that MCA had no effect on the P-450d mRNA half-life. In addition, induction of P-450d occurred both in the presence and in the absence of protein synthesis inhibitors. An analysis of nuclear precursors showed that the accumulation of the primary transcript of the P-450d gene was induced to the same extent as that of the mature mRNA after MCA treatment and that the pattern of accumulation of precursors differed between treated and control liver cells. Since P-450d induction is thought to be a receptor-mediated event, these data are consistent with a model in which a direct interaction occurs between the receptor-ligand complex and the primary transcript. Images PMID:2247082

  4. Cholesterol-based cationic liposome increases dsRNA protection of yellow head virus infection in Penaeus vannamei.

    PubMed

    Sanitt, Poohrawind; Apiratikul, Nuttapon; Niyomtham, Nattisa; Yingyongnarongkul, Boon-Ek; Assavalapsakul, Wanchai; Panyim, Sakol; Udomkit, Apinunt

    2016-06-20

    Protection of shrimp from yellow head virus (YHV) infection has been demonstrated by injection and oral delivery of dsRNA-YHV protease gene (dsYHV) or shrimp endogenous gene (dsRab7). However, to achieve complete viral suppression and to prolong dsRNA activity, the development of an effective dsRNA delivery system is required. In this study, four cationic liposomes were synthesized and tested for their ability to increase dsRNA efficiency. The results demonstrated that entrapping dsYHV in a cholesterol-based cationic liposome gave the best protection against YHV infection when compared with other cationic lipids. The cholesterol-based cationic liposome-dsYHV (Chol-dsYHV) complex conferred YHV protection in a dose-dependent manner. Injection with Chol-dsYHV at 0.05μg dsYHV/g shrimp could give comparable level of YHV protection to the injection with 1.25μg naked dsYHV/g shrimp. The shrimp injected with Chol- dsYHV at 1.25μg dsRNA/g shrimp showed only 50% mortality at 60days post injection whereas the naked dsYHV at the same concentration gave 90% mortality. Thus, the liposome-entrapped dsYHV could lower an effective dsRNA concentration in viral protection and prolong dsRNA activity. In addition, encapsulating dsRab7 in the cholesterol-based cationic liposome could protect the dsRab7 from enzymatic digestion, and continuous feeding the shrimp with the diet formulated with the liposome-entrapped dsRab7 for 4days in the total of 960μg dsRab7/g shrimp could enhance YHV protection efficiency compared with the naked dsRab7. Our studies reveal that cholesterol-based cationic liposome is a promising dsRNA carrier to enhance dsRNA efficiency in both injection and oral delivery systems.

  5. The initiator methionine tRNA drives cell migration and invasion leading to increased metastatic potential in melanoma

    PubMed Central

    Birch, Joanna; Clarke, Cassie J.; Campbell, Andrew D.; Campbell, Kirsteen; Mitchell, Louise; Liko, Dritan; Kalna, Gabriela; Strathdee, Douglas; Sansom, Owen J.; Neilson, Matthew; Blyth, Karen

    2016-01-01

    ABSTRACT The cell's repertoire of transfer RNAs (tRNAs) has been linked to cancer. Recently, the level of the initiator methionine tRNA (tRNAiMet) in stromal fibroblasts has been shown to influence extracellular matrix (ECM) secretion to drive tumour growth and angiogenesis. Here we show that increased tRNAiMet within cancer cells does not influence tumour growth, but drives cell migration and invasion via a mechanism that is independent from ECM synthesis and dependent on α5β1 integrin and levels of the translation initiation ternary complex. In vivo and ex vivo migration (but not proliferation) of melanoblasts is significantly enhanced in transgenic mice which express additional copies of the tRNAiMet gene. We show that increased tRNAiMet in melanoma drives migratory, invasive behaviour and metastatic potential without affecting cell proliferation and primary tumour growth, and that expression of RNA polymerase III-associated genes (which drive tRNA expression) are elevated in metastases by comparison with primary tumours. Thus, specific alterations to the cancer cell tRNA repertoire drive a migration/invasion programme that may lead to metastasis. PMID:27543055

  6. Increased Levels of miRNA-146a in Serum and Histologic Samples of Patients with Uveal Melanoma.

    PubMed

    Russo, Andrea; Caltabiano, Rosario; Longo, Antonio; Avitabile, Teresio; Franco, Livio M; Bonfiglio, Vincenza; Puzzo, Lidia; Reibaldi, Michele

    2016-01-01

    Purpose: To analyze MiRs expression in serum of UM patients, respect to healthy donors, and to compare this data with MiRs expressed in formalin-fixed, paraffin-embedded UM samples. Methods: Expression profile of 754 miRNAs was performed in serum of patients with uveal melanoma who underwent primary enucleation. The level of miRNAs increased in serum was individually analyzed on FFPE UM samples and compared to choroidal melanocytes from unaffected eyes. Results: Fourteen patients with uveal melanoma were included in the study. We found 8 serum miRNAs differentially expressed compared to normal controls: 2 upregulated miRNAs (miRNA-146a, miR-523); 6 downregulated miRNAs (miR-19a, miR-30d, miR-127, miR-451, miR-518f, miR-1274B). When data on upregulated miRNAs were singularly validated only a significant overexpression of miRNA-146a was found. A statistically significant upregulation of miRNA-146a was also found on FFPE UM samples, compared to choroidal melanocytes from unaffected eyes. Conclusions: miRNA-146a is increased in serum of patients with UM and in FFPE tumor samples. Further studies will show if it could be considered a potential marker of UM in the blood.

  7. Increased Levels of miRNA-146a in Serum and Histologic Samples of Patients with Uveal Melanoma

    PubMed Central

    Russo, Andrea; Caltabiano, Rosario; Longo, Antonio; Avitabile, Teresio; Franco, Livio M.; Bonfiglio, Vincenza; Puzzo, Lidia; Reibaldi, Michele

    2016-01-01

    Purpose: To analyze MiRs expression in serum of UM patients, respect to healthy donors, and to compare this data with MiRs expressed in formalin-fixed, paraffin-embedded UM samples. Methods: Expression profile of 754 miRNAs was performed in serum of patients with uveal melanoma who underwent primary enucleation. The level of miRNAs increased in serum was individually analyzed on FFPE UM samples and compared to choroidal melanocytes from unaffected eyes. Results: Fourteen patients with uveal melanoma were included in the study. We found 8 serum miRNAs differentially expressed compared to normal controls: 2 upregulated miRNAs (miRNA-146a, miR-523); 6 downregulated miRNAs (miR-19a, miR-30d, miR-127, miR-451, miR-518f, miR-1274B). When data on upregulated miRNAs were singularly validated only a significant overexpression of miRNA-146a was found. A statistically significant upregulation of miRNA-146a was also found on FFPE UM samples, compared to choroidal melanocytes from unaffected eyes. Conclusions: miRNA-146a is increased in serum of patients with UM and in FFPE tumor samples. Further studies will show if it could be considered a potential marker of UM in the blood. PMID:27895580

  8. Increased Litter Size and Suckling Intensity Stimulate mRNA of RFamide-related Peptide in Rats

    PubMed Central

    Noroozi, Atefeh; Jafarzadeh Shirazi, Mohammad Reza; Tamadon, Amin; Moghadam, Ali; Niazi, Ali

    2015-01-01

    Background RFamide-related peptide-3 (RFRP-3) inhibits gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) secretion in rats. This study evaluates the effects of litter size and suckling intensity on RFRP mRNA expression in the dorsomedial hypothalamic nucleus (DMH) of rats. Materials and Methods A total of 32 pregnant and 4 non-lactating ovariectomized (control group) Sprague-Dawley rats were used in this experimental study. Lactating rats were allotted to 8 equal groups. In 3 groups, the litter size was adjusted to 5, 10, or 15 pups upon parturition. Dams were allowed to suckle their pups continuously until 8 days postpartum. In the other 3 groups, the litter size was adjusted to 5 pups following birth. These pups were separated from the dams for 6 hours on day 8 postpartum, after which the pups were allowed to suckle for 2.5, 5, or 7.5 minutes prior to killing the dams. In 2 groups, lactating rats with 10 and 15 pups were separated from their pups for 6 hours on day 8 postpartum. In these groups, the pups were allowed to suckle their dams for 5 minutes before the dams were killed. All rats were killed on day 8 postpartum and the DMH was removed from each rat. We evaluated RFRP mRNA expression using realtime polymerase chain reaction (PCR). Results The expression of RFRP mRNA in the DMH increased with increased litter size and suckling intensity compared to the controls. The effect of suckling intensity on the expression of RFRP mRNA was more pronounced compared to the litter size. Conclusion Increased litter size and suckling intensity stimulated RFRP mRNA expression in the DMH which might contribute to lactation anestrus in rats. PMID:26644862

  9. Docosahexaenoic acid increases cellular adiponectin mRNA and secreted adiponectin protein, as well as PPARγ mRNA, in 3T3-L1 adipocytes.

    PubMed

    Oster, Richard T; Tishinsky, Justine M; Yuan, Zongfei; Robinson, Lindsay E

    2010-12-01

    Adiponectin, a protein secreted from adipose tissue, has been shown to have anti-diabetic and anti-inflammatory effects, but its regulation is not completely understood. Long-chain n-3 fatty acids eicosapentaenoic acid (20:5n-3; EPA) and docosahexaenoic acid (22:6n-3; DHA) may be involved in adiponectin regulation as they are potential ligands for peroxisome proliferator-activated receptor-γ (PPARγ), a key transcription factor for the adiponectin gene. To examine this, 3T3-L1 adipocytes were incubated with 125 µmol·L-1 EPA, DHA, palmitic, or oleic acids complexed to albumin, or with albumin alone (control) for 24 h. Adipocytes were also incubated for 24 h with EPA and DHA plus bisphenol-A-diglycidyl ether (BADGE), a PPARγ antagonist. Both EPA and DHA increased (p < 0.05) secreted adiponectin concentration compared with the control (44% and 102%, respectively), but did not affect cellular adiponectin protein content. Incubation with BADGE and DHA inhibited increases in secreted adiponectin protein, suggesting that DHA may act through a PPARγ-dependent mechanism. However, BADGE had no effect on EPA-induced increases in secreted adiponectin protein. Only DHA enhanced (p < 0.05) PPARγ and adiponectin mRNA expression compared wtih the control. Our results demonstrate that DHA increases cellular adiponectin mRNA and secreted adiponectin protein in 3T3-L1 adipocytes, possibly by a mechanism involving PPARγ. Moreover, DHA increased adiponectin concentration to a greater extent (40% more, p < 0.05) compared with EPA, emphasizing the need to consider the independent actions of EPA and DHA in adipocytes.

  10. Overexpression of microRNA OsmiR397 improves rice yield by increasing grain size and promoting panicle branching.

    PubMed

    Zhang, Yu-Chan; Yu, Yang; Wang, Cong-Ying; Li, Ze-Yuan; Liu, Qing; Xu, Jie; Liao, Jian-You; Wang, Xiao-Jing; Qu, Liang-Hu; Chen, Fan; Xin, Peiyong; Yan, Cunyu; Chu, Jinfang; Li, Hong-Qing; Chen, Yue-Qin

    2013-09-01

    Increasing grain yields is a major focus of crop breeders around the world. Here we report that overexpression of the rice microRNA (miRNA) OsmiR397, which is naturally highly expressed in young panicles and grains, enlarges grain size and promotes panicle branching, leading to an increase in overall grain yield of up to 25% in a field trial. To our knowledge, no previous report has shown a positive regulatory role of miRNA in the control of plant seed size and grain yield. We determined that OsmiR397 increases grain yield by downregulating its target, OsLAC, whose product is a laccase-like protein that we found to be involved in the sensitivity of plants to brassinosteroids. As miR397 is highly conserved across different species, our results suggest that manipulating miR397 may be useful for increasing grain yield not only in rice but also in other cereal crops.

  11. The efficiency of RNA interference for conferring stable resistance to Plum Pox Virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plum transformed with an intron hairpin RNA CP (ihRNA-CP) were resistant to PPV infection through the specific process of RNA silencing involving both small interfering -RNA interfering (siRNA) and a methylated virus transgene. This recognition process specifically targeted the triggered PPV genome...

  12. Increased litter size and suckling intensity inhibit KiSS-1 mRNA expression in rat arcuate nucleus

    PubMed Central

    Noroozi, Atefeh; Shirazi, Mohammad Reza Jafarzadeh; Zamiri, Mohammad Javad; Tamadon, Amin; Akhlaghi, Amir; Tanideh, Nader; Niazi, Ali; Moghadam, Ali

    2014-01-01

    Objective(s): The effect of litter size and suckling intensity on the expression of KiSS-1 mRNA in the arcuate nucleus (ARC) of rats were evaluated. Materials and Methods: Thirty two pregnant and four non-lactating ovariectomized (as control group) rats were used in this experiment. Lactating rats were allotted to eight equal groups. In three groups, litter size was adjusted to 5, 10, or 15 pups upon parturition and allowed to suckle their pups continuously by 8 days postpartum. In the other three groups, litter size was adjusted to five upon birth; the pups were separated from the dams for 6 hr on day 8 postpartum, after which the pups were allowed to suckle their dams for 2.5, 5, or 7.5 min prior to killing the dams. Two groups of lactating rats with either 10 or 15 pups were separated from their pups for 6 hr on day 8 postpartum, after which the pups were allowed to suckle their dams for 5 min before the dams were killed on day 8 postpartum. The ARC was removed and the expression of KiSS-1 mRNA was evaluated, using real-time PCR. Results: The expression of KiSS-1 mRNA in the ARC was decreased as the litter size and intensity of suckling stimulus were increased. The effect of suckling intensity on the expression of KiSS-1 mRNA was more pronounced than that of litter size. Conclusion: Increased litter size and suckling intensity decreased KiSS-1 mRNA expression in the ARC which may contribute to lactation anestrus in rat. PMID:25422754

  13. Isothermic and fixed-intensity heat acclimation methods elicit equal increases in Hsp72 mRNA.

    PubMed

    Gibson, O R; Mee, J A; Taylor, L; Tuttle, J A; Watt, P W; Maxwell, N S

    2015-06-01

    Thermotolerance, to which heat shock protein-72 (Hsp72) contributes, is an acquired state achieved following heat acclimation (HA), eliciting cellular adaption and protection against thermal stress. Optimal HA methods achieving the greatest heat shock response (HSR) are equivocal; therefore, investigation of methods provoking the greatest sustained HSR is required to optimize cellular adaptation. Twenty-four males performed short-term HA (STHA; five sessions) and long-term HA (LTHA; STHA plus further five sessions) utilizing fixed-intensity (FIXED; workload = 50% V ˙ O 2 p e a k ), continuous isothermic HA [ISOCONT ; target rectal temperature (Trec ) = 38.5 °C], or progressive isothermic HA (ISOPROG ; target Trec  = 38.5 °C for STHA then target Trec  = 39.0 °C for LTHA). Leukocyte Hsp72 mRNA was measured pre- and post day 1, day 5, and day 10 of HA via reverse transcription quantitative polymerase chain reaction to determine the HSR. Hsp72 mRNA increased (P < 0.05) pre- to post day 1, pre- to post day 5, and pre to post day 10 in FIXED, ISOCONT , and ISOPROG , but no differences were observed between methods (P > 0.05). The equal Hsp72 mRNA increases occurring from consistent, reduced, or increased endogenous strain following STHA and LTHA suggest that transcription occurs following attainment of sufficient endogenous criteria. These data give confidence that all reported HA methods increase Hsp72 mRNA and are capable of eliciting adaptations toward thermotolerance.

  14. Hypoxia may increase rat insulin mRNA levels by promoting binding of the polypyrimidine tract-binding protein (PTB) to the pyrimidine-rich insulin mRNA 3'-untranslated region.

    PubMed Central

    Tillmar, Linda; Welsh, Nils

    2002-01-01

    BACKGROUND: Recent reports identify the 3'-UTR of insulin mRNA as crucial for control of insulin messenger stability. This region contains a pyrimidine-rich sequence, which is similar to the hypoxia-responsive mRNA-stabilizing element of tyrosine hydroxylase. This study aimed to determine whether hypoxia affects insulin mRNA levels. MATERIALS AND METHODS: Rat islets were incubated at normoxic or hypoxic conditions and with or without hydrogen peroxide and a nitric oxide donor. Insulin mRNA was determined by Northern hybridization. Islet homogenates were used for electrophoretic mobility shift assay with an RNA-oligonucleotide, corresponding to the pyrimidine-rich sequence of the 3'-UTR of rat insulin I mRNA. The expression of reporter gene mRNA, in islets transfected with reporter gene constructs containing the wild-type or mutated insulin mRNA pyrimidine-rich sequences, was measured by semiquantitive RT-PCR. RESULTS: Insulin mRNA was increased in response to hypoxia. This was paralleled by increased binding of the polypyrimidine tract-binding protein (PTB) to the pyrimidine-rich sequence of the 3'-UTR of insulin mRNA, which was counteracted by hydrogen peroxide. The reporter gene mRNA level containing the wild-type binding site was not increased in response to hypoxia, but mutation of the site resulted in a destabilization of the mRNA. CONCLUSIONS: The complete understanding of different diabetic conditions requires the elucidation of mechanisms that control insulin gene expression. Our data show that hypoxia may increase insulin mRNA levels by promoting the binding of PTB to the insulin mRNA 3'-UTR. Hydrogen peroxide abolishes the hypoxic effect indicating involvement of reactive oxygen species and/or the redox potential in the oxygen-signaling pathway. PMID:12359957

  15. Acetylcholine receptor-inducing factor from chicken brain increases the level of mRNA encoding the receptor. alpha. subunit

    SciTech Connect

    Harris, D.A.; Falls, D.L.; Dill-Devor, R.M.; Fischbach, G.D. )

    1988-03-01

    A 42-kDa glycoprotein isolated from chicken brain, referred to as acetylcholine receptor-inducing activity (ARIA), that stimulates the rate of incorporation of acetylcholine receptors into the surface of chicken myotubes may play a role in the nerve-induced accumulation of receptors at developing neuromuscular synapses. Using nuclease-protection assays, the authors have found that ARIA causes a 2- to 16-fold increase in the level of mRNA encoding the {alpha} subunit of the receptor, with little or no change in the levels of {gamma}- and {delta}-subunit messengers. ARIA also increases the amount of a putative nuclear precursor of {alpha}-subunit mRNA, consistent with an activation of gene transcription. These results suggest that the concentration of {alpha} subunit may limit the rate of biosynthesis of the acetylcholine receptors in chicken myotubes. They also indicate that neuronal factors can regulate the expression of receptor subunit genes in a selective manner. Tetrodotoxin, 8-bromo-cAMP, and forskolin also increase the amount of {alpha}-subunit mRNA, with little change in the amount of {gamma}- and {delta}-subunit mRNAs. Unlike ARIA, however, these agents have little effect on the concentration of the {alpha}-subunit nuclear precursor.

  16. Inflammatory nociception diminishes dopamine release and increases dopamine D2 receptor mRNA in the rat's insular cortex

    PubMed Central

    2010-01-01

    Background The insular cortex (IC) receives somatosensory afferent input and has been related to nociceptive input. It has dopaminergic terminals and D1 (D1R) -excitatory- and D2 (D2R) -inhibitory- receptors. D2R activation with a selective agonist, as well as D1R blockade with antagonists in the IC, diminish neuropathic nociception in a nerve transection model. An intraplantar injection of carrageenan and acute thermonociception (plantar test) were performed to measure the response to inflammation (paw withdrawal latency, PWL). Simultaneously, a freely moving microdyalisis technique and HPLC were used to measure the release of dopamine and its metabolites in the IC. Plantar test was applied prior, one and three hours after inflammation. Also, mRNA levels of D1 and D2R's were measured in the IC after three hours of inflammation. Results The results showed a gradual decrease in the release of dopamine, Dopac and HVA after inflammation. The decrease correlates with a decrease in PWL. D2R's increased their mRNA expression compared to the controls. In regard of D1R's, there was a decrease in their mRNA levels compared to the controls. Conclusions Our results showed that the decreased extracellular levels of dopamine induced by inflammation correlated with the level of pain-related behaviour. These results also showed the increase in dopaminergic mediated inhibition by an increase in D2R's and a decrease in D1R's mRNA. There is a possible differential mechanism regarding the regulation of excitatory and inhibitory dopaminergic receptors triggered by inflammation. PMID:21050459

  17. Increased hepatic expression of miRNA-122 in patients infected with HCV genotype 3.

    PubMed

    Oliveira, Ketti G; Malta, Fernanda M; Nastri, Ana C S S; Widman, Azzo; Faria, Paola L; Santana, Rúbia A F; Alves, Venâncio A F; Carrilho, Flair J; Pinho, João R R

    2016-04-01

    Hepatitis C virus (HCV) infection affects approximately 3 % of the world population. HCV targets hepatic tissue, and most infected patients develop a chronic infection. Currently, studies have demonstrated an association between HCV-RNA replication and miR-122, the most abundant microRNA in the liver. Our aim was to evaluate liver and serum expression of miR-122 in patients infected with HCV genotypes 1 and 3, and to identify possible associations between miR-122 expression and lipid profiles, HCV viral load, apolipoproteins and liver enzymes. MicroRNAs were isolated from blood and liver tissue, and miR-122 expression was quantified by real-time PCR. HCV viral load was quantified by real-time PCR and HCV genotype, and serum biomarkers were obtained from medical report. The levels of miR-122 were higher in liver than those in blood from individuals infected with HCV genotypes 1 and 3 (p < 0.0001). The tissue levels of miR-122 were higher in subjects infected with HCV genotype 3 (6.22-fold, p < 0.001). A positive correlation was observed between the blood and hepatic levels of miR-122 in patients infected with HCV genotype 1 (r = 0.302, p = 0.026); in these patients, an inverse correlation was observed between serum apolipoprotein A-II (ApoA-II) levels and the blood (r = -0.330; p = 0.014) and hepatic (r = -0.311; p = 0.020) levels of miR-122. In patients infected with HCV genotype 3, there was a positive correlation between the hepatic miR-122 and the high-density lipoprotein-HDL (r = 0.412, p = 0.036) and insulin (r = 0.478, p = 0.044). Lipid metabolism proteins and miR-122 expression levels have different relations in HCV-3- and HCV-1-infected patients.

  18. Increase of microRNA-210, Decrease of Raptor Gene Expression and Alteration of Mammalian Target of Rapamycin Regulated Proteins following Mithramycin Treatment of Human Erythroid Cells

    PubMed Central

    Bianchi, Nicoletta; Finotti, Alessia; Ferracin, Manuela; Lampronti, Ilaria; Zuccato, Cristina; Breveglieri, Giulia; Brognara, Eleonora; Fabbri, Enrica; Borgatti, Monica; Negrini, Massimo; Gambari, Roberto

    2015-01-01

    Expression and regulation of microRNAs is an emerging issue in erythroid differentiation and globin gene expression in hemoglobin disorders. In the first part of this study microarray analysis was performed both in mithramycin-induced K562 cells and erythroid precursors from healthy subjects or β-thalassemia patients producing low or high levels of fetal hemoglobin. We demonstrated that: (a) microRNA-210 expression is higher in erythroid precursors from β-thalassemia patients with high production of fetal hemoglobin; (b) microRNA-210 increases as a consequence of mithramycin treatment of K562 cells and human erythroid progenitors both from healthy and β-thalassemia subjects; (c) this increase is associated with erythroid induction and elevated expression of γ-globin genes; (d) an anti-microRNA against microRNA-210 interferes with the mithramycin-induced changes of gene expression. In the second part of the study we have obtained convergent evidences suggesting raptor mRNA as a putative target of microRNA-210. Indeed, microRNA-210 binding sites of its 3’-UTR region were involved in expression and are targets of microRNA-210-mediated modulation in a luciferase reporter assays. Furthermore, (i) raptor mRNA and protein are down-regulated upon mithramycin-induction both in K562 cells and erythroid progenitors from healthy and β-thalassemia subjects. In addition, (ii) administration of anti-microRNA-210 to K562 cells decreased endogenous microRNA-210 and increased raptor mRNA and protein expression. Finally, (iii) treatment of K562 cells with premicroRNA-210 led to a decrease of raptor mRNA and protein. In conclusion, microRNA-210 and raptor are involved in mithramycin-mediated erythroid differentiation of K562 cells and participate to the fine-tuning and control of γ-globin gene expression in erythroid precursor cells. PMID:25849663

  19. [The influence of interfered circadian rhythm on pregnancy and neonatal rats].

    PubMed

    Chen, Wen-Jun; Sheng, Wen-Jie; Guo, Yin-Hua; Tan, Yong

    2015-10-25

    The aim of this study was to observe the influence of interfered circadian rhythm on pregnancy of rats and growth of neonatal rats, and to explore the relationship between the interfered circadian rhythm and the changes of melatonin and progesterone. Continuous light was used to inhibit melatonin secretion and therefore the interfered circadian rhythm animal model was obtained. The influence of interfered circadian rhythm on delivery of pregnant rats was observed. Serum was collected from rats during different stages of pregnancy to measure the concentrations of melatonin and progesterone. In order to observe the embryo resorption rate, half of pregnant rats were randomly selected to undergo a laparotomy, and the remainder was used to observe delivery and assess the growth of neonatal rats after delivery. The results showed that the interfered circadian rhythm induced adverse effects on pregnancy outcomes, including an increase of embryo resorption rate and a decrease in the number of live births; inhibited the secretion of melatonin along with decreased serum progesterone level; prolonged the stage of labor, but not the duration of pregnancy; and disturbed the fetal intrauterine growth and the growth of neonatal rats. The results suggest that interfered circadian rhythm condition made by continuous light could make adverse effects on both pregnant rats and neonatal rats. The results of our study may provide a way to modulate pregnant women's circadian rhythm and a possibility of application of melatonin on pregnant women.

  20. Slow-growing cells within isogenic populations have increased RNA polymerase error rates and DNA damage

    PubMed Central

    van Dijk, David; Dhar, Riddhiman; Missarova, Alsu M.; Espinar, Lorena; Blevins, William R.; Lehner, Ben; Carey, Lucas B.

    2015-01-01

    Isogenic cells show a large degree of variability in growth rate, even when cultured in the same environment. Such cell-to-cell variability in growth can alter sensitivity to antibiotics, chemotherapy and environmental stress. To characterize transcriptional differences associated with this variability, we have developed a method—FitFlow—that enables the sorting of subpopulations by growth rate. The slow-growing subpopulation shows a transcriptional stress response, but, more surprisingly, these cells have reduced RNA polymerase fidelity and exhibit a DNA damage response. As DNA damage is often caused by oxidative stress, we test the addition of an antioxidant, and find that it reduces the size of the slow-growing population. More generally, we find a significantly altered transcriptome in the slow-growing subpopulation that only partially resembles that of cells growing slowly due to environmental and culture conditions. Slow-growing cells upregulate transposons and express more chromosomal, viral and plasmid-borne transcripts, and thus explore a larger genotypic—and so phenotypic — space. PMID:26268986

  1. Increased brain radioactivity by intranasal 32P-labeled siRNA dendriplexes within in situ-forming mucoadhesive gels

    PubMed Central

    Perez, Ana Paula; Mundiña-Weilenmann, Cecilia; Romero, Eder Lilia; Morilla, Maria Jose

    2012-01-01

    Background Molecules taken up by olfactory and trigeminal nerve neurons directly access the brain by the nose-to-brain pathway. In situ-forming mucoadhesive gels would increase the residence time of intranasal material, favoring the nose-to-brain delivery. In this first approach, brain radioactivity after intranasal administration of 32P-small interference RNA (siRNA) complexed with poly(amidoamine) G7 dendrimers (siRNA dendriplexes) within in situ-forming mucoadhesive gels, was determined. Materials 32P-siRNA dendriplexes were incorporated into in situ-forming mucoadhesive gels prepared by blending thermosensitive poloxamer (23% w/w) with mucoadhesive chitosan (1% w/w, PxChi) or carbopol (0.25% w/w, PxBCP). Rheological properties, radiolabel release profile, and local toxicity in rat nasal mucosa were determined. The best-suited formulation was intranasally administered to rats, and blood absorption and brain distribution of radioactivity were measured. Results The gelation temperature of both formulations was 23°C. The PxChi liquid showed non-Newtonian pseudoplastic behavior of high consistency and difficult manipulation, and the gel retained 100% of radiolabel after 150 minutes. The PxCBP liquid showed a Newtonian behavior of low viscosity and easy manipulation, while in the gel phase showed apparent viscosity similar to that of the mucus but higher than that of aqueous solution. The gel released 35% of radiolabel and the released material showed silencing activity in vitro. Three intranasal doses of dendriplexes in PxCBP gel did not damage the rat nasal mucosa. A combination of 32P-siRNA complexation with dendrimers, incorporation of the dendriplexes into PxCBP gel, and administration of two intranasal doses was necessary to achieve higher brain radioactivity than that achieved by intravenous dendriplexes or intranasal naked siRNA. Conclusion The increased radioactivity within the olfactory bulb suggested that the combination above mentioned favored the

  2. In situ hybridization of nucleus basalis neurons shows increased. beta. -amyloid mRNA in Alzheimer disease

    SciTech Connect

    Cohen, M.L.; Golde, T.E.; Usiak, M.F.; Younkin, L.H.; Younkin, S.G.

    1988-02-01

    To determine which cells within the brain produce ..beta..-amyloid mRNA and to assess expression of the ..beta..-amyloid gene in Alzheimer disease, the authors analyzed brain tissue from Alzheimer and control patients by in situ hybridization. The results demonstrate that ..beta..-amyloid mRNA is produced by neurons in the nucleus basalis of Meynert and cerebral cortex and that nuclues basalis perikarya from Alzheimer patients consistently hybridize more ..beta..-amyloid probe than those from controls. These observations support the hypothesis that increased expression of the ..beta..-amyloid gene plays an important role in the deposition of amyloid in the brains of patients with Alzheimer disease.

  3. Increased GAD67 mRNA expression in cerebellar interneurons in autism: implications for Purkinje cell dysfunction.

    PubMed

    Yip, Jane; Soghomonian, Jean-Jacques; Blatt, Gene J

    2008-02-15

    It has been widely reported that in autism, the number of Purkinje cells (PCs) is decreased, and recently, decreased expression of glutamic acid decarboxylase 67 (GAD67) mRNA in Purkinje cells also has been observed. However, the autism literature has not addressed key GABAergic inputs into Purkinje cells. Inhibitory basket and stellate cell interneurons in the molecular layer of the cerebellar cortex provide direct key GABAergic input into Purkinje cells and could potently influence the output of Purkinje cells to deep cerebellar nuclei. We investigated the capacity for interneuronal synthesis of gamma-amino butyric acid (GABA) in both types of interneurons that innervate the remaining PCs in the posterolateral cerebellar hemisphere in autism. The level of GAD67 mRNA, one of the isoforms of the key synthesizing enzymes for GABA, was quantified at the single-cell level using in situ hybridization in brains of autistic and aged-matched controls. The National Institutes of Health imaging system showed that expression of GAD67 mRNA in basket cells was significantly up-regulated, by 28%, in eight autistic brains compared with that in eight control brains (mean +/- SEM pixels per cell, 1.03 +/- 0.05 versus 0.69 +/- 0.05, respectively; P < 0.0001 by independent t test). Stellate cells showed a trend toward a small increase in GAD67 mRNA levels, but this did not reach significance. The results suggest that basket cells likely provide increased GABAergic feed-forward inhibition to PCs in autism, directly affecting PC output to target neurons in the dentate nucleus and potentially disrupting its modulatory role in key motor and/or cognitive behaviors in autistic individuals.

  4. Stochastic and nonstochastic post-transcriptional silencing of chitinase and beta-1,3-glucanase genes involves increased RNA turnover-possible role for ribosome-independent RNA degradation.

    PubMed Central

    Holtorf, H; Schöb, H; Kunz, C; Waldvogel, R; Meins, F

    1999-01-01

    Stochastic and nonstochastic post-transcriptional gene silencing (PTGS) in Nicotiana sylvestris plants carrying tobacco class I chitinase (CHN) and beta-1,3-glucanase transgenes differs in incidence, stability, and pattern of expression. Measurements with inhibitors of RNA synthesis (cordycepin, actinomycin D, and alpha-amanitin) showed that both forms of PTGS are associated with increased sequence-specific degradation of transcripts, suggesting that increased RNA turnover may be a general feature of PTGS. The protein synthesis inhibitors cycloheximide and verrucarin A did not inhibit degradation of CHN RNA targeted for PTGS, confirming that PTGS-related RNA degradation does not depend on ongoing protein synthesis. Because verrucarin A, unlike cycloheximide, dissociates mRNA from ribosomes, our results also suggest that ribosome-associated RNA degradation pathways may not be involved in CHN PTGS. PMID:10072405

  5. Miconia sp. Increases mRNA Levels of PPAR Gamma and Inhibits Alpha Amylase and Alpha Glucosidase.

    PubMed

    Ortíz-Martinez, David Mizael; Rivas-Morales, Catalina; de la Garza-Ramos, Myriam Angelica; Verde-Star, Maria Julia; Nuñez-Gonzalez, Maria Adriana; Leos-Rivas, Catalina

    2016-01-01

    Diabetes mellitus is a public health problem worldwide. For this reason, ethanolic extract of Miconia sp. from Oaxaca, Mexico, was selected in search of an alternative against this disease. The effect of Miconia sp. on mRNA expression of PPARγ on cell line 3T3-L1, its effect on alpha amylase and alpha glucosidase, lipid accumulation during adipogenesis, and cell viability on VERO cells were evaluated. The mRNA levels of PPARγ increased on 1.393 ± 0.008 folds, lipid accumulation was increased by 29.55% with Miconia sp. extract and 34.57% with rosiglitazone, and α-amylase and α-glycosidase were inhibited with IC50 values from 28.23 ± 2.15 μg/mL and 1.95 ± 0.15 μg/mL, respectively; the IC50 on antiproliferative activity on VERO cells was 314.54 ± 45.40 μg/mL. In case of α-amylase and α-glycosidase assays, IC50 (inhibitory concentration 50) refers to necessary extract amounts to inhibit 50% of enzymatic activity. On the other hand, on antiproliferative activity, IC50 (inhibitory concentration 50) refers to necessary extract amounts to inhibit 50% of cell proliferation. It was concluded that the compounds present in Miconia sp. ethanolic extract increase mRNA expression of PPARγ, inhibit α-amylase and α-glucosidase, and increase lipid accumulation. It constitutes an alternative as adjuvant in diabetes mellitus treatment; therefore, we recommend continuing identifying the compounds responsible for its promising in vivo antidiabetic activity.

  6. Miconia sp. Increases mRNA Levels of PPAR Gamma and Inhibits Alpha Amylase and Alpha Glucosidase

    PubMed Central

    Ortíz-Martinez, David Mizael; Rivas-Morales, Catalina; de la Garza-Ramos, Myriam Angelica; Verde-Star, Maria Julia; Nuñez-Gonzalez, Maria Adriana

    2016-01-01

    Diabetes mellitus is a public health problem worldwide. For this reason, ethanolic extract of Miconia sp. from Oaxaca, Mexico, was selected in search of an alternative against this disease. The effect of Miconia sp. on mRNA expression of PPARγ on cell line 3T3-L1, its effect on alpha amylase and alpha glucosidase, lipid accumulation during adipogenesis, and cell viability on VERO cells were evaluated. The mRNA levels of PPARγ increased on 1.393 ± 0.008 folds, lipid accumulation was increased by 29.55% with Miconia sp. extract and 34.57% with rosiglitazone, and α-amylase and α-glycosidase were inhibited with IC50 values from 28.23 ± 2.15 μg/mL and 1.95 ± 0.15 μg/mL, respectively; the IC50 on antiproliferative activity on VERO cells was 314.54 ± 45.40 μg/mL. In case of α-amylase and α-glycosidase assays, IC50 (inhibitory concentration 50) refers to necessary extract amounts to inhibit 50% of enzymatic activity. On the other hand, on antiproliferative activity, IC50 (inhibitory concentration 50) refers to necessary extract amounts to inhibit 50% of cell proliferation. It was concluded that the compounds present in Miconia sp. ethanolic extract increase mRNA expression of PPARγ, inhibit α-amylase and α-glucosidase, and increase lipid accumulation. It constitutes an alternative as adjuvant in diabetes mellitus treatment; therefore, we recommend continuing identifying the compounds responsible for its promising in vivo antidiabetic activity. PMID:27478477

  7. Increased microRNA-155 and decreased microRNA-146a may promote ocular inflammation and proliferation in Graves' ophthalmopathy.

    PubMed

    Li, Kaijun; Du, Yi; Jiang, Ben-Li; He, Jian-Feng

    2014-04-18

    Graves' ophthalmopathy is an inflammatory autoimmune disease of the orbit, characterized by inflammation and proliferation of the orbital tissue caused by CD4+T cells and orbital fibroblasts. Despite recent substantial findings regarding its cellular and molecular foundations, the pathogenesis of Graves' ophthalmopathy remains unclear. Accumulating data suggest that microRNAs play important roles in the pathophysiology of autoimmunity and proliferation. Specifically, microRNA-155 (miR-155) can promote autoimmune inflammation by enhancing inflammatory T cell development. In contrast to miR-155, microRNA-146a (miR-146a) can inhibit the immune response by suppressing T cell activation. Furthermore, miR-155 and miR-146a are involved in cell proliferation, differentiation, and many other life processes. Thus, miR-155 and miR-146a, with opposite impacts on inflammatory responses carried out by T lymphocytes, appear to have multiple targets in the pathogenesis of Graves' ophthalmopathy. Our previous work showed that the expression of miR-146a was significantly decreased in peripheral blood mononuclear cells from Graves' ophthalmopathy patients compared with normal subjects. Accordingly, we proposed that the expression of miR-155 increased and the expression of miR-146a decreased in the target cells (CD4+T cells and orbital fibroblasts), thus promoting ocular inflammation and proliferation in Graves' ophthalmopathy. The proposed hypothesis warrants further investigation of the function of the differentially expressed microRNAs, which may shed new light on the pathogenesis of Graves' ophthalmopathy and lead to new strategies for its management.

  8. Age-dependent increase in miRNA-34a expression in the posterior pole of the mouse eye

    PubMed Central

    Forward, Krisztina I.; Nguyen, Anthony T.; Bordbari, Matthew H.; Oltjen, Sharon L.; Hjelmeland, Leonard M.

    2014-01-01

    Purpose MicroRNA-34a (miR-34a) has been implicated in neurodegeneration. MiR-34a belongs to a signaling network involving p53 and Sirt-1. This network responds to DNA damage with further downstream signals that induce senescence or apoptosis. Our goal was to measure the expression level of miR-34a in the mouse retina and RPE as a function of age. Methods The age-dependent change in miR-34a expression was quantified using a real-time PCR (RT-PCR) assay on microRNA isolates from eye tissue: the retina and RPE/choroid (4, 18, 24, and 32 months of age). Tissue localization of miR-34a was determined by in situ hybridization (ISH) for a series of time points. Expression of the miR-34a target gene Sirt1 was analyzed using RT-PCR and immunohistochemistry. Results MiR-34a examined with real-time PCR showed a linear increase in expression with age when compared to that of 4-month-old mice. However, the level of expression between the 24 and 32-month-old animals showed mild downregulation. An age-related increase in miR-34a expression was confirmed in the mouse eye using in situ hybridization. An inverse relationship between the levels of expression of miR-34a and its target Sirt1 mRNA was found at 18 and 24 months of age. Conclusions Our data showed that miR-34a expression increased in the retina and RPE with age. The level of DNA damage in mitochondria in the retina and RPE followed a similar time course. This suggests that miR-34a may play a role in the senescence and apoptosis of the retina and RPE cells in the aging eye. PMID:25489229

  9. RNA interference in neuroscience: progress and challenges.

    PubMed

    Miller, Victor M; Paulson, Henry L; Gonzalez-Alegre, Pedro

    2005-12-01

    1.RNA interference (RNAi) is a recently discovered biological pathway that mediates post-transcriptional gene silencing. The process of RNAi is orchestrated by an increasingly well-understood cellular machinery. 2. The common entry point for both natural and engineered RNAi are double stranded RNA molecules known as short interfering RNAs (siRNAs), that mediate the sequence-specific identification and degradation of the targeted messenger RNA (mRNA). The study and manipulation of these siRNAs has recently revolutionized biomedical research. 3. In this review, we first provide a brief overview of the process of RNAi, focusing on its potential role in brain function and involvement in neurological disease. We then describe the methods developed to manipulate RNAi in the laboratory and its applications to neuroscience. Finally, we focus on the potential therapeutic application of RNAi to neurological disease.

  10. Induction of erythroid differentiation and increased globin mRNA production with furocoumarins and their photoproducts

    PubMed Central

    Salvador, Alessia; Brognara, Eleonora; Vedaldi, Daniela; Castagliuolo, Ignazio; Brun, Paola; Zuccato, Cristina; Lampronti, Ilaria; Gambari, Roberto

    2013-01-01

    Differentiation-therapy is an important approach in the treatment of cancer, as in the case of erythroid induction in chronic myelogenous leukemia. Moreover, an important therapeutic strategy for treating beta-thalassemia and sickle-cell anemia could be the use of drugs able to induce erythroid differentiation and fetal hemoglobin (HbF) accumulation: in fact, the increased production of this type of hemoglobin can reduce the clinical symptoms and the frequency of transfusions. An important class of erythroid differentiating compounds and HbF inducers is composed by DNA-binding chemotherapeutics: however, they are not used in most instances considering their possible devastating side effects. In this contest, we approached the study of erythrodifferentiating properties of furocoumarins. In fact, upon UV-A irradiation, they are able to covalently bind DNA. Thus, the erythrodifferentiation activity of some linear and angular furocoumarins was evaluated in the experimental K562 cellular model system. Quantitative real-time reverse transcription polymerase-chain reaction assay was employed to evaluate the accumulation of different globin mRNAs. The results demonstrated that both linear and angular furocoumarins are strong inducers of erythroid differentiation of K562 cells. From a preliminary screening, we selected the most active compounds and investigated the role of DNA photodamage in their erythroid inducing activity and mechanism of action. Moreover, some cytofluorimetric experiments were carried out to better study cell cycle modifications and the mitochondrial involvement. A further development of the work was carried out studying the erythroid differentiation of photolysis products of these molecules. 5,5′-Dimethylpsoralen photoproducts induced an important increase in γ-globin gene transcription in K562 cells. PMID:23518160

  11. Increased expression of circulating miRNA-93 in women with polycystic ovary syndrome may represent a novel, non-invasive biomarker for diagnosis.

    PubMed

    Sathyapalan, T; David, R; Gooderham, N J; Atkin, S L

    2015-11-19

    MicroRNAs (miRNA) are a novel class of small noncoding single-stranded RNA molecules that regulate gene expression. There is increasing evidence of their importance in polycystic ovary syndrome (PCOS). The objective was to determine if miRNA-93 and miRNA-223 are differentially expressed in the circulation of women with PCOS compared to age matched women. A case-control study comparing women with PCOS (n = 25) to age and weight matched controls (n = 24) without PCOS was performed. MiRNA-93 and miRNA-223 were determined by total RNA reverse transcription. Both miRNA-93 and miRNA-223 were significantly increased relative to the control group (p < 0.01, p = 0.029 respectively). In both groups there was no correlation of either miRNA-93 or miRNA-223 with insulin, HOMA-IR, HOMA-β or testosterone levels. The area under the receiver operator characteristic curve for miR-223 and miR-93 was 0.66 and 0.72 respectively, suggesting miR-93 is a more efficient biomarker than miR-223 for diagnosis of PCOS. The combination of the two miRNAs together, tested using multiple logistic regression analysis, did not improve the diagnostic potential. In conclusion, circulating miRNA-93 and miRNA-223 were higher in women with PCOS compared to age and weight matched controls independent of insulin resistance and testosterone levels, and miR-93 may represent a novel diagnostic biomarker for PCOS.

  12. Wheat germ extract decreases glucose uptake and RNA ribose formation but increases fatty acid synthesis in MIA pancreatic adenocarcinoma cells.

    PubMed

    Boros, L G; Lapis, K; Szende, B; Tömösközi-Farkas, R; Balogh, A; Boren, J; Marin, S; Cascante, M; Hidvégi, M

    2001-08-01

    The fermented wheat germ extract with standardized benzoquinone composition has potent tumor propagation inhibitory properties. The authors show that this extract induces profound metabolic changes in cultured MIA pancreatic adenocarcinoma cells when the [1,2-13C2]glucose isotope is used as the single tracer with biologic gas chromatography-mass spectrometry. MIA cells treated with 0.1, 1, and 10 mg/mL wheat germ extract showed a dose-dependent decrease in cell glucose consumption. uptake of isotope into ribosomal RNA (2.4%, 9.4%, and 28.0%), and release of 13CO2. Conversely, direct glucose oxidation and ribose recycling in the pentose cycle showed a dose-dependent increase of 1.2%, 20.7%, and 93.4%. The newly synthesized fraction of cell palmitate and the 13C enrichment of acetyl units were also significantly increased with all doses of wheat germ extract. The fermented wheat germ extract controls tumor propagation primarily by regulating glucose carbon redistribution between cell proliferation-related and cell differentiation-related macromolecules. Wheat germ extract treatment is likely associated with the phosphorylation and transcriptional regulation of metabolic enzymes that are involved in glucose carbon redistribution between cell proliferation-related structural and functional macromolecules (RNA, DNA) and the direct oxidative degradation of glucose, which have devastating consequences for the proliferation and survival of pancreatic adenocarcinoma cells in culture.

  13. The long non-coding RNA HOTAIR increases tumour growth and invasion in cervical cancer by targeting the Notch pathway

    PubMed Central

    Kim, Sang Wun; Park, Sun-Ae; Chun, Kyung-Hee; Cho, Nam Hoon; Song, Yong Sang; Kim, Young Tae

    2016-01-01

    Evidence suggests that the long non-coding RNA (lncRNA), HOTAIR, is involved in cervical cancer pathogenesis. We examined serum HOTAIR expression levels in cervical cancer patients and determined the relationships between HOTAIR expression and several clinicopathological factors, including survival. We also examined the functional consequences of HOTAIR overexpression both in vitro and in vivo. Compared with control patients, HOTAIR expression was significantly greater in the serum of cervical cancer patients (P < 0.001). The results indicated that this increase was significantly associated with tumour size (P = 0.030), lymphovascular space invasion (P = 0.037), and lymph node metastasis (P = 0.043). Univariate analysis revealed that disease-free survival and overall survival times were significantly shorter in cervical cancer patients with high HOTAIR expression (hazard ratio [HR] = 4.27, 4.68 and P = 0.039, 0.031, respectively). Cell proliferation and invasion in vitro increased as a result of lentiviral-mediated HOTAIR overexpression in cervical cancer cell lines. HOTAIR knockdown inhibited these properties and increased apoptosis. In vivo xenograft experiments using the HOTAIR-overexpressing SiHa cell line revealed that HOTAIR was a strong inducer of tumour growth and modulated the expression of epithelial-mesenchymal transition and Notch-Wnt signalling pathway-related genes. This result suggested that HOTAIR overexpression promoted cell proliferation and invasion. In conclusion, increased HOTAIR expression was associated with decreased patient survival times. HOTAIR may be a useful target for treatment of cervical cancer patients. PMID:27323817

  14. Acute simulated soccer-specific training increases PGC-1α mRNA expression in human skeletal muscle.

    PubMed

    Jeong, Tae-Seok; Bartlett, Jonathan D; Joo, Chang-Hwa; Louhelainen, Jari; Close, Graeme L; Morton, James P; Drust, Barry

    2015-01-01

    The aim of the current study was to quantify oxygen uptake, heart rate and molecular responses of human skeletal muscle associated with mitochondrial biogenesis following an acute bout of simulated soccer training. Muscle biopsies (vastus lateralis) were obtained from nine active men immediately pre-completion, post-completion and 3 h post-completion of a laboratory-based soccer-specific training simulation on a motorised treadmill. The soccer-specific simulation was a similar intensity (55 ± 6% [Formula: see text]) and duration (60 min) as that observed in professional soccer training (e.g. standing 41%, walking 37%, jogging 11%, high-speed running 9% and sprinting 2%). Post-exercise, muscle glycogen decreased (Pre; 397 ± 86 mmol∙kg(-1) dw, Post; 344 ± 64 mmol∙kg(-1) dw; P = 0.03), plasma lactate increased (P < 0.001) up to ~4-5 mmol∙L(-1), non-esterified fatty acids and glycerol increased (P < 0.001) to values of 0.6 ± 0.2 mmol∙L(-1) and 145 ± 54 μmol∙L(-1), respectively. PGC-1α mRNA increased (P = 0.009) fivefold 3 h post-exercise. We provide novel data by demonstrating that soccer-specific training is associated with increases in PGC-1α mRNA. These data may have implications for practitioners in better understanding the metabolic and muscle responses to soccer-specific training protocols in the field.

  15. Down-regulation of let-7 microRNA increased K-ras expression in lung damage induced by radon.

    PubMed

    Chen, Zhihai; Wang, Dapeng; Gu, Chao; Liu, Xing; Pei, Weiwei; Li, Jianxiang; Cao, Yi; Jiao, Yang; Tong, Jian; Nie, Jihua

    2015-09-01

    Radon has long been recognized as a human carcinogen leading to lung cancer, but the underlying mechanisms remain obscure. Recent studies have shown that the let-7 microRNA and K-ras play an important role in the development of various cancers. However, the exact role between let-7 and K-ras in radon induced lung damage has not been explored so far. In the present study, wistar rats and human bronchial epithelial (HBE) cells were long-term exposed to radon, and then alterations in histological pathology of rat lung tissue, ROS, antioxidant enzymes activities and clonogenic formation in HBE cells, as well as changes in let-7 and K-ras expression were determined to observe the adverse effects induced by radon. The results showed that long-term exposure to radon produced severe lung damage in rats, significantly increased ROS production and clonogenic formation ratios and decreased SOD activities in HBE cells. In addition, an obvious down-regulation of let-7 and up-regulation of K-ras were also revealed both in mRNA and in protein level in lung tissue of rats and HBE cells exposed to radon. Furthermore, a significant down-regulation of K-ras was then confirmed in both let-7b-3p and let-7a-2-3p transfected HBE cells. Taken together, the present results propose an involvement of let-7 microRNA and K-ras in radon induced lung damage both in vivo and in vitro, which may thus be of potential value in early diagnosis and therapy of radon-induced lung tumorgenesis.

  16. Discrimination against interfering signals at the Poker Flat MST radar

    NASA Technical Reports Server (NTRS)

    Carter, D. A.

    1983-01-01

    Several on line and off line data processing techniques are used to remove interfering signals due to ground clutter, aircraft, instrumental effects, and external transmissions from the desired atmospheric echoes of Mesosphere Stratosphere, Troposphere (MST) radar. The on line, real time techniques are necessarily simple in order to minimize processing delays. This algorithm examines the individual Doppler spectra which are computed every two to four seconds (for oblique antenna beams). The total spectral power in each individual spectrum is computed by summing all the spectral points. If this integrated power increases from one spectrum to the next by a factor greater than a preselected threshold, then that spectrum is not added to the spectral sum. Succeeding spectra are compared to the last acceptable spectrum. Only a certain maximum number of spectra are allowed to be rejected in succession.

  17. 32 CFR 1903.8 - Interfering with Agency functions.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 32 National Defense 6 2012-07-01 2012-07-01 false Interfering with Agency functions. 1903.8 Section 1903.8 National Defense Other Regulations Relating to National Defense CENTRAL INTELLIGENCE AGENCY CONDUCT ON AGENCY INSTALLATIONS § 1903.8 Interfering with Agency functions. The following are...

  18. 32 CFR 1903.8 - Interfering with Agency functions.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 32 National Defense 6 2014-07-01 2014-07-01 false Interfering with Agency functions. 1903.8 Section 1903.8 National Defense Other Regulations Relating to National Defense CENTRAL INTELLIGENCE AGENCY CONDUCT ON AGENCY INSTALLATIONS § 1903.8 Interfering with Agency functions. The following are...

  19. 32 CFR 1903.8 - Interfering with Agency functions.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 32 National Defense 6 2013-07-01 2013-07-01 false Interfering with Agency functions. 1903.8 Section 1903.8 National Defense Other Regulations Relating to National Defense CENTRAL INTELLIGENCE AGENCY CONDUCT ON AGENCY INSTALLATIONS § 1903.8 Interfering with Agency functions. The following are...

  20. 32 CFR 1903.8 - Interfering with Agency functions.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 32 National Defense 6 2011-07-01 2011-07-01 false Interfering with Agency functions. 1903.8 Section 1903.8 National Defense Other Regulations Relating to National Defense CENTRAL INTELLIGENCE AGENCY CONDUCT ON AGENCY INSTALLATIONS § 1903.8 Interfering with Agency functions. The following are...

  1. 32 CFR 1903.8 - Interfering with Agency functions.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 32 National Defense 6 2010-07-01 2010-07-01 false Interfering with Agency functions. 1903.8 Section 1903.8 National Defense Other Regulations Relating to National Defense CENTRAL INTELLIGENCE AGENCY CONDUCT ON AGENCY INSTALLATIONS § 1903.8 Interfering with Agency functions. The following are...

  2. Multiple mutations and increased RNA expression in tetracycline-resistant Streptococcus pneumoniae as determined by genome-wide DNA and mRNA sequencing

    PubMed Central

    Lupien, Andréanne; Gingras, Hélène; Bergeron, Michel G.; Leprohon, Philippe; Ouellette, Marc

    2015-01-01

    Objectives The objective of this study was to characterize chromosomal mutations associated with resistance to tetracycline in Streptococcus pneumoniae. Methods Chronological appearance of mutations in two S. pneumoniae R6 mutants (R6M1TC-5 and R6M2TC-4) selected for resistance to tetracycline was determined by next-generation sequencing. A role for the mutations identified was confirmed by reconstructing resistance to tetracycline in a S. pneumoniae R6 WT background. RNA sequencing was performed on R6M1TC-5 and R6M2TC-4 and the relative expression of genes was reported according to R6. Differentially expressed genes were classified according to their ontology. Results WGS of R6M1TC-5 and R6M2TC-4 revealed mutations in the gene rpsJ coding for the ribosomal protein S10 and in the promoter region and coding sequences of the ABC genes patA and patB. These cells were cross-resistant to ciprofloxacin. Resistance reconstruction confirmed a role in resistance for the mutations in rpsJ and patA. Overexpression of the ABC transporter PatA/PatB or mutations in the coding sequence of patA contributed to resistance to tetracycline, ciprofloxacin and ethidium bromide, and was associated with a decreased accumulation of [3H]tetracycline. Comparative transcriptome profiling of the resistant mutants further revealed that, in addition to the overexpression of patA and patB, several genes of the thiamine biosynthesis and salvage pathway were increased in the two mutants, but also in clinical isolates resistant to tetracycline. This overexpression most likely contributes to the tetracycline resistance phenotype. Conclusions The combination of genomic and transcriptomic analysis coupled to functional studies has allowed the discovery of novel tetracycline resistance mutations in S. pneumoniae. PMID:25862682

  3. (Dicer)phering roles of microRNA in platelets.

    PubMed

    Boilard, Eric; Belleannée, Clémence

    2016-04-07

    In this issue of Blood, Rowley et al report that noncoding RNAs precisely regulate the messenger RNA (mRNA) profile in platelets. Interfering in this process using genetically engineered mice affects hemostatic and thrombotic functions of platelets.

  4. Increased abundance of aromatase and follicle stimulating hormone receptor mRNA and decreased insulin-like growth factor-2 receptor mRNA in small ovarian follicles of cattle selected for twin births.

    PubMed

    Echternkamp, S E; Aad, P Y; Eborn, D R; Spicer, L J

    2012-07-01

    Cattle genetically selected for twin ovulations and births (Twinner) exhibit increased ovarian follicular development, increased ovulation rate, and greater blood and follicular fluid IGF-1 concentrations compared with contemporary cattle not selected for twins (Control). Experimental objectives were to 1) assess relationships among aromatase (CYP19A1), IGF-1 (IGF1), IGF-2 receptor (IGF2R), and FSH receptor (FSHR) mRNA expression in small (≤5 mm) antral follicles and 2) determine their association with increased numbers of developing follicles in ovaries of Twinner females. Ovaries were collected from mature, cyclic (d 3 to 6) Twinner (n = 11), and Control (n = 12) cows at slaughter and pieces of cortical tissue were fixed and embedded in paraffin. Expression of mRNA was evaluated by in situ hybridization using (35)S-UTP-labeled antisense and sense probes for CYP19A1, FSHR, IGF1, and IGF2R mRNA. Silver grain density was quantified within the granulosa and theca cells of individual follicles (2 to 7 follicles/cow) by Bioquant image analysis. Follicles of Twinners tended to be smaller in diameter than Controls (1.9 ± 0.1 vs. 2.3 ± 0.1 mm; P = 0.08), but thickness of granulosa layer did not differ (P > 0.1) by genotype. Relative abundance of CYP19A1 (P < 0.01) and FSHR (P < 0.05) mRNA was greater in granulosa cells of Twinners vs. Controls, respectively, whereas IGF2R mRNA expression was less in both granulosa (P < 0.01) and theca (P < 0.05) cells in follicles of Twinners vs. Controls, respectively. Abundance of CYP19A1 mRNA in granulosa cells was correlated negatively with IGF2R mRNA expression in both granulosa (r = -0.33; P < 0.01) and theca (r = -0.21; P = 0.05) cells. Expression of IGF1 mRNA was primarily in granulosa cells, including cumulus cells, and its expression did not differ between Twinners vs. Controls (P > 0.10). Detected increases in CYP19A1 and FSHR, but not IGF1, mRNA expression along with decreases in IGF2R mRNA expression in individual

  5. Increased sensitivity of RT-PCR for Potato virus Y detection using RNA isolated by a procedure with differential centrifugation.

    PubMed

    Zhang, Jianhua; Nie, Xianzhou; Boquel, Sébastien; Al-Daoud, Fadi; Pelletier, Yvan

    2015-12-01

    The sensitivity of reverse transcription-polymerase chain reaction (RT-PCR) for virus detection is influenced by many factors such as specificity of primers and quality of templates. These factors become extremely important for successful detection when virus concentration is low. Total RNA isolated from Potato virus Y (PVY)-infected potato plants using the sodium sulfite RNA isolation method or RNeasy plant mini kit contains a high proportion of host RNA and may also contain trace amount of phenolic and polysaccharide residues, which may inhibit RT-PCR. The goal of this study was to enhance the sensitivity of PVY detection by reducing host RNA in the extract by differential centrifugation followed by extraction using an RNeasy mini kit (DCR method). One-step RT-PCR had relatively low amplification efficiency for PVY RNA when a high proportion of plant RNA was present. SYBR Green-based real time RT-PCR showed that the RNA isolated by the DCR method had a higher cycle threshold value (Ct) for the elongation factor 1-α mRNA (Ef1α) of potato than the Ct value of the RNA extracted using the RNeasy plant mini kit, indicating that the DCR method significantly reduced the proportion of potato RNA in the extract. The detectable amount of RNA extracted using the DCR method was <0.001ng when plant sap from 10 PVY-infected and PVY-free potato leaflets in a 1.5:100 fresh weight ratio was extracted, compared with 0.01 and 0.02ng of RNA using the RNeasy plant mini kit and sodium sulfite RNA isolation methods, respectively.

  6. Use of small interfering ribonucleic acids to inhibit the adipogenic effect of alcohol on human bone marrow-derived mesenchymal cells.

    PubMed

    Huang, Qiang; Zhang, Hui; Pei, Fu-xing; Chen, Zhi-yu; Wang, Guang-lin; Shen, Bin; Yang, Jing; Zhou, Zong-ke; Kong, Qing-quan

    2010-10-01

    This study tested the potential of small interfering RNAs (siRNA) targeting human peroxisome proliferator activated receptor gamma (PPARγ) to repress the adipogenic effect of alcohol on human bone marrow-derived mesenchymal cells (hBMSCs). hBMSCs were cultured from hip replacement surgery patients (n = 10). PPARγ-siRNA was transiently transfected into hBMSCs cultured in ostogenic media containing 50 mM alcohol by using a liposome-based strategy. Oil red O staining was used to test the development of differentiated adipocytes, and Alizarin red staining was used to test mineral deposition. Marker genes of adipogenesis (PPARγ2 and aP2) and osteogenesis (Osf2/Cbfa1) were examined through real time RT-PCR and Western blot, respectively. Collagen type I, alkaline phosphatase and osteocalcin protein synthesis of cultures were also assayed. Data were presented as mean ± SD. Differences between the means of the treatment groups were determined with ANOVA. PPARγ-siRNA transfection resulted in significantly lower adipocyte number, increased matrix mineralisation, repressed adipogenic gene markers, up-regulated osteogenic gene marker and bone matrix protein synthesis in the PPARγ-siRNA group compared to controls (P < 0.05). PPARγ-siRNA is a useful strategy to inhibit the adipogenic effect and the osteogenic repression of alcohol on hBMSCs. This may be a novel therapeutic intervention for osteopenic disorders in alcoholism and other conditions.

  7. Gallium nitrate increases type I collagen and fibronectin mRNA and collagen protein levels in bone and fibroblast cells.

    PubMed

    Bockman, R S; Guidon, P T; Pan, L C; Salvatori, R; Kawaguchi, A

    1993-08-01

    Gallium is a Group IIIa transitional element with therapeutic efficacy in the treatment of metabolic bone disorders. Previously described antiresorptive effects of gallium on osteoclasts are not sufficient to account for the full range of effects of gallium on bone structure and metabolism. We have recently shown that gallium nitrate inhibits osteocalcin gene expression and the synthesis of osteocalcin protein, an osteoblast-specific bone matrix protein that is thought to serve as a signal to trigger osteoclastic resorption. Here we present evidence for an additional mechanism by which gallium may function to augment bone mass by altering matrix protein synthesis by osteoblastic and fibroblastic cells. Rat calvarial explants exposed to gallium nitrate for 48 h showed increased incorporation of 3H-proline into hydroxyproline and collagenase digestible protein. In addition, gallium treatment increased steady-state mRNA levels for fibronectin and type I procollagen chains in primary rat calvarial osteoblast-enriched cultures, the ROS 17/2.8 osteoblastic osteosarcoma line, and nontransformed human dermal fibroblasts. These findings suggest that the exposure of mesenchymally-derived cells to gallium results in an altered pattern of matrix protein synthesis that would favor increased bone formation.

  8. High molecular weight RNAs and small interfering RNAs induce systemic posttranscriptional gene silencing in plants

    PubMed Central

    Klahre, Ulrich; Crété, Patrice; Leuenberger, Sabrina A.; Iglesias, Victor A.; Meins, Frederick

    2002-01-01

    Posttranscriptional gene silencing (PTGS) in transgenic plants is an epigenetic form of RNA degradation related to PTGS and RNA interference (RNAi) in fungi and animals. Evidence suggests that transgene loci and RNA viruses can generate double-stranded RNAs similar in sequence to the transcribed region of target genes, which then undergo endonucleolytic cleavage to generate small interfering RNAs (siRNA) that promote degradation of cognate RNAs. The silent state in transgenic plants and in Caenorhabditis elegans can spread systemically, implying that mobile silencing signals exist. Neither the chemical nature of these signals nor their exact source in the PTGS pathway is known. Here, we use a positive marker system and real-time monitoring of green fluorescent protein expression to show that large sense, antisense, and double-stranded RNAs as well as double-stranded siRNAs delivered biolistically into plant cells trigger silencing capable of spreading locally and systemically. Systemically silenced leaves show greatly reduced levels of target RNA and accumulate siRNAs, confirming that RNA can induce systemic PTGS. The induced siRNAs represent parts of the target RNA that are outside of the region of homology with the triggering siRNA. Our results imply that siRNAs themselves or intermediates induced by siRNAs could comprise silencing signals and that these signals induce self-amplifying production of siRNAs. PMID:12181491

  9. MicroRNA 29 Targets Nuclear Factor-κB–Repressing Factor and Claudin 1 to Increase Intestinal Permeability

    PubMed Central

    Zhou, QiQi; Costinean, Stefan; Croce, Carlo M.; Brasier, Alan R.; Merwat, Shehzad; Larson, Scott A.; Basra, Sarpreet; Verne, G. Nicholas

    2014-01-01

    BACKGROUND & AIMS Some patients with irritable bowel syndrome with diarrhea (IBS-D) have intestinal hyperpermeability, which contributes to their diarrhea and abdominal pain. MicroRNA 29 (MIR29) regulates intestinal permeability in patients with IBS-D. We investigated and searched for targets of MIR29 and investigated the effects of disrupting Mir29 in mice. METHODS We investigated expression MIR29A and B in intestinal biopsies collected during endoscopy from patients with IBS (n = 183) and without IBS (controls) (n = 36). Levels were correlated with disease phenotype. We also generated and studied Mir29−/− mice, in which expression of Mir29a and b, but not c, is lost. Colitis was induced by administration of 2,4,6-trinitrobenzenesulfonic acid; intestinal tissues were collected and permeability was assessed. Microarray analysis was performed using tissues from Mir29−/− mice. Changes in levels of target genes were measured in human colonic epithelial cells and small intestinal epithelial cells after knockdown of MIR29 with anti-MIRs. RESULTS Intestinal tissues from patients with IBS-D (but not IBS with constipation or controls) had increased levels of MIR29A and B, but reduced levels of Claudin-1 (CLDN1) and nuclear factor-κB–repressing factor (NKRF). Induction of colitis and water avoidance stress increased levels of Mir29a and Mir29b and intestinal permeability in wild-type mice; these increased intestinal permeability in colons of far fewer Mir29−/− mice. In microarray and knockdown experiments, MIR29A and B were found to reduce levels of NKRF and CLDN1 messenger RNA, and alter levels of other messenger RNAs that regulate intestinal permeability. CONCLUSIONS Based on experiments in knockout mice and analyses of intestinal tissue samples from patients with IBS-D, MIR29 targets and reduces expression of CLDN1 and NKRF to increase intestinal permeability. Strategies to block MIR29 might be developed to restore intestinal permeability in patients with

  10. The adipokine leptin increases skeletal muscle mass and significantly alters skeletal muscle miRNA expression profile in aged mice

    SciTech Connect

    Hamrick, Mark W.; Herberg, Samuel; Arounleut, Phonepasong; He, Hong-Zhi; Shiver, Austin; Qi, Rui-Qun; Zhou, Li; Isales, Carlos M.; and others

    2010-09-24

    Research highlights: {yields} Aging is associated with muscle atrophy and loss of muscle mass, known as the sarcopenia of aging. {yields} We demonstrate that age-related muscle atrophy is associated with marked changes in miRNA expression in muscle. {yields} Treating aged mice with the adipokine leptin significantly increased muscle mass and the expression of miRNAs involved in muscle repair. {yields} Recombinant leptin therapy may therefore be a novel approach for treating age-related muscle atrophy. -- Abstract: Age-associated loss of muscle mass, or sarcopenia, contributes directly to frailty and an increased risk of falls and fractures among the elderly. Aged mice and elderly adults both show decreased muscle mass as well as relatively low levels of the fat-derived hormone leptin. Here we demonstrate that loss of muscle mass and myofiber size with aging in mice is associated with significant changes in the expression of specific miRNAs. Aging altered the expression of 57 miRNAs in mouse skeletal muscle, and many of these miRNAs are now reported to be associated specifically with age-related muscle atrophy. These include miR-221, previously identified in studies of myogenesis and muscle development as playing a role in the proliferation and terminal differentiation of myogenic precursors. We also treated aged mice with recombinant leptin, to determine whether leptin therapy could improve muscle mass and alter the miRNA expression profile of aging skeletal muscle. Leptin treatment significantly increased hindlimb muscle mass and extensor digitorum longus fiber size in aged mice. Furthermore, the expression of 37 miRNAs was altered in muscles of leptin-treated mice. In particular, leptin treatment increased the expression of miR-31 and miR-223, miRNAs known to be elevated during muscle regeneration and repair. These findings suggest that aging in skeletal muscle is associated with marked changes in the expression of specific miRNAs, and that nutrient

  11. Amphiphysin I but not dynamin I nor synaptojanin mRNA expression increased after repeated methamphetamine administration in the rat cerebrum and cerebellum.

    PubMed

    Hamamura, Mitsuko; Okouchi, Jiro; Ozawa, Hidetoshi; Kimuro, Yoshihiko; Iwaki, Akiko; Fukumaki, Yasuyuki

    2013-07-01

    Dopamine increases/decreases synaptic vesicle recycling and in schizophrenia the proteins/mRNA is decreased. We isolated cDNA clone, similar to amphiphysin 1 (vesicle protein) mRNA from the neocortex of rats injected repeatedly with methamphetamine using polymerase chain reaction (PCR) differential display. This clone is highly homologous to the 3' region of the human amphiphysin gene. PCR extension study using a primer specific for the rat amphiphysin 1 gene and a primer located within the clone revealed that it is the 3' UTR region of the rat amphiphysin 1 gene. Furthermore, in situ hybridization revealed that amphiphysin 1 mRNA is expressed in the cerebrum, medial thalamus, hippocampus and cerebellum. In the cerebellum, amphiphysin mRNA expression was confined to upper granule cell layer. Repeated methamphetamine administration increased amphiphysin I mRNA expression in both anterior part of the cerebrum, and the cerebellum. However, the repeated administration did not alter mRNA expression of the other vesicle proteins, synaptotagmin I, synapsin I, synaptojanin and dynamin I, we conclude that the repeated administration selectively increased amphiphysin 1 mRNA expression. Thus, amphiphysin 1 does not work as synaptic recycling, but it is suggested, as a part of pathogenesis of brain tissue injury (under Ca²⁺ and Mg²⁺ devoid environment) in repeated methamphetamine-injected states, the gene regulate actin-asssembly, learning, cell stress signaling and cell polarity.

  12. RNA interference-mediated targeting of DKK1 gene expression in Ishikawa endometrial carcinoma cells causes increased tumor cell invasion and migration.

    PubMed

    Yi, Nuo; Liao, Qin-Ping; Li, Zhen-Hua; Xie, Bao-Jiang; Hu, Yu-Hong; Yi, Wei; Liu, Min

    2013-09-01

    The Wnt signaling pathway plays an essential role in tumor invasion and migration. DKK1 functions as an important inhibitor of the pathway and represents a promising target for cancer therapy. The aim of the present study was to determine the role of DKK1 in endometrial carcinoma (EC) cell invasion and migration using RNA interference (RNAi) technology. Ishikawa EC cells were transfected at high efficiency with specific DKK1 siRNA. RT-PCR and western blot analysis were used to determine the mRNA and protein levels of DKK1, β-catenin and metalloproteinase 14 (MMP14) in siRNA-treated and -untreated cells. In addition, the invasion and migration of the EC cells were detected by invasion and migration assays. Transient transfection of DKK1 siRNA significantly inhibited the mRNA and protein levels of DKK1. Markedly increased cell invasion and migration was observed following treatment with DKK1 siRNA when compared with the negative control siRNA-treated and siRNA-untreated cells. The knockdown of DKK1 also elevated the mRNA and protein levels of β-catenin and MMP14 involved in the Wnt signaling pathway, indicating that targeting this gene may promote intracellular Wnt signal transduction and thus, accelerate EC cell invasion and migration in vitro. The RNAi-mediated targeting of DKK1 gene expression in Ishikawa EC cells resulted in increased tumor cell invasion and migration. DKK1 was identified as an inhibitor of EC cell invasion and migration via its novel role in the Wnt signaling pathway. Targeting DKK1 may therefore represent an effective anti-invasion and -migration strategy for the treatment of EC.

  13. Knockdown of PKD1 in normal human epidermal keratinocytes increases mRNA expression of keratin 10 and involucrin: early markers of keratinocyte differentiation.

    PubMed

    Ivanova, Petya; Atanasova, Ganka; Poumay, Yves; Mitev, Vanyo

    2008-03-01

    Subconfluent normal human keratinocytes exhibit autonomous (autocrine growth factor driven) proliferation and express the specific markers for keratinocyte proliferation K5 (keratin 5) and K14 (keratin 14). Utilizing this model the effects of PKD1 (Protein kinase D1) knockdown on activation of differentiation was studied. siRNA approach was applied to achieve specific knockdown of PKD1 and the mRNA levels of different keratinocyte markers -- K14 and PCNA (markers of basal proliferating keratinocytes), involucrin and K10 (early differentiation markers) were analyzed. Treatment of cultured keratinocytes with siRNA for PKD1 resulted in reduction of mRNA levels of PKD1, altered cell phenotype and promotion of keratinocyte differentiation, demonstrated by increased expression of involucrin and K10 mRNAs. No significant changes in K14 mRNA expression levels were detected, but the expression of PCNA mRNA was markedly diminished. This study was the first to show that mRNA expression of PKD1 in subconfluent normal human keratinocytes is very low, the PKD1 mRNA levels were more than 8-fold lower than the same ones in hTert keratinocytes. These findings suggest antidifferentiative role of PKD1 in normal human keratinocytes, contrary to the prodiferentiative role of PKD1 in human hTert keratinocytes. We came to the conclusion that there are differences between transduction pathways involving PKD1 in primary human keratinocyte cultures and these in immortalized hTert keratinocytes.

  14. Green tea extract increases cyclophosphamide-induced teratogenesis by modulating the expression of cytochrome P-450 mRNA.

    PubMed

    Park, Dongsun; Jeon, Jeong Hee; Shin, Sunhee; Joo, Seong Soo; Kang, Dae-Hyuck; Moon, Seol-Hee; Jang, Min-Jung; Cho, Yeoung Mi; Kim, Jae Wook; Ji, Hyeong-Jin; Ahn, Byeongwoo; Oh, Ki-Wan; Kim, Yun-Bae

    2009-01-01

    The effects of green tea extract (GTE) on the fetal development and external, visceral and skeletal abnormalities induced by cyclophosphamide were investigated in rats. Pregnant rats were daily administered GTE (100mg/kg) by gavage for 7 d, from the 6th to 12th day of gestation, and intraperitoneally administered with cyclophosphamide (11mg/kg) 1h after the final treatment. On the 20th day of gestation, maternal and fetal abnormalities were determined by Cesarian section. Cyclophosphamide was found to reduce fetal and placental weights without increasing resorption or death. In addition, it induced malformations in live fetuses; 94.6%, 41.5% and 100% of the external (skull and limb defects), visceral (cleft palate and ureteric dilatation) and skeletal (acrania, vertebral/costal malformations and delayed ossification) abnormalities. When pre-treated with GTE, cyclophosphamide-induced body weight loss and abnormalities of fetuses were remarkably aggravated. Moreover, repeated treatment with GTE greatly increased mRNA expression and activity of hepatic cytochrome P-450 (CYP) 2B, which metabolizes cyclophosphamide into teratogenic acrolein and cytotoxic phosphoramide mustard, while reducing CYP3A expression (a detoxifying enzyme). The results suggest that repeated intake of GTE may aggravate cyclophosphamide-induced body weight loss and malformations of fetuses by modulating CYP2B and CYP3A.

  15. RNA polymerase II forms transcription networks in rye and Arabidopsis nuclei and its amount increases with endopolyploidy.

    PubMed

    Schubert, Veit

    2014-01-01

    RNA polymerase II (RNAPII) is responsible for the transcription of most eukaryotic genes. In mammalian nuclei, RNAPII is mainly localized in relatively few distinct transcription factories. In this study--applying super-resolution microscopy--it is shown that in plants, inactive (non-phosphorylated) and active (phosphorylated) RNAPII modifications compose distinct 'transcription networks' within the euchromatin. These reticulate structures sometimes attach to each other, but they are absent from heterochromatin and nucleoli. The global RNAPII distribution within nuclei is not influenced by interphase chromatin organization such as Rabl (rye) versus non-Rabl (Arabidopsis thaliana) orientation. Replication of sister chromatids without cell division causes endopolyploidy, a phenomenon widespread in plants and animals. Endopolyploidy raises the number of gene copies per nucleus. Here, it is shown that the amounts of active and inactive RNAPII enzymes in differentiated 2-32C leaf nuclei of A. thaliana proportionally increase with rising endopolyploidy. Thus, increasing the transcriptional activity of cells and tissues seems to be an important function of endopolyploidy.

  16. Specific binding of Fusarium graminearum Hex1 protein to untranslated regions of the genomic RNA of Fusarium graminearum virus 1 correlates with increased accumulation of both strands of viral RNA.

    PubMed

    Son, Moonil; Choi, Hoseong; Kim, Kook-Hyung

    2016-02-01

    The HEX1 gene of Fusarium graminearum was previously reported to be required for the efficient accumulation of Fusarium graminearum virus 1 (FgV1) RNA in its host. To investigate the molecular mechanism underlying the production of FgHEX1 and the replication of FgV1 viral RNA, we conducted electrophoretic mobility shift assays (EMSA) with recombinant FgHex1 protein and RNA sequences derived from various regions of FgV1 genomic RNA. These analyses demonstrated that FgHex1 and both the 5'- and 3'-untranslated regions of plus-strand FgV1 RNA formed complexes. To determine whether FgHex1 protein affects FgV1 replication, we quantified accumulation viral RNAs in protoplasts and showed that both (+)- and (-)-strands of FgV1 RNAs were increased in the over-expression mutant and decreased in the deletion mutant. These results indicate that the FgHex1 functions in the synthesis of both strands of FgV1 RNA and therefore in FgV1 replication probably by specifically binding to the FgV1 genomic RNA.

  17. Increased rDNA synthesis in germinated conidia of Neurospora crassa is caused by RNA primer molecules found in its culture medium

    SciTech Connect

    Dutta, S.K.; Beljanski, M.

    1984-01-01

    Purine rich small primer RNA molecules (10-15 nucleotides) were isolated from growth medium of germinated (3 hr sprout) conidia of N. crassa. These RNA-primer molecules strongly stimulated in vitro DNA synthesis in N. crassa 74A wild type, as well as in DNAs from mice spleen and lung, and quail testis. These increases of in vitro DNA synthesis was dependent on the concentration of these RNA primer molecules. In contrast, such molecules were not found in 1 or 10 hour sprouts, nor in the culture medium of mycelia (24 hr). These RNA-primer molecules could be hydrolyzed by T1 RNAse but not by pancreatic RNase. Dutta et al. reported increased (250) copies of rRNA genes in germinated conidia (3 hr sprouts) compared to 100 copies of rRNA genes in mycelial cells grown for 24 hours. These observations suggest excessive transcription of rDNAs in the germinated conidial cells which undergo cleavages by nucleates after 3-4 hours of cell growth. Some degradation products were excreted into the culture medium and acted as RNA-primers.

  18. Increased New lncRNA-mRNA Gene Pair Levels in Human Cumulus Cells Correlate With Oocyte Maturation and Embryo Development.

    PubMed

    Li, Juan; Cao, Yunxia; Xu, Xiaofeng; Xiang, Huifen; Zhang, Zhiguo; Chen, Beili; Hao, Yan; Wei, Zhaolian; Zhou, Ping; Chen, Dawei

    2015-08-01

    The close relationship between cumulus cells and oocyte indicates that the analysis of cumulus gene expression is a potential noninvasive method to aid embryo selection and in vitro fertilization outcome. Long noncoding RNAs (LncRNAs) could regulate essential pathways that contribute to human oocyte maturation, fertilization, and embryo development, which indicates that lncRNA would be valuable biomarkers. In our previous study, AK124742 is a newly detected lncRNA that was identified as being natural antisense to PSMD6, but its role in oocyte and embryo development is still not elucidated and needs to be investigated. Here, the expression of AK124742 and PSMD6 was measured in 40 pairs of cumulus cells from oocytes that result in high-quality embryos (HCCs) and from oocytes that result in poor-quality embryos (PCCs) by real-time quantitative reverse transcriptase polymerase chain reaction. The predictive value of AK124742 and PSMD6 was evaluated using a receiver-operating characteristic (ROC) curve. Notably, elevated expression levels of AK124742 and PSMD6 were observed in HCCs compared to PCCs (72.5% and 62.5%, respectively; P < .01). Expression of AK124742 was potentially positively associated with the PSMD6 levels. The relative expression levels of AK124742 and PSMD6 in the pregnancy group were significantly higher than those in the nonpregnancy group (P < .01).The area under the ROC curve of AK124742 was 0.78 (95% confidence interval: 0.64-0.93). In conclusion, AK124742 and PSMD6 as a new lncRNA-messenger RNA gene pair in human cumulus cells may be considered as potential biomarkers to aid embryo selection.

  19. Synthesis of small interfering RNAs containing acetal-type nucleoside analogs at their 3'-ends and analysis of their silencing activity and their ability to bind to the Argonaute2 PAZ domain.

    PubMed

    Inada, Natsumi; Nakamoto, Kosuke; Yokogawa, Takashi; Ueno, Yoshihito

    2015-10-20

    In this study, we aimed to create small interfering RNAs (siRNAs) with increased silencing activities and nuclease resistance properties. Therefore, we designed and synthesized five types of siRNA containing acetal-type nucleoside analogs at their 3'-dangling ends. We found that the siRNA containing 1-O-(2,2,2-trifluoroethyl)-β-D-ribofuranose at the 3'-dangling end was the most potent among the synthesized siRNAs and showed more resistance to nucleolytic degradation by a 3' exonuclease than a natural RNA did. Thus, modification of siRNAs by addition of 1-O-(2,2,2-trifluoroethyl)-β-D-ribofuranose may hold promise as a means of improving the silencing activity and nuclease resistance of siRNAs.

  20. 47 CFR 73.185 - Computation of interfering signal.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... interfering station to be considered as pertinent to transmission by one reflection. To provide for variation... angle than the pertinent angle for one reflection, the method of calculating interference will not...

  1. 47 CFR 73.185 - Computation of interfering signal.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... interfering station to be considered as pertinent to transmission by one reflection. To provide for variation... angle than the pertinent angle for one reflection, the method of calculating interference will not...

  2. 47 CFR 73.185 - Computation of interfering signal.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... interfering station to be considered as pertinent to transmission by one reflection. To provide for variation... angle than the pertinent angle for one reflection, the method of calculating interference will not...

  3. 47 CFR 73.185 - Computation of interfering signal.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... interfering station to be considered as pertinent to transmission by one reflection. To provide for variation... angle than the pertinent angle for one reflection, the method of calculating interference will not...

  4. A high level of transgenic viral small RNA is associated with broad potyvirus resistance in cucurbits.

    PubMed

    Leibman, Diana; Wolf, Dalia; Saharan, Vinod; Zelcer, Aaron; Arazi, Tzahi; Yoel, Shiboleth; Gaba, Victor; Gal-On, Amit

    2011-10-01

    Gene-silencing has been used to develop resistance against many plant viruses but little is known about the transgenic small-interfering RNA (t-siRNA) that confers this resistance. Transgenic cucumber and melon lines harboring a hairpin construct of the Zucchini yellow mosaic potyvirus (ZYMV) HC-Pro gene accumulated different levels of t-siRNA (6 to 44% of total siRNA) and exhibited resistance to systemic ZYMV infection. Resistance to Watermelon mosaic potyvirus and Papaya ring spot potyvirus-W was also observed in a cucumber line that accumulated high levels of t-siRNA (44% of total siRNA) and displayed significantly increased levels of RNA-dependent RNA (RDR)1 and Argonaute 1, as compared with the other transgenic and nontransformed plants. The majority of the t-siRNA sequences were 21 to 22 nucleotides in length and sense strand biased. The t-siRNA were not uniformly distributed throughout the transgene but concentrated in "hot spots" in a pattern resembling that of the viral siRNA peaks observed in ZYMV-infected cucumber and melon. Mutations in ZYMV at the loci associated with the siRNA peaks did not break this resistance, indicating that hot spot t-siRNA may not be essential for resistance. This study shows that resistance based on gene-silencing can be effective against related viruses and is probably correlated with t-siRNA accumulation and increased expression of RDR1.

  5. Gene expression in scrapie. Cloning of a new scrapie-responsive gene and the identification of increased levels of seven other mRNA transcripts.

    PubMed

    Dandoy-Dron, F; Guillo, F; Benboudjema, L; Deslys, J P; Lasmézas, C; Dormont, D; Tovey, M G; Dron, M

    1998-03-27

    To define genes associated with or responsible for the neurodegenerative changes observed in transmissible spongiform encephalopathies, we analyzed gene expression in scrapie-infected mouse brain using "mRNA differential display." The RNA transcripts of eight genes were increased 3-8-fold in the brains of scrapie-infected animals. Five of these genes have not previously been reported to exhibit increased expression in this disease: cathepsin S, the C1q B-chain of complement, apolipoprotein D, and two previously unidentified genes denominated scrapie-responsive gene (ScRG)-1 and ScRG-2, which are preferentially expressed in brain tissue. Increased expression of the three remaining genes, beta2 microglobulin, F4/80, and metallothionein II, has previously been reported to occur in experimental scrapie. Kinetic analysis revealed a concomitant increase in the levels of ScRG-1, cathepsin S, the C1q B-chain of complement, and beta2 microglobulin mRNA as well as glial fibrillary acidic protein and F4/80 transcripts, markers of astrocytosis and microglial activation, respectively. In contrast, the level of ScRG-2, apolipoprotein D, and metallothionein II mRNA was only increased at the terminal stage of the disease. ScRG-1 mRNA was found to be preferentially expressed in glial cells and to code for a short protein of 47 amino acids with a strong hydrophobic N-terminal region.

  6. Naked mole-rat has increased translational fidelity compared with the mouse, as well as a unique 28S ribosomal RNA cleavage.

    PubMed

    Azpurua, Jorge; Ke, Zhonghe; Chen, Iris X; Zhang, Quanwei; Ermolenko, Dmitri N; Zhang, Zhengdong D; Gorbunova, Vera; Seluanov, Andrei

    2013-10-22

    The naked mole-rat (Heterocephalus glaber) is a subterranean eusocial rodent with a markedly long lifespan and resistance to tumorigenesis. Multiple data implicate modulation of protein translation in longevity. Here we report that 28S ribosomal RNA (rRNA) of the naked mole-rat is processed into two smaller fragments of unequal size. The two breakpoints are located in the 28S rRNA divergent region 6 and excise a fragment of 263 nt. The excised fragment is unique to the naked mole-rat rRNA and does not show homology to other genomic regions. Because this hidden break site could alter ribosome structure, we investigated whether translation rate and amino acid incorporation fidelity were altered. We report that naked mole-rat fibroblasts have significantly increased translational fidelity despite having comparable translation rates with mouse fibroblasts. Although we cannot directly test whether the unique 28S rRNA structure contributes to the increased fidelity of translation, we speculate that it may change the folding or dynamics of the large ribosomal subunit, altering the rate of GTP hydrolysis and/or interaction of the large subunit with tRNA during accommodation, thus affecting the fidelity of protein synthesis. In summary, our results show that naked mole-rat cells produce fewer aberrant proteins, supporting the hypothesis that the more stable proteome of the naked mole-rat contributes to its longevity.

  7. Uncoupling protein-3 mRNA levels are increased in white adipose tissue and skeletal muscle of bezafibrate-treated rats.

    PubMed

    Cabrero, A; Llaverías, G; Roglans, N; Alegret, M; Sánchez, R; Adzet, T; Laguna, J C; Vázquez, M

    1999-07-05

    Fibrates are hypolipidemic drugs that are also able to improve glucose tolerance in animals and diabetic patients through an unknown mechanism. Since uncoupling proteins (UCP) seem to play an important role in the pathogenesis of non-insulin-dependent diabetes mellitus (NIDDM), we examined whether treatment of rats with bezafibrate for 3, 7, or 15 days modified UCP mRNA levels. Using RT-PCR, we observed a weak ectopic expression of UCP-1 and a 2-fold increase in UCP-3 mRNA levels in white adipose tissue after 7 and 15 days of treatment. Moreover, bezafibrate administration caused a 1. 7-fold induction in UCP-3 mRNA levels in skeletal muscle on day 7. Since UCP-3 mRNA levels are reduced in skeletal muscle of diabetic patients, this effect may be involved in the improvement of insulin sensitivity caused by bezafibrate in NIDDM.

  8. Nutrition-induced catch-up growth increases hypoxia inducible factor 1alpha RNA levels in the growth plate.

    PubMed

    Even-Zohar, N; Jacob, J; Amariglio, N; Rechavi, G; Potievsky, O; Phillip, M; Gat-Yablonski, G

    2008-03-01

    Although catch-up growth is a well-known phenomenon, the local pathways at the epiphyseal growth plate that govern this process remain poorly understood. To study the mechanisms governing catch-up growth in the growth plate, we subjected prepubertal rats to 10 days of 40% food restriction, followed by a renewal of the regular food supply to induce catch-up growth. The animals were weighed daily, and their humeral length was measured at sacrifice. The proximal tibial epiphyseal growth plates (EGPs) were studied, and findings were compared with EGPs from animals fed ad libitum and animals under food restriction. The gene expression profile in the growth plates was examined using DNA microarrays, and the expression levels of selected genes were validated by real-time polymerase chain reaction. To localize gene expression in different growth plate zones, microdissection was used. Protein levels and localization were examined using immunohistochemistry. We showed that the expression level of 550 genes decreased during food restriction and increased during catch-up growth, starting already one day after refeeding. HIF-1alpha, as well as several of its downstream targets, was found among these genes. Immunohistochemistry showed a similar pattern for HIF-1alpha protein abundance. Additionally, HIF-1alpha mRNA and protein levels were higher in the proliferating than in the hypertrophic zone, and this distribution was unaffected by nutritional status. These findings indicate that nutrition has a profound effect on gene expression level during growth plate growth, and suggest an important role for HIF-1alpha in the growth plate and its response to nutritional manipulation.

  9. Type I interferon signaling genes in recurrent major depression: increased expression detected by whole-blood RNA sequencing.

    PubMed

    Mostafavi, S; Battle, A; Zhu, X; Potash, J B; Weissman, M M; Shi, J; Beckman, K; Haudenschild, C; McCormick, C; Mei, R; Gameroff, M J; Gindes, H; Adams, P; Goes, F S; Mondimore, F M; MacKinnon, D F; Notes, L; Schweizer, B; Furman, D; Montgomery, S B; Urban, A E; Koller, D; Levinson, D F

    2014-12-01

    A study of genome-wide gene expression in major depressive disorder (MDD) was undertaken in a large population-based sample to determine whether altered expression levels of genes and pathways could provide insights into biological mechanisms that are relevant to this disorder. Gene expression studies have the potential to detect changes that may be because of differences in common or rare genomic sequence variation, environmental factors or their interaction. We recruited a European ancestry sample of 463 individuals with recurrent MDD and 459 controls, obtained self-report and semi-structured interview data about psychiatric and medical history and other environmental variables, sequenced RNA from whole blood and genotyped a genome-wide panel of common single-nucleotide polymorphisms. We used analytical methods to identify MDD-related genes and pathways using all of these sources of information. In analyses of association between MDD and expression levels of 13 857 single autosomal genes, accounting for multiple technical, physiological and environmental covariates, a significant excess of low P-values was observed, but there was no significant single-gene association after genome-wide correction. Pathway-based analyses of expression data detected significant association of MDD with increased expression of genes in the interferon α/β signaling pathway. This finding could not be explained by potentially confounding diseases and medications (including antidepressants) or by computationally estimated proportions of white blood cell types. Although cause-effect relationships cannot be determined from these data, the results support the hypothesis that altered immune signaling has a role in the pathogenesis, manifestation, and/or the persistence and progression of MDD.

  10. Nuclear DYW-type PPR gene families diversify with increasing RNA editing frequencies in liverwort and moss mitochondria.

    PubMed

    Rüdinger, Mareike; Volkmar, Ute; Lenz, Henning; Groth-Malonek, Milena; Knoop, Volker

    2012-02-01

    RNA editing in mitochondria and chloroplasts of land plants alters transcript sequences by site-specific conversions of cytidines into uridines. RNA editing frequencies vary extremely between land plant clades, ranging from zero in some liverworts to more than 2,000 sites in lycophytes. Unique pentatricopeptide repeat (PPR) proteins with variable domain extension (E/E+/DYW) have recently been identified as specific editing site recognition factors in model plants. The distinctive functions of these PPR protein domain additions have remained unclear, although deaminase function has been proposed for the DYW domain. To shed light on diversity of RNA editing and DYW proteins at the origin of land plant evolution, we investigated editing patterns of the mitochondrial nad5, nad4, and nad2 genes in a wide sampling of more than 100 liverworts and mosses using the recently developed PREPACT program (www.prepact.de) and exemplarily confirmed predicted RNA editing sites in selected taxa. Extreme variability in RNA editing frequency is seen both in liverworts and mosses. Only few editings exist in the liverwort Lejeunea cavifolia or the moss Pogonatum urnigerum whereas up to 20% of cytidines are edited in the liverwort Haplomitrium mnioides or the moss Takakia lepidozioides. Interestingly, the latter are taxa that branch very early within their respective clades. Amplicons targeting the E/E+/DYW domains and subsequent random clone sequencing show DYW domains among bryophytes to be highly conserved in comparison with their angiosperm counterparts and to correlate well with RNA editing frequencies regarding their diversities. We propose that DYW proteins are the key players of RNA editing at the origin of land plants.

  11. Human amniotic epithelial cell feeder layers maintain human iPS cell pluripotency via inhibited endogenous microRNA-145 and increased Sox2 expression

    SciTech Connect

    Liu, Te; Cheng, Weiwei; Huang, Yongyi; Huang, Qin; Jiang, Lizhen; Guo, Lihe

    2012-02-15

    Currently, human induced pluripotent stem (iPS) cells were generated from patient or disease-specific sources and share the same key properties as embryonic stem cells. This makes them attractive for personalized medicine, drug screens or cellular therapy. Long-term cultivation and maintenance of normal iPS cells in an undifferentiated self-renewing state are a major challenge. Our previous studies have shown that human amniotic epithelial cells (HuAECs) could provide a good source of feeder cells for mouse and human embryonic stem cells, or spermatogonial stem cells, but the mechanism for this is unknown. Here, we examined the effect of endogenous microRNA-145 regulation on Sox2 expression in human iPS cells by HuAECs feeder cells regulation, and in turn on human iPS cells pluripotency. We found that human IPS cells transfected with a microRNA-145 mutant expressed Sox2 at high levels, allowing iPS to maintain a high level of AP activity in long-term culture and form teratomas in SCID mice. Expression of stem cell markers was increased in iPS transfected with the microRNA-145 mutant, compared with iPS was transfected with microRNA-145. Besides, the expression of Drosha proteins of the microRNA-processor complex, required for the generation of precursor pre-miRNA, was significantly increased in human iPS cells cultured on MEF but not on HuAECs. Taken together, these results suggest that endogenous Sox2 expression may be regulated by microRNA-145 in human iPS cells with HuAECs feeder cells, and Sox2 is a crucial component required for maintenance of them in an undifferentiated, proliferative state capable of self-renewal. Highlights: Black-Right-Pointing-Pointer microRNA-145 inhibits Sox2 expression in human iPS cells. Black-Right-Pointing-Pointer microRNA-145 suppresses the self-renewal and pluripotency of human iPS cells. Black-Right-Pointing-Pointer HuAECs regulate expression of microRNA-145 and Sox2 in human iPS cells. Black-Right-Pointing-Pointer HuAECs feeder

  12. Increased ceramide synthase 2 and 6 mRNA levels in breast cancer tissues and correlation with sphingosine kinase expression.

    PubMed

    Erez-Roman, Racheli; Pienik, Reut; Futerman, Anthony H

    2010-01-01

    Intervention in the ceramide metabolic pathway is emerging as a novel means to regulate cancer and to modify the activity of chemotherapeutic drugs. We now study mRNA expression levels of the six ceramide synthase (CerS) genes in breast cancer tissue. CerS2 and CerS6 mRNA was significantly elevated in breast cancer tissue compared to paired normal tissue, with approximately half of the individuals showing elevated CerS2 and CerS6 mRNA. A significant correlation was found between CerS2 and CerS6 expression, and between CerS4 and CerS2/CerS6 expression. Moreover, patients that expressed higher CerS2 or 4 mRNA levels tended to show no changes in sphingosine kinase 1 levels, and likewise patients that expressed no change in CerS2 or CerS4 mRNA levels tended to express higher levels of sphingosine kinase 1. Together these results suggest an important role for the CerS genes in breast cancer etiology or diagnosis.

  13. Global tRNA misacylation induced by anaerobiosis and antibiotic exposure broadly increases stress resistance in Escherichia coli

    PubMed Central

    Schwartz, Michael H.; Waldbauer, Jacob R.; Zhang, Lichun; Pan, Tao

    2016-01-01

    High translational fidelity is commonly considered a requirement for optimal cellular health and protein function. However, recent findings have shown that inducible mistranslation specifically with methionine engendered at the tRNA charging level occurs in mammalian cells, yeast and archaea, yet it was unknown whether bacteria were capable of mounting a similar response. Here, we demonstrate that Escherichia coli misacylates non-methionyl-tRNAs with methionine in response to anaerobiosis and antibiotic exposure via the methionyl–tRNA synthetase (MetRS). Two MetRS succinyl-lysine modifications independently confer high tRNA charging fidelity to the otherwise promiscuous, unmodified enzyme. Strains incapable of tRNA mismethionylation are less adept at growth in the presence of antibiotics and stressors. The presence of tRNA mismethionylation and its potential role in mistranslation within the bacterial domain establishes this response as a pervasive biological mechanism and connects it to diverse cellular functions and modes of fitness. PMID:27672035

  14. Inhalation Properties and Stability of Nebulized Naked siRNA Solution for Pulmonary Therapy.

    PubMed

    Tahara, Kohei; Hashimoto, Wakana; Takeuchi, Hirofumi

    2016-01-01

    The use of naked unmodified small interfering RNA (N-siRNA) without vector has previously been investigated as a pulmonary therapy. However, little is known regarding stabilities and aerodynamic particle sizes of N-siRNA-containing droplets; nebulizers have not yet been optimized for N-siRNA solutions. Thus, in this study, we investigated the feasibility of inhaled N-siRNA solutions for pulmonary therapy using nebulization. Various nebulizers and N-siRNA concentrations were assessed in terms of siRNA integrity after nebulization, and inhalation properties including aerodynamic particle size were examined. In comparison with ultrasonic-, air-jet-, and vibrating-mesh nebulizers, N-siRNA integrity was not affected by nebulization. Thus, in further experiments, performances of N-siRNA aerosols with different nebulizers and N-siRNA concentrations were evaluated and screened using an aerodynamic particle sizer (APS) which employed the time-of-flight principle or a cascade impactor. Mean mass aerodynamic diameters of N-siRNA-containing droplets from vibrating-mesh nebulizers tended to decrease with increasing N-siRNA concentrations, reflecting the influence of N-siRNA solutions on surface tension, as indicated by contact angles. These data indicate the utility of APS instruments for investigating the nebulized characteristics of expensive drugs including siRNAs and may facilitate the development of N-siRNA inhalation formulations.

  15. Optimization of shRNA inhibitors by variation of the terminal loop sequence.

    PubMed

    Schopman, Nick C T; Liu, Ying Poi; Konstantinova, Pavlina; ter Brake, Olivier; Berkhout, Ben

    2010-05-01

    Gene silencing by RNA interference (RNAi) can be achieved by intracellular expression of a short hairpin RNA (shRNA) that is processed into the effective small interfering RNA (siRNA) inhibitor by the RNAi machinery. Previous studies indicate that shRNA molecules do not always reflect the activity of corresponding synthetic siRNAs that attack the same target sequence. One obvious difference between these two effector molecules is the hairpin loop of the shRNA. Most studies use the original shRNA design of the pSuper system, but no extensive study regarding optimization of the shRNA loop sequence has been performed. We tested the impact of different hairpin loop sequences, varying in size and structure, on the activity of a set of shRNAs targeting HIV-1. We were able to transform weak inhibitors into intermediate or even strong shRNA inhibitors by replacing the loop sequence. We demonstrate that the efficacy of these optimized shRNA inhibitors is improved significantly in different cell types due to increased siRNA production. These results indicate that the loop sequence is an essential part of the shRNA design. The optimized shRNA loop sequence is generally applicable for RNAi knockdown studies, and will allow us to develop a more potent gene therapy against HIV-1.

  16. Optimized siRNA-PEG conjugates for extended blood circulation and reduced urine excretion in mice.

    PubMed

    Iversen, Frank; Yang, Chuanxu; Dagnæs-Hansen, Frederik; Schaffert, David H; Kjems, Jørgen; Gao, Shan

    2013-01-01

    Some of the main concerns with in vivo application of naked small interfering RNA are rapid degradation and urinary excretion resulting in a short plasma half-life. In this study we investigated how conjugation of polyethylene glycol (PEG) with variable chain length affects siRNA pharmacokinetics and biodistribution. The PEG chains were conjugated to chemically stabilized siRNA at the 5' terminal end of the passenger strand using click chemistry. The siRNA conjugate remained functionally active and showed significantly prolonged circulation in the blood stream after intravenous injection. siRNA conjugated with 20kDa PEG (PEG20k-siRNA) was most persistent, approximately 50% PEG20k-siRNA remained 1h post-injection, while the uncoupled siRNA was rapidly removed >90% at 15min. In vivo fluorescent imaging of the living animal showed increased concentration of siRNA in peripheral tissue and delayed urine excretion when coupled to PEG 20k. Biodistribution studies by northern blotting revealed equal distribution of conjugated siRNA in liver, kidney, spleen and lung without significant degradation 24 h post-injection. Our study demonstrates that PEG conjugated siRNA can be applied as a delivery system to improve siRNA bioavailability in vivo and may potentially increase the efficiency of siRNA in therapeutic applications.

  17. MicroRNA-1 Downregulation Increases Connexin 43 Displacement and Induces Ventricular Tachyarrhythmias in Rodent Hypertrophic Hearts

    PubMed Central

    Curcio, Antonio; Torella, Daniele; Iaconetti, Claudio; Pasceri, Eugenia; Sabatino, Jolanda; Sorrentino, Sabato; Giampà, Salvatore; Micieli, Mariella; Polimeni, Alberto; Henning, Beverley J.; Leone, Angelo; Catalucci, Daniele; Ellison, Georgina M.; Condorelli, Gianluigi; Indolfi, Ciro

    2013-01-01

    Downregulation of the muscle-specific microRNA-1 (miR-1) mediates the induction of pathologic cardiac hypertrophy. Dysfunction of the gap junction protein connexin 43 (Cx43), an established miR-1 target, during cardiac hypertrophy leads to ventricular tachyarrhythmias (VT). However, it is still unknown whether miR-1 and Cx43 are interconnected in the pro-arrhythmic context of hypertrophy. Thus, in this study we investigated whether a reduction in the extent of cardiac hypertrophy could limit the pathological electrical remodeling of Cx43 and the onset of VT by modulating miR-1 levels. Wistar male rats underwent mechanical constriction of the ascending aorta to induce pathologic left ventricular hypertrophy (LVH) and afterwards were randomly assigned to receive 10mg/kg valsartan, VAL (LVH+VAL) delivered in the drinking water or placebo (LVH) for 12 weeks. Sham surgery was performed for control groups. Programmed ventricular stimulation reproducibly induced VT in LVH compared to LVH+VAL group. When compared to sham controls, rats from LVH group showed a significant decrease of miR-1 and an increase of Cx43 expression and its ERK1/2-dependent phosphorylation, which displaces Cx43 from the gap junction. Interestingly, VAL administration to rats with aortic banding significantly reduced cardiac hypertrophy and prevented miR-1 down-regulation and Cx43 up-regulation and phosphorylation. Gain- and loss-of-function experiments in neonatal cardiomyocytes (NCMs) in vitro confirmed that Cx43 is a direct target of miR-1. Accordingly, in vitro angiotensin II stimulation reduced miR-1 levels and increased Cx43 expression and phosphorylation compared to un-stimulated NCMs. Finally, in vivo miR-1 cardiac overexpression by an adenoviral vector intra-myocardial injection reduced Cx43 expression and phosphorylation in mice with isoproterenol-induced LVH. In conclusion, miR-1 regulates Cx43 expression and activity in hypertrophic cardiomyocytes in vitro and in vivo. Treatment of

  18. Dietary fiber suppresses elevations of uric acid and allantoin in serum and urine induced by dietary RNA and increases its excretion to feces in rats.

    PubMed

    Koguchi, Takashi; Nakajima, Hisao; Wada, Masahiro; Yamamoto, Yuji; Innami, Satoshi; Maekawa, Akio; Tadokor, Tadahiro

    2002-06-01

    This study was performed to examine the effects of several kinds of dietary fibers (DF) with different physical properties on dietary RNA metabolism. Male Wistar strain rats, 4 wk old, were fed diets with or without a 3% yeast RNA and a 5% DF (cellulose, chitin, chitosan, inulin, and xanthan gum) for 20 d (Experiment 1) or 5 d (Experiment 2). Feeding DF tested lowered the serum uric acid and allantoin concentrations and the urinary excretions of their compounds and increased the amount of RNA excreted into the feces compared with fiber-free. The water-holding capacity and nucleotide adsorption of chitin and chitosan in acidic solutions were higher than those of cellulose. The digestion rate of RNA by RNase A in vitro was found to be lower in the DF tested than in fiber-free. The decrease was remarkable in chitosan and xanthan gum. The uptakes of 14C-labeled adenosine and adenosine 5'-monophosphate (5'-AMP) in the rat jejunum were markedly decreased in regard to chitosan and xanthan gum in comparison with the fiber-free. These phenomena suggest that DF with high viscosity is more strongly associated with the suppression of RNA digestion by RNase A and the depression of the uptake of purine compounds to jejunum. The present results reveal that the elevation of serum uric acid concentration induced by dietary RNA can be suppressed by DF in rats.

  19. Antioxidant supplementation enhances the exercise-induced increase in mitochondrial uncoupling protein 3 and endothelial nitric oxide synthase mRNA content in human skeletal muscle.

    PubMed

    Hellsten, Ylva; Nielsen, Jens J; Lykkesfeldt, Jens; Bruhn, Maria; Silveira, Leonardo; Pilegaard, Henriette; Bangsbo, Jens

    2007-08-01

    The effects of acute exercise on the mRNA content of selected genes were examined during control conditions and after oral intake of antioxidants. In addition, to provide evidence for formation of reactive oxygen species (ROS) in human skeletal muscle during exercise, cytochrome c reduction was measured in microdialysate from the muscle. For the study on the effects of antioxidants on mRNA content, seven healthy, habitually active, male subjects participated in a double-blinded experimental design in which they, on one occasion, received a placebo and, on another, a mixture of antioxidants containing 1500 mg vitamin C, 120 mg coenzyme Q, and 345 mg alpha-tocopherol every day for 7 days before the experiment. On the experimental day the subjects cycled for 90 min and muscle biopsies were taken preexercise and at 1, 3, and 5 h after exercise. Exercise induced an increase in the eNOS, UCP3, PGC-1alpha, VEGF, Hsp72, and HO-1 mRNA content (p < 0.001), whereas there was no change in the Hsc70 mRNA level. Prior antioxidant treatment further enhanced (p < 0.05) the eNOS and UCP3 mRNA content after exercise. Moreover, the overall level of Hsc70 mRNA tended (p = 0.07) to be higher after antioxidant treatment. In another group of healthy male subjects, cytochrome c reduction was determined in microdialysate from the thigh muscle at rest and during knee extensor exercise to determine ROS formation. There was a significant increase in cytochrome c reduction with exercise both at 14 ( approximately 25%) and at 30 W ( approximately 50%). The data show that ROS are formed within skeletal muscle during exercise and that oral intake of antioxidants can enhance the exercise-induced adaptive mRNA responses of eNOS and UCP3.

  20. Increased mRNA Levels of TCF7L2 and MYC of the Wnt Pathway in Tg-ArcSwe Mice and Alzheimer's Disease Brain

    PubMed Central

    Blom, Elin S.; Wang, Yijing; Skoglund, Lena; Hansson, Anita C.; Ubaldi, Massimo; Lourdusamy, Anbarasu; Sommer, Wolfgang H.; Mielke, Matthew; Hyman, Bradley T.; Heilig, Markus; Lannfelt, Lars; Nilsson, Lars N. G.; Ingelsson, Martin

    2011-01-01

    Several components in the Wnt pathway, including β-catenin and glycogen synthase kinase 3 beta, have been implied in AD pathogenesis. Here, mRNA brain levels from five-month-old tg-ArcSwe and nontransgenic mice were compared using Affymetrix microarray analysis. With surprisingly small overall changes, Wnt signaling was the most affected pathway with altered expression of nine genes in tg-ArcSwe mice. When analyzing mRNA levels of these genes in human brain, transcription factor 7-like 2 (TCF7L2) and v-myc myelocytomatosis viral oncogene homolog (MYC), were increased in Alzheimer's disease (AD) (P < .05). Furthermore, no clear differences in TCF7L2 and MYC mRNA were found in brains with frontotemporal lobar degeneration, suggesting that altered regulation of these Wnt-related genes could be specific to AD. Finally, mRNA levels of three neurogenesis markers were analyzed. Increased mRNA levels of dihydropyrimidinase-like 3 were observed in AD brain, suggesting that altered Wnt pathway regulation may signify synaptic rearrangement or neurogenesis. PMID:21234373

  1. Increased abundance of frost mRNA during recovery from cold stress is not essential for cold tolerance in adult Drosophila melanogaster.

    PubMed

    Udaka, H; Percival-Smith, A; Sinclair, B J

    2013-10-01

    Frost (Fst) is a candidate gene associated with the response to cold in Drosophila melanogaster because Fst mRNA accumulation increases during recovery from low temperature exposure. We investigated the contribution of Fst expression to chill-coma recovery time, acute cold tolerance and rapid cold hardening (RCH) in adult D. melanogaster by knocking down Fst mRNA expression using GAL4/UAS-mediated RNA interference. In this experiment, four UAS-Fst and one tubulin-GAL4 lines were used. We predicted that if Fst is essential for cold tolerance phenotypes, flies with low Fst mRNA levels should be less cold tolerant than flies with normal levels of cold-induced Fst mRNA. Cold-induced Fst abundance and recovery time from chill-coma were not negatively correlated in male or female flies. Survival of 2 h exposures to sub-zero temperatures in Fst knockdown lines was not lower than that in a control line. Moreover, a low temperature pretreatment increased survival of severe cold exposure in flies regardless of Fst abundance level during recovery from cold stress, suggesting that Fst expression is not essential for RCH. Thus, cold-induced Fst accumulation is not essential for cold tolerance measured as chill-coma recovery time, survival to acute cold stress and RCH response in adult D. melanogaster.

  2. Serum concentration and increased temperature alter Mayaro virus RNA and protein synthesis in Aedes albopictus (mosquito)-infected cells.

    PubMed

    Motta, M C; Fournier, M V; Carvalho, M G

    1995-01-01

    We have previously shown the inhibition of Mayaro virus multiplication in Aedes albopictus-infected cells maintained at a supraoptimal temperature for growth (37 degrees C) and a stimulation of virus production in response to high serum concentrations in the incubation medium. In the present study, we addressed the question of how the effect of continuous heat stress and high serum concentration soon after infection interfere with virus macromolecule synthesis. Cells maintained at 28 degrees C in the presence of 2% serum synthesized a viral genomic RNA of 12 kb and a subgenomic RNA of 5.2 kb 6 h postinfection. Analysis of the protein profile showed the presence of the viral nucleocapsid protein of 34 kDa (P34). However, if infected cells were maintained at 37 degrees C, a smear starting immediately below the 5.2-kb RNA was noticed and the viral P34 was not detected by SDS-PAGE. Addition of 10% serum to the growth medium of infected cells maintained at 37 degrees C results in a viral RNA profile and protein synthesis similar to those observed in cultures kept at 28 degrees C, i.e., the smear was not observed and the P34 protein was detected. The results suggest that the inhibition of virus multiplication by temperature may be related to the inhibition of viral nonstructural protein synthesis early during infection. The presence of high serum levels in the incubation medium protects macromolecule synthesis against heat stress.

  3. Skeletal muscle myostatin mRNA expression is fiber-type specific and increases during hindlimb unloading

    NASA Technical Reports Server (NTRS)

    Carlson, C. J.; Booth, F. W.; Gordon, S. E.

    1999-01-01

    Transgenic mice lacking a functional myostatin (MSTN) gene demonstrate greater skeletal muscle mass resulting from muscle fiber hypertrophy and hyperplasia (McPherron, A. C., A. M. Lawler, and S. -J. Lee. Nature 387: 83-90, 1997). Therefore, we hypothesized that, in normal mice, MSTN may act as a negative regulator of muscle mass. Specifically, we hypothesized that the predominately slow (type I) soleus muscle, which demonstrates greater atrophy than the fast (type II) gastrocnemius-plantaris complex (Gast/PLT), would show more elevation in MSTN mRNA abundance during hindlimb unloading (HU). Surprisingly, MSTN mRNA was not detectable in weight-bearing or HU soleus muscle, which atrophied 42% by the 7th day of HU in female ICR mice. In contrast, MSTN mRNA was present in weight-bearing Gast/PLT muscle and was significantly elevated (67%) at 1 day but not at 3 or 7 days of HU. However, the Gast/PLT muscle had only atrophied 17% by the 7th day of HU. Because the soleus is composed only of type I and IIa fibers, whereas the Gast/PLT expresses type IId/x and IIb in addition to type I and IIa, it was necessary to perform a more careful analysis of the relationship between MSTN mRNA levels and myosin heavy-chain (MHC) isoform expression (as a marker of fiber type). A significant correlation (r = 0.725, P < 0. 0005) was noted between the percentage of MHC isoform IIb expression and MSTN mRNA abundance in several muscles of the mouse hindlimb. These results indicate that MSTN expression is not strongly associated with muscle atrophy induced by HU; however, it is strongly associated with MHC isoform IIb expression in normal muscle.

  4. Efficient inhibition of the formation of joint adhesions by ERK2 small interfering RNAs

    SciTech Connect

    Li, Fengfeng; Ruan, Hongjiang; Fan, Cunyi; Zeng, Bingfang; Wang, Chunyang; Wang, Xiang

    2010-01-01

    Transforming growth factor-{beta}1 and fibroblast growth factor-2 play very important roles in fibroblast proliferation and collagen expression. These processes lead to the formation of joint adhesions through the SMAD and MAPK pathways, in which extracellular signal-regulated kinase (ERK)2 is considered to be crucial. Based on these theories, we examined the effects of a lentivirus-mediated small interfering RNA (siRNA) targeting ERK2 on the suppression of joint adhesion formation in vivo. The effects were assessed in vivo from different aspects including the adhesion score, histology and joint contracture angle. We found that the adhesions in the ERK2 siRNA group became soft and weak, and were easily stretched. Accordingly, the flexion contracture angles in the ERK2 siRNA group were also reduced (P < 0.05 compared with the control group). The animals appeared healthy, with no signs of impaired wound healing. In conclusion, local delivery of a lentivirus-mediated siRNA targeting ERK2 can ameliorate joint adhesion formation effectively and safely.

  5. Persistent CSF but not plasma HIV RNA is associated with increased risk of new-onset moderate-to-severe depressive symptoms; a prospective cohort study.

    PubMed

    Hammond, Edward R; Crum, Rosa M; Treisman, Glenn J; Mehta, Shruti H; Clifford, David B; Ellis, Ronald J; Gelman, Benjamin B; Grant, Igor; Letendre, Scott L; Marra, Christina M; Morgello, Susan; Simpson, David M; Mcarthur, Justin C

    2016-08-01

    Major depressive disorder is the most common neuropsychiatric complication in human immunodeficiency virus (HIV) infections and is associated with worse clinical outcomes. We determined if detectable cerebrospinal fluid (CSF) HIV ribonucleic acid (RNA) at threshold ≥50 copies/ml is associated with increased risk of depression. The CNS HIV Anti-Retroviral Therapy Effects Research (CHARTER) cohort is a six-center US-based prospective cohort with bi-annual follow-up of 674 participants. We fit linear mixed models (N = 233) and discrete-time survival models (N = 154; 832 observations) to evaluate trajectories of Beck Depression Inventory (BDI) II scores and the incidence of new-onset moderate-to-severe depressive symptoms (BDI ≥ 17) among participants on combination antiretroviral therapy (cART), who were free of depression at study entry and received a minimum of three CSF examinations over 2496 person-months follow-up. Detectable CSF HIV RNA (threshold ≥50 copies/ml) at any visit was associated with a 4.7-fold increase in new-onset depression at subsequent visits adjusted for plasma HIV RNA and treatment adherence; hazard ratio (HR) = 4.76, (95 % CI 1.58-14.3); P = 0.006. Depression (BDI) scores were 2.53 points higher (95 % CI 0.47-4.60; P = 0.02) over 6 months if CSF HIV RNA was detectable at a prior study visit in fully adjusted models including age, sex, race, education, plasma HIV RNA, duration and adherence of CART, and lifetime depression diagnosis by Diagnostic Statistical Manual (DSM-IV) criteria. Persistent CSF but not plasma HIV RNA is associated with an increased risk for new-onset depression. Further research evaluating the role of immune activation and inflammatory markers may improve our understanding of this association.

  6. Modulation of somatosensory evoked magnetic fields by intensity of interfering stimuli in human somatosensory cortex: an MEG study.

    PubMed

    Lim, Manyoel; Kim, June Sic; Chung, Chun Kee

    2012-07-02

    Somatosensory evoked responses are known to be modulated by previous interfering stimuli. Here, we first investigated the modulatory effects of interfering stimuli with different intensities on somatosensory evoked magnetic field in human primary (S1) and secondary (S2) somatosensory cortices. In the control condition of the study, test stimulus, set to strong intensity, was delivered to the left median nerve. Interfering stimuli with three different levels of intensity from weak (WI) through moderate (MI) and finally to strong (SI) were interspersed to the left median nerve between the test stimuli in each interfering condition. The cortical responses to the test stimulus were modeled with equivalent current dipoles in the contralateral S1 and bilateral S2 cortices from 17 subjects. The amplitude of the N20m deflection from the S1 was not changed by any interfering stimuli, whereas the amplitude of later P35m deflection was reduced by MI stimulus. The amplitude of P60m deflection was reduced by MI and SI stimuli. The extent of amplitude reduction of the bilateral S2 response was markedly increased as intensity of interfering stimuli increased from weak to moderate, but further reduction by the SI stimuli compared to MI stimuli was not observed. Those results indicated that somatosensory cortical activation in the S1 (P35m and P60m) and S2 were modulated by intensity of interfering stimuli. Our findings of a greater gating effect on the bilateral S2 compared to the contralateral S1 indicate that S2 may play an important role in temporal integration of different intensity levels of somatosensory inputs.

  7. Serum IL8 and mRNA level of CD11b in circulating neutrophils are increased in clinically amyopathic dermatomyositis with active interstitial lung disease.

    PubMed

    Zou, Jing; Chen, Jie; Yan, Qingran; Guo, Qiang; Bao, Chunde

    2016-01-01

    The objective of this study is to assess serum IL8 and the potential activity of circulating neutrophils on relative messenger RNA (mRNA) levels and their relationship with disease activity in clinically amyopathic dermatomyositis (CADM) associated with interstitial lung disease (ILD). We studied 18 CADM patients and compared them with 18 classic dermatomyositis (DM) patients and 18 healthy control subjects. Serum IL8 level and mRNA expressions of neutrophils (chemokine (C-X-C motif) receptor 1 (CXCR1), cluster of differentiation molecule 11b (CD11b), cluster of differentiation 64 (CD64), myeloid cell leukemia 1 (MCL1), interleukin-18 (IL18)) were detected. The overproduction of serum IL8 level was most significant in the CADM group with active period. The mRNA expressions of CD11b, IL18, and MCL1 were greatly increased in the neutrophils in patients with CADM compared with DM or healthy controls. Up-expressions of CD11b, IL18, and MCL1 were detected in the neutrophils in CADM patients of active period compared with remission period. A positive correlation was found between CD11b mRNA level and high-resolution computed tomography (HRCT) score, in CADM associated with ILD. Serum IL8 level and mRNA levels of CD11b, MCL1, and IL18 in circulating neutrophils are related with the disease activity of CADM-ILD. The mRNA level of CD11b is positively correlated with HRCT score in CADM-ILD.

  8. Inhibition of CD147 expression by RNA interference reduces proliferation, invasion and increases chemosensitivity in cancer stem cell-like HT-29 cells.

    PubMed

    Chen, Jie; Pan, Yuqin; He, Bangshun; Ying, Houqun; Wang, Feng; Sun, Huiling; Deng, Qiwen; Liu, Xian; Lin, Kang; Peng, Hongxin; Cho, William C; Wang, Shukui

    2015-10-01

    The association between CD147 and cancer stem cells (CSCs) provides a new angle for cancer treatments. The aim of this study was to investigate the biological roles of CD147 in colorectal CSCs. The Oct4-green fluorescent protein (GFP) vector was used to isolate CSCs and pYr-mir30-shRNA was used to generate short hairpin RNA (shRNA) specifically for CD147. After RNA interference (RNAi), CD147 was evaluated by reverse transcription‑quantitative PCR and western blot analysis, and its biological functions were assessed by MTT and invasion assays. The results showed that the differentiation of isolated CSC-like HT-29 cells was blocked and these cells were highly positive for CD44 and CD147. RNAi-mediated CD147 silencing reduced the expression of CD147 at both mRNA and protein levels. Moreover, the activities of proliferation and invasion were decreased obviously in CSCs. Knockdown of CD147 increased the chemosensitivity of CSC-like cells to gemcitabine, cisplatin, docetaxel at 0.1, 1 and 10 µM respectively, however, there was no significant difference among the three groups to paclitaxel at 10 µM. In conclusion, these results suggest that CD147 plays an important role in colorectal CSCs and might be regarded as a novel CSC-specific targeted strategy against colorectal cancer.

  9. A histone methyltransferase inhibits seed germination by increasing PIF1 mRNA expression in imbibed seeds.

    PubMed

    Lee, Nayoung; Kang, Hyojin; Lee, Daeyoup; Choi, Giltsu

    2014-04-01

    Phytochrome-interacting factor 1 (PIF1) inhibits light-dependent seed germination. The specific function of PIF1 in seed germination is partly due to its high level of expression in imbibed seeds, but the associated regulatory factors have not been identified. Here we show that mutation of the early flowering in short days (EFS) gene, encoding an H3K4 and H3K36 methyltransferase, decreases the level of H3K36me2 and H3K36me3 but not H3K4me3 at the PIF1 locus, reduces the targeting of RNA polymerase II to the PIF1 locus, and reduces mRNA expression of PIF1 in imbibed seeds. Consistently, the efs mutant geminated even under the phyBoff condition, and had an expression profile of PIF1 target genes similar to that of the pif1 mutant. Introduction of an EFS transgene into the efs mutant restored the level of H3K36me2 and H3K36me3 at the PIF1 locus, the high-level expression of PIF1 mRNA, the expression pattern of PIF1 target genes, and the light-dependent germination of these seeds. Introduction of a PIF1 transgene into the efs mutant also restored the expression pattern of PIF1 target genes and light-dependent germination in imbibed seeds, but did not restore the flowering phenotype. Taken together, our results indicate that EFS is necessary for high-level expression of PIF1 mRNA in imbibed seeds.

  10. Increased shelterin mRNA expression in peripheral blood mononuclear cells and skeletal muscle following an ultra-long-distance running event.

    PubMed

    Laye, Matthew J; Solomon, Thomas P J; Karstoft, Kristian; Pedersen, Karin K; Nielsen, Susanne D; Pedersen, Bente K

    2012-03-01

    Located at the end of chromosomes, telomeres are progressively shortened with each replication of DNA during aging. Integral to the regulation of telomere length is a group of proteins making up the shelterin complex, whose tissue-specific function during physiological stress is not well understood. In this study, we examine the mRNA and protein levels of proteins within and associated with the shelterin complex in subjects (n = 8, mean age = 44 yr) who completed a physiological stress of seven marathons in 7 days. Twenty-two to 24 h after the last marathon, subjects had increased mRNA levels of DNA repair enzymes Ku70 and Ku80 (P < 0.05) in both skeletal muscle and peripheral blood mononuclear cells (PBMCs). Additionally, the PBMCs displayed an increment in three shelterin protein mRNA levels (TRF1, TRF2, and Pot-1, P < 0.05) following the event. Seven days of ultrarunning did not result in changes in mean telomere length, telomerase activity, hTert mRNA, or hterc mRNAs found in PBMCs. Higher protein concentrations of TRF2 were found in skeletal muscle vs. PBMCs at rest. Mean telomere length in skeletal muscle did not change and did not contain detectable levels of htert mRNA or telomerase activity. Furthermore, changes in the PBMCs could not be attributed to changes in the proportion of subtypes of CD4(+) or CD8(+) cells. We have provided the first evidence that, in humans, proteins within and associated with the shelterin complex increase at the mRNA level in response to a physiological stress differentially in PBMCs and skeletal muscle.

  11. Sphingosine kinase-1, S1P transporter spinster homolog 2 and S1P2 mRNA expressions are increased in liver with advanced fibrosis in human.

    PubMed

    Sato, Masaya; Ikeda, Hitoshi; Uranbileg, Baasanjav; Kurano, Makoto; Saigusa, Daisuke; Aoki, Junken; Maki, Harufumi; Kudo, Hiroki; Hasegawa, Kiyoshi; Kokudo, Norihiro; Yatomi, Yutaka

    2016-08-26

    The role of sphingosine 1-phosphate (S1P) in liver fibrosis or inflammation was not fully examined in human. Controversy exists which S1P receptors, S1P1 and S1P3 vs S1P2, would be importantly involved in its mechanism. To clarify these matters, 80 patients who received liver resection for hepatocellular carcinoma and 9 patients for metastatic liver tumor were enrolled. S1P metabolism was analyzed in background, non-tumorous liver tissue. mRNA levels of sphingosine kinase 1 (SK1) but not SK2 were increased in livers with fibrosis stages 3-4 compared to those with 0-2 and to normal liver. However, S1P was not increased in advanced fibrotic liver, where mRNA levels of S1P transporter spinster homolog 2 (SPNS2) but not S1P-degrading enzymes were enhanced. Furthermore, mRNA levels of S1P2 but not S1P1 or S1P3 were increased in advanced fibrotic liver. These increased mRNA levels of SK1, SPNS2 and S1P2 in fibrotic liver were correlated with α-smooth muscle actin mRNA levels in liver, and with serum ALT levels. In conclusion, S1P may be actively generated, transported to outside the cells, and bind to its specific receptor in human liver to play a role in fibrosis or inflammation. Altered S1P metabolism in fibrotic liver may be their therapeutic target.

  12. 36 CFR 2.32 - Interfering with agency functions.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 36 Parks, Forests, and Public Property 1 2010-07-01 2010-07-01 false Interfering with agency functions. 2.32 Section 2.32 Parks, Forests, and Public Property NATIONAL PARK SERVICE, DEPARTMENT OF THE... authorized to maintain order and control public access and movement during fire fighting operations,...

  13. 36 CFR 1002.32 - Interfering with agency functions.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 36 Parks, Forests, and Public Property 3 2010-07-01 2010-07-01 false Interfering with agency functions. 1002.32 Section 1002.32 Parks, Forests, and Public Property PRESIDIO TRUST RESOURCE PROTECTION... maintain order and control public access and movement during fire fighting operations, search and...

  14. Hepatitis B virus X protein induces RNA polymerase III-dependent gene transcription and increases cellular TATA-binding protein by activating the Ras signaling pathway.

    PubMed

    Wang, H D; Trivedi, A; Johnson, D L

    1997-12-01

    Our previous studies have shown that the hepatitis B virus protein, X, activates all three classes of RNA polymerase III (pol III)-dependent promoters by increasing the cellular level of TATA-binding protein (TBP) (H.-D. Wang et al., Mol. Cell. Biol. 15:6720-6728, 1995), a limiting transcription component (A. Trivedi et al., Mol. Cell. Biol. 16:6909-6916, 1996). We have investigated whether these X-mediated events are dependent on the activation of the Ras/Raf-1 signaling pathway. Transient expression of a dominant-negative mutant Ras gene (Ras-ala15) in a Drosophila S-2 stable cell line expressing X (X-S2), or incubation of the cells with a Ras farnesylation inhibitor, specifically blocked both the X-dependent activation of a cotransfected tRNA gene and the increase in cellular TBP levels. Transient expression of a constitutively activated form of Ras (Ras-val12) in control S2 cells produced both an increase in tRNA gene transcription and an increase in cellular TBP levels. These events are not cell type specific since X-mediated gene induction was also shown to be dependent on Ras activation in a stable rat 1A cell line expressing X. Furthermore, increases in RNA pol III-dependent gene activity and TBP levels could be restored in X-S2 cells expressing Ras-ala15 by coexpressing a constitutively activated form of Raf-1. These events are serum dependent, and when the cells are serum deprived, the X-mediated effects are augmented. Together, these results demonstrate that the X-mediated induction of RNA pol III-dependent genes and increase in TBP are both dependent on the activation of the Ras/Raf-1 signaling cascade. In addition, these studies define two new and important consequences mediated by the activation of the Ras signal transduction pathway: an increase in the central transcription factor, TBP, and the induction of RNA pol III-dependent gene activity.

  15. Comparative analysis of RNA silencing suppression activities between viral suppressors and an endogenous plant RNA-dependent RNA polymerase.

    PubMed

    Yoon, Ju-Yeon; Han, Kyoung-Sik; Park, Han-Yong; Choi, Seung-Kook

    2012-06-01

    RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in eukaryotes, including higher plants. To counteract this, several plant viruses express silencing suppressors that inhibit RNA silencing in host plants. Here, we show that both 2b protein from peanut stunt virus (PSV) and a hairpin construct (designated hp-RDR6) that silences endogenous RNA-dependent RNA polymerase 6 (RDR6) strongly suppress RNA silencing. The Agrobacterium infiltration system was used to demonstrate that both PSV 2b and hp-RDR6 suppressed local RNA silencing as strongly as helper component (HC-Pro) from potato virus Y (PVY) and P19 from tomato bush stunt virus (TBSV). The 2b protein from PSV eliminated the small-interfering RNAs (siRNAs) associated with RNA silencing and prevented systemic silencing, similar to 2b protein from cucumber mosaic virus (CMV). On the other hand, hp-RDR6 suppressed RNA silencing by inhibiting the generation of secondary siRNAs. The small coat protein (SCP) of squash mosaic virus (SqMV) also displayed weak suppression activity of RNA silencing. Agrobacterium-mediated gene transfer was used to investigate whether viral silencing suppressors or hp-RDR6 enhanced accumulations of green fluorescence protein (GFP) and β-glucuronidase (GUS) as markers of expression in leaf tissues of Nicotina benthamiana. Expression of both GFP and GUS was significantly enhanced in the presence of PSV 2b or CMV 2b, compared to no suppression or the weak SqMV SCP suppressor. Co-expression with hp-RDR6 also significantly increased the expression of GFP and GUS to levels similar to those induced by PVY HC-Pro and TBSV P19.

  16. Short peptides interfering with signaling pathways as new therapeutic tools for cancer treatment.

    PubMed

    Ellert-Miklaszewska, Aleksandra; Poleszak, Katarzyna; Kaminska, Bozena

    2017-01-01

    Short peptides have many advantages, such as low molecular weight, selectivity for a specific target, organelles or cells with minimal toxicity. We describe properties of short peptides, which interfere with communication networks in tumor cells and within microenvironment of malignant gliomas, the most common brain tumors. We focus on ligand/receptor axes and intracellular signaling pathways critical for gliomagenesis that could be targeted with interfering peptides. We review structures and efficacy of organelle-specific and cell-penetrating peptides and describe diverse chemical modifications increasing proteolytic stability and protecting synthetic peptides against degradation. We report results of application of short peptides in glioma therapy clinical trials, their rises and falls. The most advanced examples of therapeutics such as short interfering peptides combined with cell-penetrating peptides that show good effectiveness in disease models are presented. It is foreseen that identification of peptides with better clinical properties may improve their success rates in clinical trials.

  17. Iron chelation down-regulates dopamine transporter expression by decreasing mRNA stability and increasing endocytosis in N2a cells.

    PubMed

    Hegde, Narasimha V; Jensen, Gordon L; Unger, Erica L

    2011-02-15

    Cell surface expression of the dopamine transporter (DAT) is determined by the relative rates of its internalization and recycling. Changes in the cellular labile iron pool (LIP) affect many cellular mechanisms including those that regulate DAT trafficking. In this study, we analyzed DAT expression and posttranslational modifications in response to changes in cellular iron in transfected neuroblastoma cells (N2a). Iron chelation by desferrioxamine (DFO) altered DAT protein levels by decreasing the stability of DAT mRNA. Increased phosphorylation and ubiquitination of this transporter protein following DFO treatment were also observed. Cellular iron depletion elevated protein levels of the early endosomal marker Rab5. Moreover, confocal microscopy studies showed increased localization of DAT into the endosomal compartment in DFO-treated cells compared to control. Together, these findings suggest that cellular iron depletion regulates DAT expression through reducing mRNA stability as well as an increasing in endocytosis.

  18. Increased interleukin-1beta mRNA expression in skin biopsies of horses with Culicoides hypersensitivity following challenge with Culicoides nubeculosus extract.

    PubMed

    Kolm, Gabriela; Knapp, Elzbieta; Wagner, Regina; Klein, Dieter

    2006-09-15

    Interleukin-1beta (IL-1beta) is a primary cytokine of the skin that has a pivotal role in keratinocyte differentiation, epidermal wound healing and host defense. Pathological increase of cutaneous IL-1beta is associated with edema formation, epidermal hyperproliferation and atopic dermatitis in humans. However, in horses the role of cutaneous IL-1beta in edema formation and allergic skin disease has not been characterised so far. Particularly in Culicoides hypersensitivity (CHS), intradermal injection of Culicoides extract may be associated with enhanced transcription of local IL-1beta. To examine the mRNA expression of IL-1beta and its receptor antagonist IL-1RA in the skin of horses, biopsy specimens of horses affected and non-affected by CHS prior and following intradermal challenge with a commercial C. nubeculosus extract were examined. Our hypothesis was that cutaneous IL-1beta mRNA was significantly upregulated in horses with CHS in response to Culicoides allergen. Biopsies were taken from sites prior to and 4 h following intradermal challenge with C. nubeculosus extract. In order to obtain reliable data, real time PCR was performed and genes of interest were normalized using three different housekeeping genes, beta-actin, GAPDH, beta-2-microglobulin. No significant difference was detected in non-challenged cutaneous IL-1beta mRNA and IL-1RA mRNA levels between CHS affected and non-affected horses. Intradermal injection of C. nubeculosus extract resulted in local upregulation of IL-1beta mRNA both in horses with typical history, characteristic clinical signs for CHS and a positive intradermal skin test (IDT), and non-affected horses with a negative IDT. However, the difference in prior and post challenged site IL-1beta mRNA levels only reached statistical significance in the affected horses (p=0.01 versus 0.7). In contrast, IL-1RA mRNA levels did not demonstrate any modification following intradermal injection with C. nubeculosus in either group. In contrast

  19. Replication of a pathogenic non-coding RNA increases DNA methylation in plants associated with a bromodomain-containing viroid-binding protein

    PubMed Central

    Lv, Dian-Qiu; Liu, Shang-Wu; Zhao, Jian-Hua; Zhou, Bang-Jun; Wang, Shao-Peng; Guo, Hui-Shan; Fang, Yuan-Yuan

    2016-01-01

    Viroids are plant-pathogenic molecules made up of single-stranded circular non-coding RNAs. How replicating viroids interfere with host silencing remains largely unknown. In this study, we investigated the effects of a nuclear-replicating Potato spindle tuber viroid (PSTVd) on interference with plant RNA silencing. Using transient induction of silencing in GFP transgenic Nicotiana benthamiana plants (line 16c), we found that PSTVd replication accelerated GFP silencing and increased Virp1 mRNA, which encodes bromodomain-containing viroid-binding protein 1 and is required for PSTVd replication. DNA methylation was increased in the GFP transgene promoter of PSTVd-replicating plants, indicating involvement of transcriptional gene silencing. Consistently, accelerated GFP silencing and increased DNA methylation in the of GFP transgene promoter were detected in plants transiently expressing Virp1. Virp1 mRNA was also increased upon PSTVd infection in natural host potato plants. Reduced transcript levels of certain endogenous genes were also consistent with increases in DNA methylation in related gene promoters in PSTVd-infected potato plants. Together, our data demonstrate that PSTVd replication interferes with the nuclear silencing pathway in that host plant, and this is at least partially attributable to Virp1. This study provides new insights into the plant-viroid interaction on viroid pathogenicity by subverting the plant cell silencing machinery. PMID:27767195

  20. Contraction-induced increases in Na+-K+-ATPase mRNA levels in human skeletal muscle are not amplified by activation of additional muscle mass.

    PubMed

    Nordsborg, Nikolai; Thomassen, Martin; Lundby, Carsten; Pilegaard, Henriette; Bangsbo, Jens

    2005-07-01

    The present study tested the hypothesis that exercise with a large compared with a small active muscle mass results in a higher contraction-induced increase in Na(+)-K(+)-ATPase mRNA expression due to greater hormonal responses. Furthermore, the relative abundance of Na(+)-K(+)-ATPase subunit alpha(1), alpha(2), alpha(3), alpha(4), beta(1), beta(2), and beta(3) mRNA in human skeletal muscle was investigated. On two occasions, eight subjects performed one-legged knee extension exercise (L) or combined one-legged knee extension and bilateral arm cranking (AL) for 5.00, 4.25, 3.50, 2.75, and 2.00 min separated by 3 min of rest. Leg exercise power output was the same in AL and L, but heart rate at the end of each exercise interval was higher in AL compared with L. One minute after exercise, arm venous blood lactate was higher in AL than in L. A higher level of blood epinephrine and norepinephrine was evident 3 min after exercise in AL compared with L. Nevertheless, none of the exercise-induced increases in alpha(1), alpha(2), beta(1), and beta(3) mRNA expression levels were higher in AL compared with L. The most abundant Na(+)-K(+)-ATPase subunit at the mRNA level was beta(1), which was expressed 3.4 times than alpha(2). Expression of alpha(1), beta(2), and beta(3) was less than 5% of the alpha(2) expression, and no reliable detection of alpha(3) and alpha(4) was possible. In conclusion, activation of additional muscle mass does not result in a higher exercise-induced increase in Na(+)-K(+)-ATPase subunit-specific mRNA.

  1. Interleukin 4 or oncostatin M induces a prolonged increase in P- selectin mRNA and protein in human endothelial cells

    PubMed Central

    1996-01-01

    During acute inflammation, P-selectin is transiently mobilized from Weibel-Palade bodies to the surface of histamine-activated endothelial cells, where it mediates rolling adhesion of neutrophils under hydrodynamic flow. During chronic or allergic inflammation, sustained expression of P-selectin on the endothelial cell surface has been observed. We found that the cytokines interleukin 4 (IL-4) or oncostatin M (OSM) induced a five- to ninefold increase in P-selectin messenger RNA (mRNA) in human umbilical vein endothelial cells (HUVEC) that persisted as long as 72 h. IL-4 elevated P-selectin mRNA by increasing its transcription rate rather than by prolonging its already long half-life. Stimulation of P-selectin transcription by IL-4 or OSM required new protein synthesis and tyrosine phosphorylation of cellular proteins. Tumor necrosis factor alpha, IL-1 beta, lipopolysaccharide, or IL-3 did not increase P-selectin mRNA in HUVEC, and did not augment the IL-4-induced increase in P-selectin transcripts. IL-4 or OSM increased P-selectin protein on the cell surface as well as in Weibel- Palade bodies. Under flow conditions, neutrophils rolled on P-selectin expressed by IL-4-treated HUVEC, and even more neutrophils rolled on P- selectin after IL-4-treated HUVEC were stimulated with histamine. These data demonstrate that IL-4 or OSM stimulates endothelial cells to synthesize more P-selectin over prolonged periods. The increased expression of P-selectin may facilitate the emigration of leukocytes into sites of chronic or allergic inflammation. PMID:8691152

  2. Spin O decay angular distribution for interfering mesons in electroproduction

    SciTech Connect

    Funsten, H.; Gilfoyle, G.

    1994-04-01

    Self analyzing meson electroproduction experiments are currently being planned for the CEBAF CLAS detector. These experiments deduce the spin polarization of outgoing unstable spin s (?)0 mesons from their decay angular distribution, W({theta},{psi}). The large angular acceptance of the CLAS detector permits kinematic tracking of a sufficient number of these events to accurately determine electroproduction amplitudes from the deduced polarization. Maximum polarization information is obtained from W({theta},{psi}) for decay into spin 0 daughters. The helicity of the decaying meson is transferred to the daughter`s relative orbital angular momentum m-projection; none is {open_quotes}absorbed{close_quotes} into daughter helicities. The decaying meson`s helicity maximally appears in W({theta},{psi}). W({theta},{psi}) for spin 0 daughters has been derived for (1) vector meson electroproduction and (2) general interfering mesons produced by incident pions. This paper derives W({theta},{psi}) for electroproduction of two interfering mesons that decay into spin 0 daughters. An application is made to the case of interfering scalar and vector mesons. The derivation is an extension of work by Schil using the general decay formalism of Martin. The expressions can be easily extended to the case of N interfering mesons since interference occurs pairwise in the observable W ({theta},{psi}), a quadratic function of the meson amplitudes. The derivation uses the virtual photon density matrix of Schil which is transformed by a meson electroproduction transition operator, T. The resulting density matrix for the interfering mesons is then converted into a corresponding statistical tensor and contracted into the efficiency tensor for spin 0 daughters.

  3. 2-Amino-alpha-2'-deoxyadenosine increased duplex stability of methoxyethylphosphoramidate alpha-oligodeoxynucleotides with RNA target.

    PubMed

    Naval, Magali; Michel, Thibaut; Vasseur, Jean-Jacques; Debart, Françoise

    2002-06-03

    A new efficient synthesis of 2-amino-alpha-2'-deoxyadenosine and its incorporation into methoxyethylphosphoramidate alpha-oligodeoxynucleotides (ODNs) via H-phosphonate chemistry were reported. Thermal denaturation experiments demonstrated a significant stabilization of the complexes formed between these analogues and their RNA target (+2 degrees C/NH2A) relative to adenosine-containing phosphoramidate alpha-oligonucleotides. Concerning the binding specificity of these modified ODNs, unlike natural ODNs, discrimination against G pairing is higher and against C pairing is lower.

  4. Mechanism of Action of Glucagon-Like Peptide-2 to Increase IGF-I mRNA in Intestinal Subepithelial Fibroblasts

    PubMed Central

    Leen, Jason L. S.; Izzo, Angelo; Upadhyay, Chandani; Rowland, Katherine J.; Dubé, Philip E.; Gu, Steven; Heximer, Scott P.; Rhodes, Christopher J.; Storm, Daniel R.; Lund, P. Kay

    2011-01-01

    IGF-I, a known secretory product of intestinal subepithelial myofibroblasts (ISEMFs), is essential for the intestinotropic effects of glucagon-like peptide-2 (GLP-2). Furthermore, GLP-2 increases IGF-I mRNA transcript levels in vitro in heterogeneous fetal rat intestinal cultures, as well as in vivo in the rodent small intestine. To determine the mechanism underlying the stimulatory effect of GLP-2 on intestinal IGF-I mRNA, murine ISEMF cells were placed into primary culture. Immunocytochemistry showed that the ISEMF cells appropriately expressed α-smooth muscle actin and vimentin but not desmin. The cells also expressed GLP-2 receptor and IGF-I mRNA transcripts. Treatment of ISEMF cells with (Gly2)GLP-2 induced IGF-I mRNA transcripts by up to 5-fold of basal levels after treatment with 10−8 m GLP-2 for 2 h (P < 0.05) but did not increase transcript levels for other intestinal growth factors, such as ErbB family members. Immunoblot revealed a 1.6-fold increase in phospho (p)-Akt/total-(t)Akt with 10−8 m GLP-2 treatment (P < 0.05) but no changes in cAMP, cAMP-dependent β-galactosidase expression, pcAMP response element-binding protein/tcAMP response element-binding protein, pErk1/2/tErk1/2, or intracellular calcium. Furthermore, pretreatment of ISEMF cells with the phosphatidylinositol 3 kinase (PI3K) inhibitors, LY294002 and wortmannin, abrogated the IGF-I mRNA response to GLP-2, as did overexpression of kinase-dead Akt. The role of PI3K/Akt in GLP-2-induced IGF-I mRNA levels in the murine jejunum was also confirmed in vivo. These findings implicate the PI3K/Akt pathway in the stimulatory effects of GLP-2 to enhance intestinal IGF-I mRNA transcript levels and provide further evidence in support of a role for IGF-I produced by the ISEMF cells in the intestinotropic effects of GLP-2. PMID:21159855

  5. Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

    NASA Astrophysics Data System (ADS)

    Kallehauge, Thomas Beuchert; Li, Shangzhong; Pedersen, Lasse Ebdrup; Ha, Tae Kwang; Ley, Daniel; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup; Lee, Gyun Min; Lewis, Nathan E.

    2017-01-01

    Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production.

  6. Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

    PubMed Central

    Kallehauge, Thomas Beuchert; Li, Shangzhong; Pedersen, Lasse Ebdrup; Ha, Tae Kwang; Ley, Daniel; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup; Lee, Gyun Min; Lewis, Nathan E.

    2017-01-01

    Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production. PMID:28091612

  7. Increased micro-RNA 29b in the aged brain correlates with the reduction of insulin-like growth factor-1 and fractalkine ligand.

    PubMed

    Fenn, Ashley M; Smith, Kristen M; Lovett-Racke, Amy E; Guerau-de-Arellano, Mireia; Whitacre, Caroline C; Godbout, Jonathan P

    2013-12-01

    Microglia develop an inflammatory phenotype during normal aging. The mechanism by which this occurs is not well understood, but might be related to impairments in several key immunoregulatory systems. Here we show that micro-RNA (miR)-29a and miR-29b, 2 immunoregulatory micro-RNAs, were increased in the brain of aged BALB/c mice compared with adults. Insulin-like growth factor-1 (IGF-1) and fractalkine ligand (CX3CL1) are negative modulators of microglial activation and were identified as targets of miR-29a and miR-29b using luciferase assay and primary microglia transfection. Indeed, higher expression of miR-29b in the brain of aged mice was associated with reduced messenger RNA (mRNA) levels of IGF-1 and CX3CL1. Parallel to these results in mice, miR-29a and miR-29b were also markedly increased in cortical brain tissue of older individuals (mean, 77 years) compared with middle-aged adults (mean, 45 years). Moreover, increased expression of miR-29b in human cortical tissue was negatively correlated with IGF-1 and CX3CL1 expression. Collectively, these data indicate that an age-associated increase in miR-29 corresponded with the reduction of 2 important regulators of microglia, IGF-1 and CX3CL1.

  8. Warm temperatures induce transgenerational epigenetic release of RNA silencing by inhibiting siRNA biogenesis in Arabidopsis.

    PubMed

    Zhong, Si-Hui; Liu, Jun-Zhong; Jin, Hua; Lin, Lin; Li, Qun; Chen, Ying; Yuan, Yue-Xing; Wang, Zhi-Yong; Huang, Hai; Qi, Yi-Jun; Chen, Xiao-Ya; Vaucheret, Hervé; Chory, Joanne; Li, Jianming; He, Zu-Hua

    2013-05-28

    Owing to their sessile nature, plants have evolved sophisticated genetic and epigenetic regulatory systems to respond quickly and reversibly to daily and seasonal temperature changes. However, our knowledge of how plants sense and respond to warming ambient temperatures is rather limited. Here we show that an increase in growth temperature from 22 °C to 30 °C effectively inhibited transgene-induced posttranscriptional gene silencing (PTGS) in Arabidopsis. Interestingly, warmth-induced PTGS release exhibited transgenerational epigenetic inheritance. We discovered that the warmth-induced PTGS release occurred during a critical step that leads to the formation of double-stranded RNA (dsRNA) for producing small interfering RNAs (siRNAs). Deep sequencing of small RNAs and RNA blot analysis indicated that the 22-30 °C increase resulted in a significant reduction in the abundance of many trans-acting siRNAs that require dsRNA for biogenesis. We discovered that the temperature increase reduced the protein abundance of SUPPRESSOR OF GENE SILENCING 3, as a consequence, attenuating the formation of stable dsRNAs required for siRNA biogenesis. Importantly, SUPPRESSOR OF GENE SILENCING 3 overexpression released the warmth-triggered inhibition of siRNA biogenesis and reduced the transgenerational epigenetic memory. Thus, our study reveals a previously undescribed association between warming temperatures, an epigenetic system, and siRNA biogenesis.

  9. The Influence of Interfering Substances on the Antimicrobial Activity of Selected Quaternary Ammonium Compounds.

    PubMed

    Araújo, Paula A; Lemos, Madalena; Mergulhão, Filipe; Melo, Luís; Simões, Manuel

    2013-01-01

    Standard cleaning processes may not remove all the soiling typically found in food industry, such as carbohydrates, fats, or proteins. Contaminants have a high impact in disinfection as their presence may reduce the activity of disinfectants. The influence of alginic acid, bovine serum albumin, yeast extract, and humic acids was assessed on the antimicrobial activities of benzalkonium chloride and cetyltrimethyl ammonium bromide against Bacillus cereus vegetative cells and Pseudomonas fluorescens. The bacteria (single and consortium) were exposed to surfactants (single and combined) in the absence and presence of potential disinfection interfering substances. The antimicrobial effects of the surfactants were assessed based on the bacterial respiratory activity measured by oxygen uptake rate due to glucose oxidation. The tested surfactants were efficient against both bacteria (single and consortium) with minimum bactericidal concentrations ranging from 3 to 35 mg·L(-1). The strongest effect was caused by humic acids that severely quenched antimicrobial action, increasing the minimum bactericidal concentration of the surfactants on P. fluorescens and the consortium. The inclusion of the other interfering substances resulted in mild interferences in the antibacterial activity. This study clearly demonstrates that humic acids should be considered as an antimicrobial interfering substance in the development of disinfection strategies.

  10. Pitfall in cannabinoid analysis--detection of a previously unrecognized interfering compound in human serum.

    PubMed

    Toennes, Stefan W; Hanisch, Stephanie; Pogoda, Werner; Wunder, Cora; Paulke, Alexander

    2015-01-01

    In clinical and forensic toxicology, high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS) is increasingly used since it allows the development of sensitive and fast drug analysis procedures. During development of a LC-MS/MS method for determination of the psychoactive cannabinoid Δ(9)-tetrahydrocannabinol (THC) and of its two metabolites 11-hydroxy-THC (THCOH) and 11-nor-9-carboxy-THC (THCCOOH) in serum, a previously unrecognized interfering compound was detected. Extending the fast gradient elution program by an isocratic phase leads to sufficient separation of the interfering compound, initially co-eluting with THCCOOH and exhibiting the same fragments. For characterization, product ion scans and precursor ion scans were performed. Samples from cannabis users were analyzed to estimate the abundance of the interfering compound. The mass spectrometric experiments showed that the interfering compound exhibited the same molecular mass as THCCOOH and a similar fragmentation pattern except for relative fragment intensities. This compound was exclusively detectable in authentic samples. Concentrations were in the range of 4.5 to 51 % (median 14.6 %, n = 73) of those of THCCOOH. After further optimization of the gradient, the method was sufficiently selective and sensitive and validation parameters were within acceptance limits. A new compound related to cannabis use was detected in human serum, and data suggest an isomeric structure to THCCOOH. Considering the rather high amounts observed, it was surprising that this compound had not been detected previously. Further studies on its structure and origin are necessary.

  11. Effect of combination therapy of siRNA targeting growth hormone receptor and 5-fluorouracil in hepatic metastasis of colon cancer

    PubMed Central

    ZHOU, DONG; ZHANG, YI; LIANG, DAOMING; YUAN, YONG; ZENG, DEMIAO; CHEN, JIAYONG; YANG, JIE

    2015-01-01

    The aim of this study was to investigate the effects of small interfering RNA (siRNA) targeting human growth hormone receptor (hGHR) combined with 5-fluorouracil (5-FU) on the hepatic metastasis of colon cancer. The animal model of liver metastases using human SW480 colon cancer cells was established on BALB/c mice and the siRNA interfering plasmid targeting hGHR gene was constructed. The tumor-bearing mice were randomly divided into the saline control, plasmid, growth hormone (GH), 5-FU, 5-FU+plasmid and 5-FU+plasmid+GH groups. The liver metastasis in each group was observed. All the animals showed liver metastases and using siRNA-interfering plasmid treatment the incidence of liver metastases was significantly reduced in the tumor groups compared to the saline or GH group. The combined treatment of interfering plasmid and 5-FU slightly decreased the incidence of liver metastases in the tumor groups compared to the plasmid alone or 5-FU alone treatment, although the findings were not statistically significant. On the basis of the combination of interfering plasmid and 5-FU, the additional GH did not increase the incidence of liver metastases (P>0.05), but improved the weight loss of the mice (P<0.05) induced by the inhibition of GHR and toxicity of 5-FU. The present results showed that siRNA targeting hGHR is able to reduce the incidence of liver metastases of human SW480 colon cancer cells in mice. Thus, GHR may be important in tumor metastasis. PMID:26788158

  12. Expression of cytokines by human astrocytomas following stimulation by C3a and C5a anaphylatoxins: specific increase in interleukin-6 mRNA expression.

    PubMed

    Sayah, S; Ischenko, A M; Zhakhov, A; Bonnard, A S; Fontaine, M

    1999-06-01

    C3a and C5a anaphylatoxins are two proinflammatory peptides generated during complement activation that act through distinct Gi protein-coupled receptors named C3aR and C5aR, respectively. We have demonstrated previously that human astrocytes expressed C3aR and C5aR constitutively and were able to produce a functional complement. In this study, we examined the effect of an anaphylatoxin stimulation on cytokine expression by human astrocyte cell lines. Interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha, and transforming growth factor-beta mRNA expression was studied by quantitative RT-PCR. Whereas IL-1beta, tumor necrosis factor-alpha, and transforming growth factor-beta mRNA levels remained unchanged, stimulation of astrocytoma cells (T98G, CB193, U118MG) by C3a, C5a, and peptidic C3aR and C5aR agonists induced an increase in the IL-6 mRNA level. The amount of IL-6 was markedly increased at 3 and 6 h and returned to the basal level at 9 h of stimulation. This response was specific, because pretreatment of cells with pertussis toxin or with polyclonal anti-C3aR or anti-C5aR antibodies completely blocked the IL-6 mRNA increase. The IL-6 response was also investigated at the protein level, but IL-6 protein was detected neither in cell lysates nor in supernatants of stimulated cells. The anaphylatoxin-mediated transcriptional activation of IL-6 gene suggests that C3a and C5a could play a role in priming glial cells during the inflammatory process in the brain.

  13. Low-level light-emitting diode therapy increases mRNA expressions of IL-10 and type I and III collagens on Achilles tendinitis in rats.

    PubMed

    Xavier, Murilo; de Souza, Renato Aparecido; Pires, Viviane Araújo; Santos, Ana Paula; Aimbire, Flávio; Silva, José Antônio; Albertini, Regiane; Villaverde, Antonio Balbin

    2014-01-01

    The present study investigated the effects of low-level light-emitting diode (LED) therapy (880 ± 10 nm) on interleukin (IL)-10 and type I and III collagen in an experimental model of Achilles tendinitis. Thirty male Wistar rats were separated into six groups (n = 5), three groups in the experimental period of 7 days, control group, tendinitis-induced group, and LED therapy group, and three groups in the experimental period of 14 days, tendinitis group, LED therapy group, and LED group with the therapy starting at the 7th day after tendinitis induction (LEDT delay). Tendinitis was induced in the right Achilles tendon using an intratendinous injection of 100 μL of collagenase. The LED parameters were: optical power of 22 mW, spot area size of 0.5 cm(2), and irradiation time of 170 s, corresponding to 7.5 J/cm(2) of energy density. The therapy was initiated 12 h after the tendinitis induction, with a 48-h interval between irradiations. The IL-10 and type I and III collagen mRNA expression were evaluated by real-time polymerase chain reaction at the 7th and 14th days after tendinitis induction. The results showed that LED irradiation increased IL-10 (p < 0.001) in treated group on 7-day experimental period and increased type I and III collagen mRNA expression in both treated groups of 7- and 14-day experimental periods (p < 0.05), except by type I collagen mRNA expression in LEDT delay group. LED (880 nm) was effective in increasing mRNA expression of IL-10 and type I and III collagen. Therefore, LED therapy may have potentially therapeutic effects on Achilles tendon injuries.

  14. Schistocephalus solidus infections increase gonadotropins and gonadotropin releasing hormone (GnRH3) mRNA levels in the three-spined stickleback, Gasterosteus aculeatus.

    PubMed

    Shao, Yi Ta; Tseng, Yung Che; Trombley, Susanne; Hwang, Pung Pung; Schmitz, Monika; Borg, Bertil

    2012-09-01

    Parasites often impair the reproduction of their hosts, one well known case being the cestode Schistocephalus solidus which is a common parasite in three-spined sticklebacks, Gasterosteus aculeatus. One of the possible ways that this could be exerted is by suppression on the brain-pituitary-gonadal (BPG) axis. In this study, mRNA levels of FSH-β and LH-β and of GnRH2 (cGnRH II) and GnRH3 (sGnRH) were measured via Q-PCR in infected and uninfected fish sampled from the field a few weeks before the onset of breeding. The pituitary mRNA levels of both FSH-β and LH-β were higher in infected males than in uninfected males. Also in females, FSH-β mRNA levels were higher in infected individuals than in others, whereas there was no significant difference found in LH-β expression. Brain mRNA levels of GnRH3 were higher in infected fish than in uninfected fish in both sexes, but no difference was found in GnRH2 mRNA levels. Thus, infection by S. solidus was able to alter the expressions not only of gonadotropins (GtHs), but also of GnRH which has not been observed previously. However, the effects are opposite to what should be expected if the parasite suppressed reproduction via actions on the brain-pituitary level. The gonads are perhaps more likely to be impaired by the parasites in other ways, and changed feedbacks on the BPG axis could then lead to the increases in GtHs and GnRH.

  15. Feeding a DHA-enriched diet increases skeletal muscle protein synthesis in growing pigs: association with increased skeletal muscle insulin action and local mRNA expression of insulin-like growth factor 1.

    PubMed

    Wei, Hong-Kui; Zhou, Yuanfei; Jiang, Shuzhong; Tao, Ya-Xiong; Sun, Haiqing; Peng, Jian; Jiang, Siwen

    2013-08-01

    Dietary n-3 PUFA have been demonstrated to promote muscle growth in growing animals. In the present study, fractional protein synthesis rates (FSR) in the skeletal muscle of growing pigs fed a DHA-enriched (DE) diet (DE treatment) or a soyabean oil (SO) diet (SO treatment) were evaluated in the fed and feed-deprived states. Feeding-induced increases in muscle FSR, as well as the activation of the mammalian target of rapamycin and protein kinase B, were higher in the DE treatment as indicated by the positive interaction between diet and feeding. In the fed state, the activation of eIF4E-binding protein 1 in the skeletal muscle of pigs on the DE diet was higher than that in pigs on the SO diet (P<0·05). Feeding the DE diet increased muscle insulin-like growth factor 1 (IGF-1) expression (P<0·05) and insulin action (as demonstrated by increased insulin receptor (IR) phosphorylation, P<0·05), resulting in increased IR substrate 1 activation in the fed state. However, no difference in plasma IGF-1 concentration or hepatic IGF-1 expression between the two treatments was associated. The increased IGF-1 expression in the DE treatment was associated with increased mRNA expression of the signal transducer and activator of transcription 5A and decreased mRNA expression of protein tyrosine phosphatase, non-receptor type 3 in skeletal muscle. Moreover, mRNA expression of protein tyrosine phosphatase, non-receptor type 1 (PTPN1), the activation of PTPN1 and the activation of NF-κB in muscle were significantly lower in the DE treatment (P<0·05). The results of the present study suggest that feeding a DE diet increased feeding-induced muscle protein synthesis in growing pigs, and muscle IGF-1 expression and insulin action were involved in this action.

  16. Hyperglycaemia suppresses microRNA expression in platelets to increase P2RY12 and SELP levels in type 2 diabetes mellitus.

    PubMed

    Fejes, Zsolt; Póliska, Szilárd; Czimmerer, Zsolt; Káplár, Miklós; Penyige, András; Gál Szabó, Gabriella; Beke Debreceni, Ildikó; Kunapuli, Satya P; Kappelmayer, János; Nagy, Béla

    2017-02-28

    Megakaryocyte (MK)-derived miRNAs have been detected in platelets. Here, we analysed the expression of platelet and circulating miR-223, miR-26b, miR-126 and miR-140 that might be altered with their target mRNAs in type 2 diabetes mellitus (DM2). MiRNAs were isolated from leukocyte-depleted platelets and plasma samples obtained from 28 obese DM2, 19 non-DM obese and 23 healthy individuals. The effect of hyperglycaemia on miRNAs was also evaluated in MKs using MEG-01 and K562 cells under hyperglycaemic conditions after 8 hours up to four weeks. Quantitation of mature miRNA, pre-miRNAs and target mRNA levels (P2RY12 and SELP) were measured by RT-qPCR. To prove the association of miR-26b and miR-140 with SELP (P-selectin) mRNA level, overexpression or inhibition of these miRNAs in MEG-01 MKs was performed using mimics or anti-miRNAs, respectively. The contribution of calpain substrate Dicer to modulation of miRNAs was studied by calpain inhibition. Platelet activation was evaluated via surface P-selectin by flow cytometry. Mature and pre-forms of investigated miRNAs were significantly reduced in DM2, and platelet P2RY12 and SELP mRNA levels were elevated by two-fold at increased platelet activation compared to controls. Significantly blunted miRNA expressions were observed by hyperglycaemia in MEG-01 and K562-MK cells versus baseline values, while the manipulation of miR-26b and miR-140 expression affected SELP mRNA level. Calpeptin pretreatment restored miRNA levels in hyperglycaemic MKs. Overall, miR-223, miR-26b, miR-126 and miR-140 are expressed at a lower level in platelets and MKs in DM2 causing upregulation of P2RY12 and SELP mRNAs that may contribute to adverse platelet function.

  17. Prenatal ethanol increases ethanol intake throughout adolescence, alters ethanol-mediated aversive learning, and affects μ but not δ or κ opioid receptor mRNA expression.

    PubMed

    Fabio, María Carolina; Macchione, Ana Fabiola; Nizhnikov, Michael E; Pautassi, Ricardo Marcos

    2015-06-01

    Animal models of prenatal ethanol exposure (PEE) have indicated a facilitatory effect of PEE on adolescent ethanol intake, but few studies have assessed the effects of moderate PEE throughout adolescence. The mechanisms underlying this facilitatory effect remain largely unknown. In the present study, we analysed ethanol intake in male and female Wistar rats with or without PEE (2.0 g/kg, gestational days 17-20) from postnatal days 37 to 62. The results revealed greater ethanol consumption in PEE rats than in controls, which persisted throughout adolescence. By the end of testing, ethanol ingestion in PEE rats was nearly 6.0 g/kg. PEE was associated with insensitivity to ethanol-induced aversion. PEE and control rats were further analysed for levels of μ, δ and κ opioid receptor mRNA in the infralimbic cortex, nucleus accumbens shell, and ventral tegmental area. Similar levels of mRNA were observed across most areas and opioid receptors, but μ receptor mRNA in the ventral tegmental area was significantly increased by PEE. Unlike previous studies that assessed the effects of PEE on ethanol intake close to birth, or in only a few sessions during adolescence, the present study observed a facilitatory effect of PEE that lasted throughout adolescence. PEE was associated with insensitivity to the aversive effect of ethanol, and increased levels of μ opioid receptor transcripts. PEE is a prominent vulnerability factor that probably favors the engagement of adolescents in risky trajectories of ethanol use.

  18. RNA Sequence Analysis of Human Huntington Disease Brain Reveals an Extensive Increase in Inflammatory and Developmental Gene Expression

    PubMed Central

    Labadorf, Adam; Hoss, Andrew G.; Lagomarsino, Valentina; Latourelle, Jeanne C.; Hadzi, Tiffany C.; Bregu, Joli; MacDonald, Marcy E.; Gusella, James F.; Chen, Jiang-Fan; Akbarian, Schahram; Weng, Zhiping; Myers, Richard H.

    2015-01-01

    Huntington’s Disease (HD) is a devastating neurodegenerative disorder that is caused by an expanded CAG trinucleotide repeat in the Huntingtin (HTT) gene. Transcriptional dysregulation in the human HD brain has been documented but is incompletely understood. Here we present a genome-wide analysis of mRNA expression in human prefrontal cortex from 20 HD and 49 neuropathologically normal controls using next generation high-throughput sequencing. Surprisingly, 19% (5,480) of the 28,087 confidently detected genes are differentially expressed (FDR<0.05) and are predominantly up-regulated. A novel hypothesis-free geneset enrichment method that dissects large gene lists into functionally and transcriptionally related groups discovers that the differentially expressed genes are enriched for immune response, neuroinflammation, and developmental genes. Markers for all major brain cell types are observed, suggesting that HD invokes a systemic response in the brain area studied. Unexpectedly, the most strongly differentially expressed genes are a homeotic gene set (represented by Hox and other homeobox genes), that are almost exclusively expressed in HD, a profile not widely implicated in HD pathogenesis. The significance of transcriptional changes of developmental processes in the HD brain is poorly understood and warrants further investigation. The role of inflammation and the significance of non-neuronal involvement in HD pathogenesis suggest anti-inflammatory therapeutics may offer important opportunities in treating HD. PMID:26636579

  19. Androgens regulate ovarian follicular development by increasing follicle stimulating hormone receptor and microRNA-125b expression.

    PubMed

    Sen, Aritro; Prizant, Hen; Light, Allison; Biswas, Anindita; Hayes, Emily; Lee, Ho-Joon; Barad, David; Gleicher, Norbert; Hammes, Stephen R

    2014-02-25

    Although androgen excess is considered detrimental to women's health and fertility, global and ovarian granulosa cell-specific androgen-receptor (AR) knockout mouse models have been used to show that androgen actions through ARs are actually necessary for normal ovarian function and female fertility. Here we describe two AR-mediated pathways in granulosa cells that regulate ovarian follicular development and therefore female fertility. First, we show that androgens attenuate follicular atresia through nuclear and extranuclear signaling pathways by enhancing expression of the microRNA (miR) miR-125b, which in turn suppresses proapoptotic protein expression. Second, we demonstrate that, independent of transcription, androgens enhance follicle-stimulating hormone (FSH) receptor expression, which then augments FSH-mediated follicle growth and development. Interestingly, we find that the scaffold molecule paxillin regulates both processes, making it a critical regulator of AR actions in the ovary. Finally, we report that low doses of exogenous androgens enhance gonadotropin-induced ovulation in mice, further demonstrating the critical role that androgens play in follicular development and fertility. These data may explain reported positive effects of androgens on ovulation rates in women with diminished ovarian reserve. Furthermore, this study demonstrates mechanisms that might contribute to the unregulated follicle growth seen in diseases of excess androgens such as polycystic ovary syndrome.

  20. Kisspeptin mRNA expression is increased in the posterior hypothalamus in the rat model of polycystic ovary syndrome.

    PubMed

    Matsuzaki, Toshiya; Tungalagsuvd, Altankhuu; Iwasa, Takeshi; Munkhzaya, Munkhsaikhan; Yanagihara, Rie; Tokui, Takako; Yano, Kiyohito; Mayila, Yiliyasi; Kato, Takeshi; Kuwahara, Akira; Matsui, Sumika; Irahara, Minoru

    2017-01-30

    Hypersecretion of luteinizing hormone (LH) is a common endocrinological finding of polycystic ovary syndrome (PCOS). This derangement might have a close relationship with hypothalamic kisspeptin expression that is thought to be a key regulator of gonadotropin-releasing hormone (GnRH). We evaluated the relationship between the hypothalamic-pituitary-gonadal axis (HPG axis) and kisspeptin using a rat model of PCOS induced by letrozole. Letrozole pellets (0.4 mg/day) and control pellets were placed subcutaneously onto the backs of 3-week-old female Wistar rats. Body weight, vaginal opening and vaginal smear were checked daily. Blood and tissues of ovary, uterus and brain were collected at 12-weeks of age. An hypothalamic block was cut into anterior and posterior blocks, which included the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC), respectively, in order to estimate hypothalamic kisspeptin expression in each area. The letrozole group showed a similar phenotype to human PCOS such as heavier body weight, heavier ovary, persistent anovulatory state, multiple enlarged follicles with no corpus luteum and higher LH and testosterone (T) levels compared to the control group. Kisspeptin mRNA expression in the posterior hypothalamic block including ARC was higher in the letrozole group than in the control group although its expression in the anterior hypothalamic block was similar between groups. These results suggest that enhanced KNDy neuron activity in ARC contributes to hypersecretion of LH in PCOS and might be a therapeutic target to rescue ovulatory disorder of PCOS in the future.

  1. Nanocarrier mediated Delivery of siRNA/miRNA in Combination with Chemotherapeutic Agents for Cancer Therapy: Current Progress and Advances

    PubMed Central

    Gandhi, Nishant S.; Tekade, Rakesh K.; Chougule, Mahavir B.

    2014-01-01

    Chemotherapeutic agents have certain limitations when it comes to treating cancer, the most important being severe side effects along with multidrug resistance developed against them. Tumor cells exhibits drug resistance due to activation of various cellular level processes viz. activation of drug efflux pumps, anti-apoptotic defense mechanisms etc. Currently, RNA interference (RNAi) based therapeutic approaches are under vibrant scrutinization to seek cancer cure. Especially small interfering RNA (siRNA) and micro RNA (miRNA), are able to knock down the carcinogenic genes by targeting the mRNA expression, which underlies the uniqueness of this therapeutic approach. Recent research focus in the regime of cancer therapy involves the engagement of targeted delivery of siRNA/miRNA in combinations with other therapeutic agents (such as gene, DNA or chemotherapeutic drug) for targeting permeability glycoprotein (P-gp), Multidrug resistant protein 1(MRP-1), B-cell lymphoma (BCL-2) and other targets that are mainly responsible for resistance in cancer therapy. RNAi-chemotherapeutic drug combinations have also been found to be effective against different molecular targets as well and can increase the sensitization of cancer cells to therapy several folds. However, due to stability issues associated with siRNA/miRNA suitable protective carrier is needed and nanotechnology based approaches have been widely explored to overcome these drawbacks. Furthermore, it has been univocally advocated that the co-delivery of siRNA/miRNA with other chemodrugs significantly enhances their capability to overcome cancer resistance compared to naked counterparts. The objective of this article is to review recent nanocarrier based approaches adopted for the delivery of siRNA/miRNA combinations with other anticancer agents (siRNA/miRNA/pDNA/chemodrugs) to treat cancer. PMID:25204288

  2. Delivery of small interfering RNAs in human cervical cancer cells by polyethylenimine-functionalized carbon nanotubes.

    PubMed

    Huang, Yuan-Pin; Lin, I-Jou; Chen, Chih-Chen; Hsu, Yi-Chiang; Chang, Chi-Chang; Lee, Mon-Juan

    2013-06-06

    Carbon nanotubes are capable of penetrating the cell membrane and are widely considered as potential carriers for gene or drug delivery. Because the C-C and C=C bonds in carbon nanotubes are nonpolar, functionalization is required for carbon nanotubes to interact with genes or drugs as well as to improve their biocompatibility. In this study, polyethylenimine (PEI)-functionalized single-wall (PEI-NH-SWNTs) and multiwall carbon nanotubes (PEI-NH-MWNTs) were produced by direct amination method. PEI functionalization increased the positive charge on the surface of SWNTs and MWNTs, allowing carbon nanotubes to interact electrostatically with the negatively charged small interfering RNAs (siRNAs) and to serve as nonviral gene delivery reagents. PEI-NH-MWNTs and PEI-NH-SWNTs had a better solubility in water than pristine carbon nanotubes, and further removal of large aggregates by centrifugation produced a stable suspension of reduced particle size and improved homogeneity and dispersity. The amount of grafted PEI estimated by thermogravimetric analysis was 5.08% (w/w) and 5.28% (w/w) for PEI-NH-SWNTs and PEI-NH-MWNTs, respectively. For the assessment of cytotoxicity, various concentrations of PEI-NH-SWNTs and PEI-NH-MWNTs were incubated with human cervical cancer cells, HeLa-S3, for 48 h. PEI-NH-SWNTs and PEI-NH-MWNTs induced cell deaths in a dose-dependent manner but were less cytotoxic compared to pure PEI. As determined by electrophoretic mobility shift assay, siRNAs directed against glyceraldehyde-3-phosphate dehydrogenase (siGAPDH) were completely associated with PEI-NH-SWNTs or PEI-NH-MWNTs at a PEI-NH-SWNT/siGAPDH or PEI-NH-MWNT/siGAPDH mass ratio of 80:1 or 160:1, respectively. Furthermore, PEI-NH-SWNTs and PEI-NH-MWNTs successfully delivered siGAPDH into HeLa-S3 cells at PEI-NH-SWNT/siGAPDH and PEI-NH-MWNT/siGAPDH mass ratios of 1:1 to 20:1, resulting in suppression of the mRNA level of GAPDH to an extent similar to that of DharmaFECT, a common transfection

  3. Delivery of small interfering RNAs in human cervical cancer cells by polyethylenimine-functionalized carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Huang, Yuan-Pin; Lin, I.-Jou; Chen, Chih-Chen; Hsu, Yi-Chiang; Chang, Chi-Chang; Lee, Mon-Juan

    2013-06-01

    Carbon nanotubes are capable of penetrating the cell membrane and are widely considered as potential carriers for gene or drug delivery. Because the C-C and C=C bonds in carbon nanotubes are nonpolar, functionalization is required for carbon nanotubes to interact with genes or drugs as well as to improve their biocompatibility. In this study, polyethylenimine (PEI)-functionalized single-wall (PEI-NH-SWNTs) and multiwall carbon nanotubes (PEI-NH-MWNTs) were produced by direct amination method. PEI functionalization increased the positive charge on the surface of SWNTs and MWNTs, allowing carbon nanotubes to interact electrostatically with the negatively charged small interfering RNAs (siRNAs) and to serve as nonviral gene delivery reagents. PEI-NH-MWNTs and PEI-NH-SWNTs had a better solubility in water than pristine carbon nanotubes, and further removal of large aggregates by centrifugation produced a stable suspension of reduced particle size and improved homogeneity and dispersity. The amount of grafted PEI estimated by thermogravimetric analysis was 5.08% ( w/ w) and 5.28% ( w/ w) for PEI-NH-SWNTs and PEI-NH-MWNTs, respectively. For the assessment of cytotoxicity, various concentrations of PEI-NH-SWNTs and PEI-NH-MWNTs were incubated with human cervical cancer cells, HeLa-S3, for 48 h. PEI-NH-SWNTs and PEI-NH-MWNTs induced cell deaths in a dose-dependent manner but were less cytotoxic compared to pure PEI. As determined by electrophoretic mobility shift assay, siRNAs directed against glyceraldehyde-3-phosphate dehydrogenase (siGAPDH) were completely associated with PEI-NH-SWNTs or PEI-NH-MWNTs at a PEI-NH-SWNT/siGAPDH or PEI-NH-MWNT/siGAPDH mass ratio of 80:1 or 160:1, respectively. Furthermore, PEI-NH-SWNTs and PEI-NH-MWNTs successfully delivered siGAPDH into HeLa-S3 cells at PEI-NH-SWNT/siGAPDH and PEI-NH-MWNT/siGAPDH mass ratios of 1:1 to 20:1, resulting in suppression of the mRNA level of GAPDH to an extent similar to that of DharmaFECT, a common transfection

  4. Structure-Guided Control of siRNA Off-Target Effects.

    PubMed

    Suter, Scott R; Sheu-Gruttadauria, Jessica; Schirle, Nicole T; Valenzuela, Rachel; Ball-Jones, Alexi A; Onizuka, Kazumitsu; MacRae, Ian J; Beal, Peter A

    2016-07-20

    Short interfering RNAs (siRNAs) are promising therapeutics that make use of the RNA interference (RNAi) pathway, but liabilities arising from the native RNA structure necessitate chemical modification for drug development. Advances in the structural characterization of components of the human RNAi pathway have enabled structure-guided optimization of siRNA properties. Here we report the 2.3 Å resolution crystal structure of human Argonaute 2 (hAgo2), a key nuclease in the RNAi pathway, bound to an siRNA guide strand bearing an unnatural triazolyl nucleotide at position 1 (g1). Unlike natural nucleotides, this analogue inserts deeply into hAgo2's central RNA binding cleft and thus is able to modulate pairing between guide and target RNAs. The affinity of the hAgo2-siRNA complex for a seed-only matched target was significantly reduced by the triazolyl modification, while the affinity for a fully matched target was unchanged. In addition, siRNA potency for off-target repression was reduced (4-fold increase in IC50) by the modification, while on-target knockdown was improved (2-fold reduction in IC50). Controlling siRNA on-target versus microRNA (miRNA)-like off-target potency by projection of substituent groups into the hAgo2 central cleft from g1 is a new approach to enhance siRNA selectivity with a strong structural rationale.

  5. Disruption of microRNA-21 by TALEN leads to diminished cell transformation and increased expression of cell-environment interaction genes

    PubMed Central

    Wu, Xiwei; Wang, Xiaoling; Wang, Yingjia; Lin, Ting-Yu; Kurata, Jessica; Wu, Jun; Vonderfecht, Steven; Sun, Guihua; Huang, He; Yee, Jiing-Kuan; Hu, Jianda; Lin, Ren-Jang

    2015-01-01

    MicroRNA-21 is dysregulated in many cancers and fibrotic diseases. Since miR-21 suppresses several tumor suppressor and anti-apoptotic genes, it is considered a cancer therapeutic target. Antisense oligonucleotides are commonly used to inhibit a miRNA; however, blocking miRNA function via an antagomir is temporary, often only achieves a partial knock-down, and may be complicated by off-target effects. Here, we used transcription activator-like effector nucleases (TALENs) to disrupt miR-21 in cancerous cells. Individual deletion clones were screened and isolated without drug selection. Sequencing and quantitative RT-PCR identified clones with no miR-21 expression. The loss of miR-21 led to subtle but global increases of mRNAs containing miR-21 target sequences. Cells without miR-21 became more sensitive to cisplatin and less transformed in culture and in mouse xenografts. In addition to the increase of PDCD4 and PTEN protein, mRNAs for COL4A1, JAG1, SERPINB5/Maspin, SMAD7, and TGFBI – all are miR-21 targets and involved in TGFβ and fibrosis regulation – were significantly upregulated in miR-21 knockout cells. Gene ontology and pathway analysis suggested that cell-environment interactions involving extracellular matrix can be an important miR-21 pathogenic mechanism. The study also demonstrates the value of using TALEN-mediated microRNA gene disruption in human pathobiological studies. PMID:25304376

  6. Nerve Growth Factor Increases mRNA Levels for the Prion Protein and the β -amyloid Protein Precursor in Developing Hamster Brain

    NASA Astrophysics Data System (ADS)

    Mobley, William C.; Neve, Rachael L.; Prusiner, Stanley B.; McKinley, Michael P.

    1988-12-01

    Deposition of amyloid filaments serves as a pathologic hallmark for some neurodegenerative disorders. The prion protein (PrP) is found in amyloid of animals with scrapie and humans with Creutzfeldt-Jakob disease; the β protein is present in amyloid deposits in Alzheimer disease and Down syndrome patients. These two proteins are derived from precursors that in the brain are expressed primarily in neurons and are membrane bound. We found that gene expression for PrP and the β -protein precursor (β -PP) is regulated in developing hamster brain. Specific brain regions showed distinct patterns of ontogenesis for PrP and β -PP mRNAs. The increases in PrP and β -PP mRNAs in developing basal forebrain coincided with an increase in choline acetyltransferase activity, raising the possibility that these markers might be coordinately controlled in cholinergic neurons and regulated by nerve growth factor (NGF). Injections of NGF into the brains of neonatal hamsters increased both PrP and β -PP mRNA levels. Increased PrP and β -PP mRNA levels induced by NGF were confined to regions that contain NGF-responsive cholinergic neurons and were accompanied by elevations in choline acetyltransferase. It remains to be established whether or not exogenous NGF acts to increase PrP and β -PP gene expression selectively in forebrain cholinergic neurons in the developing hamster and endogenous NGF regulates expression of these genes.

  7. Artificial microRNAs and synthetic trans-acting small interfering RNAs interfere with viroid infection.

    PubMed

    Carbonell, Alberto; Daròs, José-Antonio

    2016-12-27

    Artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are two classes of artificial small RNAs (sRNAs) engineered to silence endogenous transcripts as well as viral RNAs in plants. Here, we explore the possibility of using amiRNAs and syn-tasiRNAs to specifically interfere with infections by viroids, small (250-400 nt) non-coding circular RNAs with compact secondary structure infecting a wide range of plant species. The combined use of recent high-throughput methods for artificial sRNA construct generation and of the Potato spindle tuber viroid (PSTVd)/Nicotiana benthamiana pathosystem allowed for the simple and time-effective screening of multiple artificial sRNAs targeting sites distributed along PSTVd RNAs of (+) or (-) polarity. The majority of amiRNAs were highly active in agroinfiltrated leaves when co-expressed with an infectious PSTVd transcript, as were syn-tasiRNAs derived from a construct including the five most effective amiRNA sequences. A comparative analysis showed that the effects of the most effective amiRNA and of the syn-tasiRNAs were similar in agroinfiltrated leaves, as well as in upper non-agroinfiltrated leaves where PSTVd accumulation was significantly delayed. These results suggest that amiRNAs and syn-tasiRNAs can be used effectively to control viroid infections in plants. This article is protected by copyright. All rights reserved.

  8. Cell type-specific delivery of short interfering RNAs by dye-functionalised theranostic nanoparticles

    NASA Astrophysics Data System (ADS)

    Press, Adrian T.; Traeger, Anja; Pietsch, Christian; Mosig, Alexander; Wagner, Michael; Clemens, Mark G.; Jbeily, Nayla; Koch, Nicole; Gottschaldt, Michael; Bézière, Nicolas; Ermolayev, Volodymyr; Ntziachristos, Vasilis; Popp, Jürgen; Kessels, Michael M.; Qualmann, Britta; Schubert, Ulrich S.; Bauer, Michael

    2014-12-01

    Efficient delivery of short interfering RNAs reflects a prerequisite for the development of RNA interference therapeutics. Here, we describe highly specific nanoparticles, based on near infrared fluorescent polymethine dye-derived targeting moieties coupled to biodegradable polymers. The fluorescent dye, even when coupled to a nanoparticle, mimics a ligand for hepatic parenchymal uptake transporters resulting in hepatobiliary clearance of approximately 95% of the dye within 45 min. Body distribution, hepatocyte uptake and excretion into bile of the dye itself, or dye-coupled nanoparticles can be tracked by intravital microscopy or even non-invasively by multispectral optoacoustic tomography. Efficacy of delivery is demonstrated in vivo using 3-hydroxy-3-methyl-glutaryl-CoA reductase siRNA as an active payload resulting in a reduction of plasma cholesterol levels if siRNA was formulated into dye-functionalised nanoparticles. This suggests that organ-selective uptake of a near infrared dye can be efficiently transferred to theranostic nanoparticles allowing novel possibilities for personalised silencing of disease-associated genes.

  9. Short-interfering RNAs Induce Retinal Degeneration via TLR3 and IRF3

    PubMed Central

    Kleinman, Mark E; Kaneko, Hiroki; Cho, Won Gil; Dridi, Sami; Fowler, Benjamin J; Blandford, Alexander D; Albuquerque, Romulo JC; Hirano, Yoshio; Terasaki, Hiroko; Kondo, Mineo; Fujita, Takashi; Ambati, Balamurali K; Tarallo, Valeria; Gelfand, Bradley D; Bogdanovich, Sasha; Baffi, Judit Z; Ambati, Jayakrishna

    2012-01-01

    The discovery of sequence-specific gene silencing by endogenous double-stranded RNAs (dsRNA) has propelled synthetic short-interfering RNAs (siRNAs) to the forefront of targeted pharmaceutical engineering. The first clinical trials utilized 21-nucleotide (nt) siRNAs for the treatment of neovascular age-related macular degeneration (AMD). Surprisingly, these compounds were not formulated for cell permeation, which is required for bona fide RNA interference (RNAi). We showed that these “naked” siRNAs suppress neovascularization in mice not via RNAi but via sequence-independent activation of cell surface Toll-like receptor-3 (TLR3). Here, we demonstrate that noninternalized siRNAs induce retinal degeneration in mice by activating surface TLR3 on retinal pigmented epithelial cells. Cholesterol conjugated siRNAs capable of cell permeation and triggering RNAi also induce the same phenotype. Retinal degeneration was not observed after treatment with siRNAs shorter than 21-nts. Other cytosolic dsRNA sensors are not critical to this response. TLR3 activation triggers caspase-3-mediated apoptotic death of the retinal pigment epithelium (RPE) via nuclear translocation of interferon regulatory factor-3. While this unexpected adverse effect of siRNAs has implications for future clinical trials, these findings also introduce a new preclinical model of geographic atrophy (GA), a late stage of dry AMD that causes blindness in millions worldwide. PMID:21988875

  10. Alcohol dysregulates corticotropin-releasing-hormone (CRH) promoter activity by interfering with the negative glucocorticoid response element (nGRE).

    PubMed

    Przybycien-Szymanska, Magdalena M; Mott, Natasha N; Pak, Toni R

    2011-01-01

    EtOH exposure in male rats increases corticotropin-releasing hormone (CRH) mRNA in the paraventricular nucleus of the hypothalamus (PVN), a brain region responsible for coordinating stress and anxiety responses. In this study we identified the molecular mechanisms involved in mediating these effects by examining the direct effects of EtOH on CRH promoter activity in a neuronal cell line derived from the PVN (IVB). In addition, we investigated the potential interactions of EtOH and glucocorticoids on the CRH promoter by concomitantly treating cells with EtOH and the glucocorticoid receptor (GR) antagonist RU486, and by sequentially deleting GR binding sites within glucocorticoid response element (GRE) on the CRH promoter. Cells were transiently transfected with a firefly luciferase reporter construct containing 2.5 kb of the rat wild type (WT) or mutated CRH promoter. Our results showed that EtOH treatment induced a biphasic response in CRH promoter activity. EtOH exposure for 0.5 h significantly decreased promoter activity compared to vehicle treated controls, whereas promoter activity was significantly increased after 2.0 h of EtOH exposure. Treatment with RU486, or deletion of the GR binding sites 1 and 2 within the GRE, abolished the EtOH-induced increase in the promoter activity, however did not affect EtOH-induced decrease in CRH promoter activity at an earlier time point. Overall, our data suggest that alcohol exposure directly regulates CRH promoter activity by interfering with the normal feedback mechanisms of glucocorticoids mediated by GR signaling at the GRE site of the CRH promoter.

  11. Increases in transient receptor potential vanilloid-1 mRNA and protein in primary afferent neurons stimulated by protein kinase C and their possible role in neurogenic inflammation

    PubMed Central

    Xu, Xijin; Wang, Peng; Zou, Xiaoju; Li, Dingge; Fang, Li; Lin, Qing

    2008-01-01

    A recent study by our group demonstrates pharmacologically that the transient receptor potential vanilloid-1 (TRPV1) is activated by intradermal injection of capsaicin to initiate neurogenic inflammation by the release of neuropeptides in the periphery. In this study, expression of TRPV1, phosphorylated protein kinase C (p-PKC) and calcitonin gene-related peptide (CGRP) in dorsal root ganglion (DRG) neurons were visualized using immunofluorescence, real-time PCR and Western blots to examine whether increases in TRPV1 mRNA and protein levels evoked by capsaicin injection are subject to modulation by the activation of PKC and to analyze the role of this process in the pathogenesis of neurogenic inflammation. Capsaicin injection into the hindpaw skin of anesthetized rats evoked increases in the expression of TRPV1, CGRP and p-PKC in mRNA and/or protein levels and in the number of single labeled TRPV1, p-PKC and CGRP neurons in ipsilateral L4–5 DRGs. Co-expressions of TRPV1 with p-PKC and/or CGRP in DRG neurons were also significantly increased after CAP injection. These evoked expressions both at molecular and cellular levels were significantly inhibited after TRPV1 receptors were blocked by 5′-iodoresiniferatoxin (5 μg) or PKC was inhibited by chelerythrine chloride (5 μg). Taken together, these results provide evidence that up-regulation of TRPV1 mRNA and protein levels under inflammatory conditions evoked by capsaicin injection is subject to modulation by the PKC cascade in which increased CGRP level in DRG neurons may be related to the initiation of neurogenic inflammation. Thus, up-regulation of TRPV1 receptors in DRG neurons seems critical for initiating acute neurogenic inflammation. PMID:18752301

  12. Prolonged food deprivation increases mRNA expression of deiodinase 1 and 2, and thyroid hormone receptor β-1 in a fasting-adapted mammal.

    PubMed

    Martinez, Bridget; Soñanez-Organis, José G; Vázquez-Medina, José Pablo; Viscarra, Jose A; MacKenzie, Duncan S; Crocker, Daniel E; Ortiz, Rudy M

    2013-12-15

    Food deprivation in mammals is typically associated with reduced thyroid hormone (TH) concentrations and deiodinase content and activity to suppress metabolism. However, in prolonged-fasted, metabolically active elephant seal pups, TH levels are maintained, if not elevated. The functional relevance of this apparent paradox is unknown and demonstrates variability in the regulation of TH levels, metabolism and function in food-deprived mammals. To address our hypothesis that cellular TH-mediated activity is upregulated with fasting duration, we quantified the mRNA expression and protein content of adipose and muscle deiodinase type I (DI1) and type II (DI2), and TH receptor beta-1 (THrβ-1) after 1, 3 and 7 weeks of fasting in northern elephant seal pups (N=5-7 per week). Fasting did not decrease the concentrations of plasma thyroid stimulating hormone, total triiodothyronine (tT3), free T3, total thyroxine (tT4) or free T4, suggesting that the hypothalamic-pituitary-thyroid axis is not suppressed, but rather maintained during fasting. Mean mRNA expression of adipose DI1 and DI2 increased threefold and fourfold, respectively, and 20- and 30-fold, respectively, in muscle. With the exception of adipose DI1, protein expression of adipose DI2 and muscle DI1 and DI2 increased twofold to fourfold. Fasting also increased adipose (fivefold) and muscle (fourfold) THrβ-1 mRNA expression, suggesting that the mechanisms mediating cellular TH activity are upregulated with prolonged fasting. The data demonstrate a unique, atypical mechanism of TH activity and regulation in mammals adapted to prolonged food deprivation in which the potential responsiveness of peripheral tissues and cellular TH activity are increased, which may contribute to their lipid-based metabolism.

  13. The Cytokine-mRNA Increase Induced by Withdrawal from Chronic Ethanol in the Sterile Environment of Brain is mediated by CRF and HMGB1 Release

    PubMed Central

    Whitman, Buddy A.; Knapp, Darin J.; Werner, David F.; Crews, Fulton T.; Breese, George R.

    2013-01-01

    Background Many neurobiological factors may initiate and sustain alcoholism. Recently, dysregulation of the neuroimmune-system by chronic-ethanol (CE) has implicated toll-like receptor-4 (TLR4)-activation. Even though TLR4s are linked to CE-initiation of brain cytokine-mRNAs, the means by which CE influences neuroimmune signaling in the sterile environment of brain remains uncertain. Therefore, the hypothesis is tested that release of an endogenous TLR4 agonist, high-mobility group box 1 (HMGB1) and/or CRF during CE-withdrawal are responsible for CE-protocols increasing cytokine-mRNAs. Methods Acute-ethanol 2.75g/kg) and acute-LPS (lipopolysaccharide)(250μg/kg) dosing on cytokine-mRNAs are first compared. Then, the effects of chronic-LPS exposure (250 μg/kg for 10-days) on cytokine-mRNAs are compared to changes induced by CE-protocols [15-days of continuous 7% ethanol-diet (CE-protocol) or three-intermittent 5-day cycles of 7%-ethanol-diet (CIE-protocol)]. Additionally, TLR4-, HMGB1- and down-stream effector mRNAs are assessed after CE, CIE, and chronic-LPS. To test whether HMGB1 and/or CRF support the CE-withdrawal increase in cytokine-mRNAs, the HMGB1-antagonists, glycyrrhizin and ethyl-pyruvate, and a CRF1-receptor-antagonist (CRF1RA) are administered during 24-hours of CE-withdrawal. Results While cytokine-mRNAs were not increased following acute-ethanol, acute-LPS increased all cytokine-mRNAs 4-hours after injection. CE produced no change in cytokine-mRNAs prior to CE-removal; however, the CE- and CIE-protocols increased cytokine-mRNAs by 24-hours after withdrawal. In contrast, chronic-LPS produced no cytokine-mRNA changes 24-hours after LPS-dosing. TLR4-mRNA was elevated 24-hours following both CE-protocols and chronic-LPS exposure. While chronic-LPS had no effect on HMGB1-mRNA, withdrawal from CE-protocols significantly elevated HMGB1-mRNA. Systemic administration of HMGB1-antagonists or a CRF1RA significantly reduced the cytokine-mRNA increase following

  14. Expression of antisense of microRNA-26a-5p in mesenchymal stem cells increases their therapeutic effects against cirrhosis

    PubMed Central

    Chen, Li; Zeng, Wenhuan; Yang, Bo; Cui, Xiang; Feng, Cong; Wang, Lili; Wang, Hao; Zhou, Xuan; Li, Peng; Lv, Faqin; Li, Tanshi

    2017-01-01

    Hepatocyte growth factor (HGF) is a potent mitogen for mature hepatocytes, and has been shown to prevent cirrhosis during liver regeneration. Transplantation of mesenchymal stem cells (MSCs) reduces the development of cirrhosis after liver injury. However, the production and secretion of transplanted MSCs in liver were not studied yet. Here we found that the MSCs expressed low levels of HGF protein, but surprisingly high levels of HGF mRNA. Further investigation using bioinformatics analyses and luciferase reporter assay showed that MSCs expressed high levels of microRNA-26a-5p (miR-26a-5p), which targeted 3’-UTR of HGF mRNA to inhibit its protein translation. In vivo, miR-26a-5p-depleted MSCs were transplanted into mice with carbon tetrachloride (CCl4)-induced cirrhosis. We found that suppression of miR-26a-5p in MSCs further ameliorated the severity of liver fibrosis, reduced the portal hypertension and sodium retention, compared to transplantation of control MSCs. Hence, our study suggests that suppression of miR-26a-5p in MSCs may improve their therapeutic effects against cirrhosis through increasing HGF production. PMID:28386375

  15. Antidepressant dose of taurine increases mRNA expression of GABAA receptor α2 subunit and BDNF in the hippocampus of diabetic rats.

    PubMed

    Caletti, Greice; Almeida, Felipe Borges; Agnes, Grasiela; Nin, Maurício Schüler; Barros, Helena Maria Tannhauser; Gomez, Rosane

    2015-04-15

    Diabetes mellitus is a metabolic disorder associated with higher risk for depression. Diabetic rats present depressive-like behaviors and taurine, one of the most abundant free amino acids in the brain, reverses this depressive behaviors. Because taurine is a GABAA agonist modulator, we hypothesize that its antidepressant effect results from the interaction on this system by changing α2 GABAA receptor subunit expression, beside changes on BDNF mRNA, and memory in diabetic rats. Streptozotocin-diabetic and non-diabetic Wistar rats were daily injected with 100mg/kg of taurine or saline, intraperitoneally, for 30 days. At the end of the experiment, rats were exposed to the novel object recognition memory. Later they were euthanized, the brains were weighed, and the hippocampus was dissected for α2 GABAA subunit and BDNF mRNA expression. Real-time quantitative PCR (qPCR) showed that diabetic rats presented lower α2 GABAA subunit and BDNF mRNA expression than non-diabetic rats and taurine increased both parameters in these sick rats. Taurine also reversed the lower brain weight and improved the short-term memory in diabetic rats. Thus, the taurine antidepressant effect may be explained by interference with the GABA system, in line to its neuroprotective effect showed here by preventing brain weight loss and improving memory in diabetic rats.

  16. Increased duodenal DMT-1 expression and unchanged HFE mRNA levels in HFE-associated hereditary hemochromatosis and iron deficiency.

    PubMed

    Byrnes, V; Barrett, S; Ryan, E; Kelleher, T; O'Keane, C; Coughlan, B; Crowe, J

    2002-01-01

    HFE-associated hereditary hemochromatosis is characterized by imbalances of iron homeostasis and alterations in intestinal iron absorption. The identification of the HFE gene and the apical iron transporter divalent metal transporter-1, DMT-1, provide a direct method to address the mechanisms of iron overload in this disease. The aim of this study was to evaluate the regulation of duodenal HFE and DMT-1 gene expression in HFE-associated hereditary hemochromatosis. Small bowel biopsies and serum iron indices were obtained from a total of 33 patients. The study population comprised 13 patients with hereditary hemochromatosis (C282Y homozygous), 10 patients with iron deficiency anemia, and 10 apparently healthy controls, all of whom were genotyped for the two common mutations in the HFE gene (C282Y and H63D). Total RNA was isolated from tissue and amplified via RT-PCR for HFE, DMT-1, and the internal control GAPDH. DMT-1 protein expression was additionally assessed by immunohistochemistry. Levels of HFE mRNA did not differ significantly between patient groups (P = 0.09), specifically between C282Y homozygotes and iron deficiency anemic patients, when compared to controls (P = 0.09, P = 0.9, respectively). In contrast, DMT-1 mRNA levels were at least twofold greater in patients with hereditary hemochromatosis and iron deficiency anemia when compared to controls (P = 0.02, P = 0.01, respectively). Heightened DMT-1 protein expression correlated with mRNA levels in all patients. Loss of HFE function in hereditary hemochromatosis is not derived from inhibition of its gene expression. DMT-1 expression in C282Y homozygote subjects is consistent with the hypothesis of a "paradoxical" duodenal iron deficiency in hereditary hemochromatosis. The observed twofold upregulation of the DMT-1 is consistent with the slow but steady increase in body iron stores observed in those presenting with clinical features of hereditary hemochromatosis.

  17. The Exosome and Trans-Acting Small Interfering RNAs Regulate Cuticular Wax Biosynthesis during Arabidopsis Inflorescence Stem Development1[OPEN

    PubMed Central

    Lam, Patricia; Zhao, Lifang; Eveleigh, Nathan; Yu, Yu; Chen, Xuemei

    2015-01-01

    The primary aerial surfaces of land plants are covered with a cuticle, a protective layer composed of the cutin polyester matrix and cuticular waxes. Previously, we discovered a unique mechanism of regulating cuticular wax biosynthesis during Arabidopsis (Arabidopsis thaliana) stem elongation that involves ECERIFERUM7 (CER7), a core subunit of the exosome. Because loss-of-function mutations in CER7 result in reduced expression of the wax biosynthetic gene CER3, we proposed that CER7 is involved in degrading a messenger RNA encoding a CER3 repressor. To identify this putative repressor, we performed a cer7 suppressor screen that resulted in the isolation of the posttranscriptional gene-silencing components RNA-DEPENDENT RNA POLYMERASE1 and SUPPRESSOR OF GENE SILENCING3, indicating that small RNAs regulate CER3 expression. To establish the identity of the effector RNA species and determine whether these RNAs control CER3 transcript levels directly, we cloned additional genes identified in our suppressor screen and performed next-generation sequencing of small RNA populations that differentially accumulate in the cer7 mutant in comparison with the wild type. Our results demonstrate that the trans-acting small interfering RNA class of small RNAs are the effector molecules involved in direct silencing of CER3 and that the expression of five additional genes (EARLY RESPONSE TO DEHYDRATION14, AUXIN RESISTANT1, a translation initiation factor SUI1 family protein, and two genes of unknown function) is controlled by both CER7 and trans-acting small interfering RNAs. PMID:25502190

  18. The use of small interfering RNAs to inhibit adipocyte differentiation in human preadipocytes and fetal-femur-derived mesenchymal cells

    SciTech Connect

    Xu, Y.; Mirmalek-Sani, S.-H.; Yang, X.; Zhang, J.; Oreffo, R.O.C. . E-mail: roco@soton.ac.uk

    2006-06-10

    RNA interference (RNAi) has been used in functional genomics and offers innovative approaches in the development of novel therapeutics. Human mesenchymal stem cells offer a unique cell source for tissue engineering/regeneration strategies. The current study examined the potential of small interfering RNAs (siRNA) against human peroxisome proliferator activated receptor gamma (PPAR{gamma}) to suppress adipocyte differentiation (adipogenesis) in human preadipocytes and fetal-femur-derived mesenchymal cells. Adipogenesis was investigated using cellular and biochemical analysis. Transient transfection with PPAR{gamma}-siRNA using a liposomal-based strategy resulted in a significant inhibition of adipogenesis in human preadipocytes and fetal-femur-derived mesenchymal cells, compared to controls (cell, liposomal and negative siRNA). The inhibitory effect of PPAR{gamma}-siRNA was supported by testing human PPAR{gamma} mRNA and adipogenic associated genes using reverse transcription polymerase chain reaction (RT-PCR) to adiponectin receptor 1 and 2 as well as examination of fatty acid binding protein 3 (FABP{sub 3}) expression, an adipocyte-specific marker. The current studies indicate that PPAR{gamma}-siRNA is a useful tool to study adipogenesis in human cells, with potential applications both therapeutic and in the elucidation of mesenchymal cell differentiation in the modulation of cell differentiation in human mesenchymal cells.

  19. Increased BDNF and trk-B mRNA expression in cortical and limbic regions following formation of a social recognition memory.

    PubMed

    Broad, Kevin D; Mimmack, Mike L; Keverne, E Barry; Kendrick, Keith M

    2002-12-01

    Brain-derived neurotrophic factor (BDNF) and its receptor, tyrosine receptor kinase (trk-B), play important roles in neural plasticity, long-term potentiation and memory formation. Sheep form a selective recognition memory for their lambs within 2 h of birth. Initially, this memory is exclusively based on olfactory cues; however, as it consolidates over a 12-h recognition period it extends to incorporate visual cues. We investigated whether changes in BDNF and trk-B mRNA expression occurred in both olfactory and visual processing systems at 4.5 h postpartum, 2-3 h after the behavioural manifestations of an olfactory recognition memory were found. Animals that formed a recognition memory showed increased BDNF mRNA expression in the inferior part of the temporal cortex, subfield CA1 of the hippocampus, the diagonal band, basolateral amygdala and the anterior cingulate, medial frontal, entorhinal and pyriform cortices. No increases were observed in either the olfactory bulbs or the dentate gyrus. Expression of trk-B mRNA was significantly increased only in the medial temporal, entorhinal and pyriform cortices. These findings demonstrate that by 2-3 h following the initial formation of olfactory recognition memory there are BDNF/trk-B-mediated plasticity changes in brain areas involved in the consolidation of olfactory memory (the pyriform and entorhinal cortices). However, similar changes also occur in areas of the brain involved in visual memory, face and object recognition (the temporal cortex, entorhinal cortex, hippocampal subfield CA1 and basolateral amygdala), and in areas of the brain with integrative and attentional functions (the medial frontal and anterior cingulate cortices and diagonal band). This suggests that reorganization of neural circuits underlying the visual recognition of lambs or the integration of olfactory/visual information is occurring even at this time even though accurate behavioural recognition at this stage can only be made using

  20. Increased expression of Hsp70 and Hsp90 mRNA as biomarkers of thermal stress in loggerhead turtle embryos (Caretta Caretta).

    PubMed

    Tedeschi, J N; Kennington, W J; Berry, O; Whiting, S; Meekan, M; Mitchell, N J

    2015-01-01

    The survival and viability of sea turtle embryos is dependent upon favourable nest temperatures throughout the incubation period. Consequently, future generations of sea turtles may be at risk from increasing nest temperatures due to climate change, but little is known about how embryos respond to heat stress. Heat shock genes are likely to be important in this process because they code for proteins that prevent cellular damage in response to environmental stressors. This study provides the first evidence of an expression response in the heat shock genes of embryos of loggerhead sea turtles (Caretta caretta) exposed to realistic and near-lethal temperatures (34°C and 36°C) for 1 or 3 hours. We investigated changes in Heat shock protein 60 (Hsp60), Hsp70, and Hsp90 mRNA in heart (n=24) and brain tissue (n=29) in response to heat stress. Under the most extreme treatment (36°C, 3h), Hsp70 increased mRNA expression by a factor of 38.8 in heart tissue and 15.7 in brain tissue, while Hsp90 mRNA expression increased by a factor of 98.3 in heart tissue and 14.7 in brain tissue. Hence, both Hsp70 and Hsp90 are useful biomarkers for assessing heat stress in the late-stage embryos of sea turtles. The method we developed can be used as a platform for future studies on variation in the thermotolerance response from the clutch to population scale, and can help us anticipate the resilience of reptile embryos to extreme heating events.

  1. Differential gene expression in liver and small intestine from lactating rats compared to age-matched virgin controls detects increased mRNA of cholesterol biosynthetic genes

    PubMed Central

    2011-01-01

    Background Lactation increases energy demands four- to five-fold, leading to a two- to three-fold increase in food consumption, requiring a proportional adjustment in the ability of the lactating dam to absorb nutrients and to synthesize critical biomolecules, such as cholesterol, to meet the dietary needs of both the offspring and the dam. The size and hydrophobicity of the bile acid pool increases during lactation, implying an increased absorption and disposition of lipids, sterols, nutrients, and xenobiotics. In order to investigate changes at the transcriptomics level, we utilized an exon array and calculated expression levels to investigate changes in gene expression in the liver, duodenum, jejunum, and ileum of lactating dams when compared against age-matched virgin controls. Results A two-way mixed models ANOVA was applied to detect differentially expressed genes. Significance calls were defined as a p < 0.05 for the overall physiologic state effect (lactation vs. control), and a within tissue pairwise comparison of p < 0.01. The proportion of false positives, an estimate of the ratio of false positives in the list of differentially expressed genes, was calculated for each tissue. The number of differentially expressed genes was 420 in the liver, 337 in the duodenum, 402 in the jejunum, and 523 in the ileum. The list of differentially expressed genes was in turn analyzed by Ingenuity Pathways Analysis (IPA) to detect biological pathways that were overrepresented. In all tissues, sterol regulatory element binding protein (Srebp)-regulated genes involved in cholesterol synthesis showed increased mRNA expression, with the fewest changes detected in the jejunum. We detected increased Scap mRNA in the liver only, suggesting an explanation for the difference in response to lactation between the liver and small intestine. Expression of Cyp7a1, which catalyzes the rate limiting step in the bile acid biosynthetic pathway, was also significantly increased in liver. In

  2. Low temperature inhibits RNA silencing-mediated defence by the control of siRNA generation

    PubMed Central

    Szittya, György; Silhavy, Dániel; Molnár, Attila; Havelda, Zoltán; Lovas, Ágnes; Lakatos, Lóránt; Bánfalvi, Zsófia; Burgyán, József

    2003-01-01

    Temperature dramatically affects plant–virus interactions. Outbreaks of virus diseases are frequently associated with low temperature, while at high temperature viral symptoms are often attenuated (heat masking) and plants rapidly recover from virus diseases. However, the underlying mechanisms of these well-known observations are not yet understood. RNA silencing is a conserved defence system of eukaryotic cells, which operates against molecular parasites including viruses and transgenes. Here we show that at low temperature both virus and transgene triggered RNA silencing are inhibited. Therefore, in cold, plants become more susceptible to viruses, and RNA silencing-based phenotypes of transgenic plants are lost. Consistently, the levels of virus- and transgene-derived small (21–26 nucleotide) interfering (si) RNAs—the central molecules of RNA silencing-mediated defence pathways—are dramatically reduced at low temperature. In contrast, RNA silencing was activated and the amount of siRNAs gradually increased with rising temperature. However, temperature does not influence the accumulation of micro (mi) RNAs, which play a role in developmental regulation, suggesting that the two classes of small (si and mi) RNAs are generated by different nuclease complexes. PMID:12554663

  3. Construction of p66Shc gene interfering lentivirus vectors and its effects on alveolar epithelial cells apoptosis induced by hyperoxia

    PubMed Central

    Zhang, Chan; Dong, Wen-Bin; Zhao, Shuai; Li, Qing-Ping; Kang, Lan; Lei, Xiao-Ping; Guo, Lin; Zhai, Xue-Song

    2016-01-01

    Background The aim of this study is to observe the inhibitive effects of p66Shc gene interfering lentivirus vectors on the expression of p66Shc, and to explore its effects on alveolar epithelial cells apoptosis induced by hyperoxia. Methods The gene sequences were cloned into the pLenR-GPH-shRNA lentiviral vector, which was selected by Genebank searches. The pLenR-GPH-shRNA and lentiviral vector packaging plasmid mix were cotransfected into 293T cells to package lentiviral particles. Culture virus supernatant was harvested, and then the virus titer was determined by serial dilution assay. A549 cells were transduced with the constructed lentiviral vectors, and real-time polymerase chain reaction (RT-PCR) and Western blot were used to evaluate p66Shc expression. This study is divided into a control group, a hyperoxia group, an A549-p66ShcshRNA hyperoxia group, and a negative lentivirus group. Cell apoptosis was detected by flow cytometry after 24 hours; the expression of X-linked inhibitor of apoptosis protein (XIAP) and caspase-9 were detected by immunohistochemistry assay. The production of reactive oxygen species and cellular mitochondria membrane potential (ΔΨm) were determined by fluorescence microscopy. Results We successfully established the p66Shc gene interfering lentivirus vectors, A549-p66ShcshRNA. The A549-p66ShcshRNA was transfected into alveolar epithelial cells, and the inhibitive effects on the expression of p66Shc were observed. Both RT-PCR and Western blot demonstrated downregulation of p66Shc expression in A549 cells. In the A549-p66ShcshRNA hyperoxia group, we found dampened oxidative stress. A549-p66ShcshRNA can cause p66Shc gene silencing, reduce mitochondrial reactive oxygen species generation, reduce membrane potential decrease, reduce the apoptosis of A549 cells, and reduce alveolar epithelial cell injury, while the lentiviral empty vector group had no such changes. Conclusion p66Shc gene interfering lentivirus vector can affect the

  4. Dietary fat elevates hepatic apoA-I production by increasing the fraction of apolipoprotein A-I mRNA in the translating pool.

    PubMed

    Azrolan, N; Odaka, H; Breslow, J L; Fisher, E A

    1995-08-25

    Elevated plasma high density lipoprotein cholesterol (HDL-C) levels are associated with a decreased risk for coronary heart disease. Ironically, diets enriched in saturated fat and cholesterol (HF/HC diets), which tend to accelerate atherosclerotic processes by increasing LDL cholesterol levels, also raise HDL-C. We have recently reported, using a human apoA-I (hapoA-1) transgenic mouse model, that the elevation of HDL-C by a HF/HC diet is attributable, in part, to an increase in the hepatic production of hapoA-1. To further define the hepatocellular processes associated with this induction, we have prepared primary hepatocytes from hapoA-1 transgenic mice. Rates of hapoA-1 secretion were 40% greater from cells prepared from animals fed the HF/HC relative to a low fat-low cholesterol (LF/LC) control diet. The abundance of hapoA-1 mRNA in these cells was similar between hepatocytes prepared from the HF/HC and LF/LC diet fed animals, suggesting a post-transcriptional mechanism that does not involve mRNA stability. Inhibition of secretion using brefeldin A revealed an increase in cellular hapoA-1 accumulation. Thus, the HF/HC diet apparently affects hepatic hapoA-1 production via a mechanism that is manifest prior to the exit of newly synthesized hapoA-1 from the Golgi. Pulse-chase experiments revealed a 39% greater peak hapoA-1 synthesis, with no difference in the degradation of total labeled hapoA-1 protein, as a result of the HF/HC diet feeding. Finally, resolution of liver S10 extracts via sucrose density sedimentation and metrizamide density equilibrium gradient centrifugation analyses both revealed similar increases (31 and 24%, respectively) in the relative percentage of hapoA-1 mRNA associated with the translating polysomal fractions as a result of the HF/HC feeding. Together, these data suggest that the HF/HC diet affects hepatic hapoA-1 production via a specific modulation in the relative amount of hapoA-1 mRNA in the polysomal pool. These observations

  5. Increased plasma VEGF levels following ischemic preconditioning are associated with downregulation of miRNA-762 and miR-3072-5p

    PubMed Central

    Ueno, Koji; Samura, Makoto; Nakamura, Tamami; Tanaka, Yuya; Takeuchi, Yuriko; Kawamura, Daichi; Takahashi, Masaya; Hosoyama, Tohru; Morikage, Noriyasu; Hamano, Kimikazu

    2016-01-01

    Ischemic preconditioning (IPC) has protective effects against ischemia-perfusion injury of organs. In the present study, we investigated the associated mechanisms after performing remote IPC (rIPC) of lower limbs by clamping abdominal aorta in mice. Subsequent experiments showed decreased damage and paralysis of lower limbs following spinal cord injury (SCI). Concomitantly, plasma vascular endothelial growth factor (VEGF) levels were increased 24 h after rIPC compared with those in sham-operated animals. In subsequent microRNA analyses, thirteen microRNAs were downregulated in exosomes 24 h after rIPC. Further studies of femoral CD34-positive bone marrow (BM) cells confirmed downregulation of these seven microRNAs 24 h after rIPC compared with those in sham-operated controls. Subsequent algorithm-based database searches suggested that two of the seven microRNAs bind to the 3′ UTR of VEGF mRNA, and following transfection into CD34-positive BM cells, anti-miR-762, and anti-miR-3072-5p inhibitors led to increased VEGF concentrations. The present data suggest that rIPC transiently increases plasma VEGF levels by downregulating miR-762 and miR-3072-5p in CD34-positive BM cells, leading to protection against organ ischemia. PMID:27905554

  6. 1,25-Dihydroxyvitamin D3 and its analogues increase catalase at the mRNA, protein and activity level in a canine transitional carcinoma cell line.

    PubMed

    Middleton, R P; Nelson, R; Li, Q; Blanton, A; Labuda, J A; Vitt, J; Inpanbutr, N

    2015-12-01

    Antioxidant enzymes, such as catalase, superoxide dismutases (SOD), MnSOD and Cu/ZnSOD, protect cells by scavenging reactive oxygen species (ROS). Numerous studies have reported the anti-cancer effects of 1,25-dihydroxyvitamin D3 (calcitriol) and its related analogues, seocalcitol and analogue V. In this study, canine bladder transitional cell carcinoma (cbTCC) cells were used to determine effects of calcitriol and its related analogues on antioxidant enzyme gene expression, protein expression and activity. Catalase mRNA was increased in response to calcitriol (10(-7) M), and seocalcitol (10(-7) and 10(-9) M). MnSOD mRNA was decreased in response to calcitriol at 10(-7) M. Catalase was significantly increased in response to calcitriol (10(-7) and 10(-9) M), and seocalcitol (10(-9) M). Catalase enzymatic activity increased in response to calcitriol, seocalcitol and analogue V (10(-9) M). In addition, global gene expression analysis identified the involvement of mitogen-activated protein kinase (MAPK) signalling in cbTCC's response to calcitriol and seocalcitol treatment.

  7. Potential applications of RNA interference-based therapeutics in the treatment of cardiovascular disease.

    PubMed

    Hassan, Ali

    2006-06-01

    RNA interference (RNAi) in eukaryotes is a recently identified phenomenon in which small double stranded RNA molecules called short interfering RNA (siRNA) interact with messenger RNA (mRNA) containing homologous sequences in a sequence-specific manner. Ultimately, this interaction results in degradation of the target mRNA. Because of the high sequence specificity of the RNAi process, and the apparently ubiquitous expression of the endogenous protein components necessary for RNAi, there appears to be little limitation to the genes that can be targeted for silencing by RNAi. Thus, RNAi has enormous potential, both as a research tool and as a mode of therapy. Several recent patents have described advances in RNAi technology that are likely to lead to new treatments for cardiovascular disease. These patents have described methods for increased delivery of siRNA to cardiovascular target tissues, chemical modifications of siRNA that improve their pharmacokinetic characteristics, and expression vectors capable of expressing RNAi effectors in situ. Though RNAi has only recently been demonstrated to occur in mammalian tissues, work has advanced rapidly in the development of RNAi-based therapeutics. Recently, therapeutic silencing of apoliporotein B, the ligand for the low density lipoprotein receptor, has been demonstrated in adult mice by systemic administration of chemically modified siRNA. This demonstrates the potential for RNAi-based therapeutics, and suggests that the future for RNAi in the treatment of cardiovascular disease is bright.

  8. Cationic derivatives of biocompatible hyaluronic acids for delivery of siRNA and antisense oligonucleotides.

    PubMed

    Han, Su-Eun; Kang, Hyungu; Shim, Ga Yong; Kim, Sun Jae; Choi, Han-Gon; Kim, Jiseok; Hahn, Sei Kwang; Oh, Yu-Kyoung

    2009-02-01

    In this study, we tested the use of cationic polymer derivatives of biocompatible hyaluronic acid (HA) as a delivery system of siRNA and antisense oligonucleotides. HA was modified with cationic polymer polyethylenimine (PEI). When compared with PEI alone, cationic PEI derivatives of HA (HA-PEI) provided increased cellular delivery of Small interfering RNA (siRNA) in B16F1, A549, HeLa, and Hep3B tumor cells. Indeed, more than 95% of the cells were positive for siRNA following its delivery with HA-PEI. A survivin-specific siRNA that was delivered using HA-PEI potently reduced the mRNA expression levels of the target gene in all of the cell lines. By contrast, survivin-specific siRNA delivered by PEI alone did not induce a significant reduction in mRNA levels. In green fluorescent protein (GFP)-expressing 293 T cells, a loss of GFP expression was evident in the cells that had been treated with GFP-specific siRNA and HA-PEI complex. The inhibition of target gene expression by antisense oligonucleotide G3139 was also enhanced after delivery with HA-PEI. Moreover, HA-PEI displayed lower cytotoxicity than PEI alone. These results suggest that HA-PEI could be further developed as biocompatible delivery systems of siRNA and antisense oligonucleotides for enhanced cellular uptake and inhibition of target gene expression.

  9. Increased readthrough transcription across the simian virus 5 M-F gene junction leads to growth defects and a global inhibition of viral mRNA synthesis.

    PubMed

    Parks, G D; Ward, K R; Rassa, J C

    2001-03-01

    Recombinant simian virus 5 (rSV5) mutants containing substitutions in the M-F intergenic region were generated to determine the effect of increased readthrough transcription on the paramyxovirus growth cycle. We have previously shown, using an SV5 dicistronic minigenome, that replacement of the 22-base M-F intergenic region with a foreign sequence results in a template (Rep22) that directs very high levels of M-F readthrough transcription. An rSV5 containing the Rep22 substitution grew slower and to final titers that were 50- to 80-fold lower than those of wild-type (WT) rSV5. Cells infected with the Rep22 virus produced very low levels of monocistronic M and F mRNA, consistent with the M-F readthrough phenotype. Surprisingly, Rep22 virus-infected cells also displayed a global decrease in the accumulation of viral mRNA from genes located upstream and downstream of the M-F junction, and overall viral protein synthesis was reduced. Second-site revertants of the Rep22 virus that had regained WT transcription and growth properties contained a single base substitution that increased the M gene end U tract from four to eight residues, suggesting that the growth defects originated from higher-than-normal M-F readthrough transcription. Thus, the primary growth defect for the Rep22 virus appears to be in viral RNA synthesis and not in morphogenesis. A second rSV5 virus (G14), which contained a different foreign M-F intergenic sequence, grew to similar or slightly higher titers than WT rSV5 in some cell types and produced ~1.5- to 2-fold more mRNA and viral protein. The data support the hypothesis that inhibition of Rep22 virus growth is due to increased access by the polymerase to the 5' end of the genome and to the resulting overexpression of L protein. We propose that the elevated naturally occurring M-F readthrough which is characteristic of many paramyxoviruses serves as a mechanism to fine-tune the level of polymerase that is optimal for virus growth.

  10. Deflazacort increases osteoclast formation in mouse bone marrow culture and the ratio of RANKL/OPG mRNA expression in marrow stromal cells.

    PubMed Central

    Chung, H.; Kang, Y. S.; Hwang, C. S.; Moon, I. K.; Yim, C. H.; Choi, K. H.; Han, K. O.; Jang, H. C.; Yoon, H. K.; Han, I. K.

    2001-01-01

    Information on precise effects of deflazacort on bone cell function, especially osteoclasts, is quite limited. Therefore, the present study was undertaken to test effects of deflazacort on osteoclast-like cell formation in mouse bone marrow cultures and on the regulation of osteoprotegerin (OPG) and its ligand (RANKL) mRNA expressions by RT-PCR in the ST2 marrow stromal cells. TRAP-positive mononuclear cells increased after the treatment of deflazacort at 10(-9) to 10(-7) M alone for 6 days in a dose-dependent manner. Number of TRAP-positive multi-nucleated cells (MNCs) increased significantly with combined treatment of deflazacort at 10(-7) M and 1,25-(OH)2D3 at 10(-9) M compared to that of cultures treated with 1,25-(OH)2D3 alone (p<0.05). Exposure to deflazacort at 10(-7) M in the presence of 1,25-(OH)2D3 at 10(-9) M in the last 3-day culture had greater stimulatory effect on osteoclast-like cell formation than that of the first 3-day culture did. Deflazacort at 10(-10) -10(-6) M downregulated OPG and upregulated RANKL in mRNA levels in a dose-dependent manner. These observations suggest that deflazacort stimulate osteoclast precursor in the absence of 1,25-(OH)2D3 and enhance differentiation of osteoclasts in the presence of 1,25-(OH)2D3. These effects are, in part, thought to be mediated by the regulation of the expression of OPG and RANKL mRNA in marrow stromal cells. PMID:11748360

  11. Increased bioplastic production with an RNA polymerase sigma factor SigE during nitrogen starvation in Synechocystis sp. PCC 6803.

    PubMed

    Osanai, Takashi; Numata, Keiji; Oikawa, Akira; Kuwahara, Ayuko; Iijima, Hiroko; Doi, Yoshiharu; Tanaka, Kan; Saito, Kazuki; Hirai, Masami Yokota

    2013-12-01

    Because cyanobacteria directly harvest CO2 and light energy, their carbon metabolism is important for both basic and applied sciences. Here, we show that overexpression of the sigma factor sigE in Synechocystis sp. PCC 6803 widely changes sugar catabolism and increases production of the biodegradable polyester polyhydroxybutyrate (PHB) during nitrogen starvation. sigE overexpression elevates the levels of proteins implicated in glycogen catabolism, the oxidative pentose phosphate pathway, and polyhydroxyalkanoate biosynthesis. PHB accumulation is enhanced by sigE overexpression under nitrogen-limited conditions, yet the molecular weights of PHBs synthesized by the parental glucose-tolerant and sigE overexpression strain are similar. Although gene expression induced by nitrogen starvation is changed and other metabolites (such as GDP-mannose and citrate) accumulate under sigE overexpression, genetic engineering of this sigma factor altered the metabolic pathway from glycogen to PHB during nitrogen starvation.

  12. Sonic hedgehog increases the skin wound-healing ability of mouse embryonic stem cells through the microRNA 200 family

    PubMed Central

    Suh, Han Na; Han, Ho Jae

    2015-01-01

    Background and Purpose To use stem cell therapy effectively, it is important to enhance the therapeutic potential of stem cells with soluble factors. Although sonic hedgehog (shh) is important in maintaining the stem cell, the recovery effect of mouse embryonic stem cells (mESCs) with shh has not yet been elucidated. The present study investigated the effect of mESCs with shh in skin recovery in vivo as well as the related intracellular signal pathways in vitro. Experimental Approach The healing effect of mESCs with shh on skin wounds was examined in vivo in ICR mice. The involvement of Smads, the microRNA (miR)-200 family, zinc finger E-box-binding homeobox (ZEBs) and E-cadherin on shh-induced mESC migration and self-renewal was determined in vitro. Key Results The mESCs with shh increased re-epithelialization and VEGF expression in skin wounds. Shh-treated mESCs increased both secreted and intracellular levels of VEGF. Shh induced dephosphorylation of glycogen synthase kinase 3β through the Smoothened receptor and increased the phosphorylation of Smad1 and Smad2/3 in mESCs. Shh-induced decrease of the mmu-miR-141, -200c, -200a, -200b and -429 expression levels was significantly reversed by Smad4 siRNA. Shh increased nuclear expression of ZEB1/ZEB2 and decreased E-cadherin expression while increasing cell migration and skin wound healing. Both these effects were reversed by mmu-miR-141 and -200b mimics. Conclusions and Implications Mouse ESCs accelerated skin wound healing by shh through down-regulating E-cadherin, an effect dependent on mmu-miR-141 and -200b. Our data provides evidence for the effectiveness of shh in stem cell-based therapy in vivo. PMID:25257936

  13. Global effects of the CSR-1 RNA interference pathway on the transcriptional landscape.

    PubMed

    Cecere, Germano; Hoersch, Sebastian; O'Keeffe, Sean; Sachidanandam, Ravi; Grishok, Alla

    2014-04-01

    Argonaute proteins and their small RNA cofactors short interfering RNAs are known to inhibit gene expression at the transcriptional and post-transcriptional levels. In Caenorhabditis elegans, the Argonaute CSR-1 binds thousands of endogenous siRNAs (endo-siRNAs) that are antisense to germline transcripts. However, its role in gene expression regulation remains controversial. Here we used genome-wide profiling of nascent RNA transcripts and found that the CSR-1 RNA interference pathway promoted sense-oriented RNA polymerase II transcription. Moreover, a loss of CSR-1 function resulted in global increase in antisense transcription and ectopic transcription of silent chromatin domains, which led to reduced chromatin incorporation of centromere-specific histone H3. On the basis of these findings, we propose that the CSR-1 pathway helps maintain the directionality of active transcription, thereby propagating the distinction between transcriptionally active and silent genomic regions.

  14. Aminoglycosides: Molecular Insights on the Recognition of RNA and Aminoglycoside Mimics

    PubMed Central

    Chittapragada, Maruthi; Roberts, Sarah; Ham, Young Wan

    2009-01-01

    RNA is increasingly recognized for its significant functions in biological systems and has recently become an important molecular target for therapeutics development. Aminoglycosides, a large class of clinically significant antibiotics, exert their biological functions by binding to prokaryotic ribosomal RNA (rRNA) and interfering with protein translation, resulting in bacterial cell death. They are also known to bind to viral mRNAs such as HIV-1 RRE and TAR. Consequently, aminoglycosides are accepted as the single most important model in understanding the principles that govern small molecule-RNA recognition, which is essential for the development of novel antibacterial, antiviral or even anti-oncogenic agents. This review outlines the chemical structures and mechanisms of molecular recognition and antibacterial activity of aminoglycosides and various aminoglycoside mimics that have recently been devised to improve biological efficacy, binding affinity and selectivity, or to circumvent bacterial resistance. PMID:19812740

  15. Screening for N-AHSL-Based-Signaling Interfering Enzymes.

    PubMed

    Uroz, Stéphane; Oger, Phil M

    2017-01-01

    Quorum sensing (QS)-based signaling is a widespread pathway used by bacteria for the regulation of functions involved in their relation to the environment or their host. QS relies upon the production, accumulation and perception of small diffusable molecules by the bacterial population, hence linking high gene expression with high cell population densities. Among the different QS signal molecules, an important class of signal molecules is the N-acyl homoserine lactone (N-AHSL). In pathogens such as Erwinia or Pseudomonas, N-AHSL based QS is crucial to overcome the host defenses and ensure a successful infection. Interfering with QS-regulation allows the algae Delisea pulcra to avoid surface colonization by bacteria. Thus, interfering the QS-regulation of pathogenic bacteria is a promising antibiotic-free antibacterial therapeutic strategy. To date, two N-AHSL lactonases and one amidohydrolase families of N-ASHL degradation enzymes have been characterized and have proven to be efficient in vitro to control N-AHSL-based QS-regulated functions in pathogens. In this chapter, we provide methods to screen individual clones or bacterial strains as well as pool of clones for genomic and metagenomic libraries, that can be used to identify strains or clones carrying N-ASHL degradation enzymes.

  16. RNA interference suppression of lignin biosynthesis increases fermentable sugar yields for biofuel production from field-grown sugarcane.

    PubMed

    Jung, Je Hyeong; Vermerris, Wilfred; Gallo, Maria; Fedenko, Jeffrey R; Erickson, John E; Altpeter, Fredy

    2013-08-01

    The agronomic performance, cell wall characteristics and enzymatic saccharification efficiency of transgenic sugarcane plants with modified lignin were evaluated under replicated field conditions. Caffeic acid O-methyltransferase (COMT) was stably suppressed by RNAi in the field, resulting in transcript reduction of 80%-91%. Along with COMT suppression, total lignin content was reduced by 6%-12% in different transgenic lines. Suppression of COMT also altered lignin composition by reducing syringyl units and p-coumarate incorporation into lignin. Reduction in total lignin by 6% improved saccharification efficiency by 19%-23% with no significant difference in biomass yield, plant height, stalk diameter, tiller number, total structural carbohydrates or brix value when compared with nontransgenic tissue culture-derived or transgenic control plants. Lignin reduction of 8%-12% compromised biomass yield, but increased saccharification efficiency by 28%-32% compared with control plants. Biomass from transgenic sugarcane lines that have 6%-12% less lignin requires approximately one-third of the hydrolysis time or 3- to 4-fold less enzyme to release an equal or greater amount of fermentable sugar than nontransgenic plants. Reducing the recalcitrance of lignocellulosic biomass to saccharification by modifying lignin biosynthesis is expected to greatly benefit the economic competitiveness of sugarcane as a biofuel feedstock.

  17. Increased Serum Level of MicroRNA-663 Is Correlated with Poor Prognosis of Patients with Nasopharyngeal Carcinoma

    PubMed Central

    Liang, Shaoqiang; Deng, Yanming; Chen, Lusi; Zhang, Yang; Zheng, Zhenhe; Luo, Weijun; Lv, Zhiqian; Li, Shaoen; Xun, Tao

    2016-01-01

    MicroRNAs (miRs) play crucial roles in the carcinogenesis and malignant progression of human cancers including nasopharyngeal carcinoma (NPC). In this study, we aimed to investigate the association of serum miR-663 levels with the clinical factors and prognosis of NPC patients. Real-time PCR was performed to examine the amount of miR-663 in serum in NPC patients and healthy controls. Our data showed that the amount of miR-663 in serum was significantly higher in NPC patients than in healthy controls. Moreover, the serum levels of miR-663 were significantly correlated with the grade, lymph node metastasis, and clinical stage of NPC. Furthermore, higher serum miR-663 levels were closely associated with worse 5-year overall survival (OS) and relapse-free survival (RFS) of patients with NPC, and the serum level of miR-663 was found to be an independent predicator for the prognosis of NPC. In addition, after receiving chemoradiotherapy, the serum levels of miR-663 were significantly reduced in NPC patients. In summary, miR-663 was upregulated in the serum of NPC patients, which was downregulated after chemoradiotherapy, and its increased levels were closely associated with malignant progression and poor prognosis in NPC patients. Therefore, the amount of miR-663 in serum may become a potential predicator for the clinical outcome of NPC patients. PMID:27667893

  18. Muscle Glycogen Depletion Following 75-km of Cycling Is Not Linked to Increased Muscle IL-6, IL-8, and MCP-1 mRNA Expression and Protein Content

    PubMed Central

    Nieman, David C.; Zwetsloot, Kevin A.; Lomiwes, Dominic D.; Meaney, Mary P.; Hurst, Roger D.

    2016-01-01

    The cytokine response to heavy exertion varies widely for unknown reasons, and this study evaluated the relative importance of glycogen depletion, muscle damage, and stress hormone changes on blood and muscle cytokine measures. Cyclists (N = 20) participated in a 75-km cycling time trial (168 ± 26.0 min), with blood and vastus lateralis muscle samples collected before and after. Muscle glycogen decreased 77.2 ± 17.4%, muscle IL-6, IL-8, and MCP-1 mRNA increased 18.5 ± 2.8−, 45.3 ± 7.8−, and 8.25 ± 1.75-fold, and muscle IL-6, IL-8, and MCP-1 protein increased 70.5 ± 14.1%, 347 ± 68.1%, and 148 ± 21.3%, respectively (all, P < 0.001). Serum myoglobin and cortisol increased 32.1 ± 3.3 to 242 ± 48.3 mg/mL, and 295 ± 27.6 to 784 ± 63.5 nmol/L, respectively (both P < 0.001). Plasma IL-6, IL-8, and MCP-1 increased 0.42 ± 0.07 to 18.5 ± 3.8, 4.07 ± 0.37 to 17.0 ± 1.8, and 96.5 ± 3.7 to 240 ± 21.6 pg/mL, respectively (all P < 0.001). Increases in muscle IL-6, IL-8, and MCP-1 mRNA were unrelated to any of the outcome measures. Muscle glycogen depletion was related to change in plasma IL-6 (r = 0.462, P = 0.040), with change in myoglobin related to plasma IL-8 (r = 0.582, P = 0.007) and plasma MCP-1 (r = 0.457, P = 0.043), and muscle MCP-1 protein (r = 0.588, P = 0.017); cortisol was related to plasma IL-8 (r = 0.613, P = 0.004), muscle IL-8 protein (r = 0.681, P = 0.004), and plasma MCP-1 (r = 0.442, P = 0.050). In summary, this study showed that muscle IL-6, IL-8, and MCP-1 mRNA expression after 75-km cycling was unrelated to glycogen depletion and muscle damage, with change in muscle glycogen related to plasma IL-6, and changes in serum myoglobin and cortisol related to the chemotactic cytokines IL-8 and MCP-1. PMID:27729872

  19. In plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs.

    PubMed

    Martínez de Alba, Angel Emilio; Moreno, Ana Beatriz; Gabriel, Marc; Mallory, Allison C; Christ, Aurélie; Bounon, Rémi; Balzergue, Sandrine; Aubourg, Sebastien; Gautheret, Daniel; Crespi, Martin D; Vaucheret, Hervé; Maizel, Alexis

    2015-03-11

    Cytoplasmic degradation of endogenous RNAs is an integral part of RNA quality control (RQC) and often relies on the removal of the 5' cap structure and their subsequent 5' to 3' degradation in cytoplasmic processing (P-)bodies. In parallel, many eukaryotes degrade exogenous and selected endogenous RNAs through post-transcriptional gene silencing (PTGS). In plants, PTGS depends on small interfering (si)RNAs produced after the conversion of single-stranded RNAs to double-stranded RNAs by the cellular RNA-dependent RNA polymerase 6 (RDR6) in cytoplasmic siRNA-bodies. PTGS and RQC compete for transgene-derived RNAs, but it is unknown whether this competition also occurs for endogenous transcripts. We show that the lethality of decapping mutants is suppressed by impairing RDR6 activity. We establish that upon decapping impairment hundreds of endogenous mRNAs give rise to a new class of rqc-siRNAs, that over-accumulate when RQC processes are impaired, a subset of which depending on RDR6 for their production. We observe that P- and siRNA-bodies often are dynamically juxtaposed, potentially allowing for cross-talk of the two machineries. Our results suggest that the decapping of endogenous RNA limits their entry into the PTGS pathway. We anticipate that the rqc-siRNAs identified in decapping mutants represent a subset of a larger ensemble of endogenous siRNAs.

  20. In plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs

    PubMed Central

    Martínez de Alba, Angel Emilio; Moreno, Ana Beatriz; Gabriel, Marc; Mallory, Allison C.; Christ, Aurélie; Bounon, Rémi; Balzergue, Sandrine; Aubourg, Sebastien; Gautheret, Daniel; Crespi, Martin D.; Vaucheret, Hervé; Maizel, Alexis

    2015-01-01

    Cytoplasmic degradation of endogenous RNAs is an integral part of RNA quality control (RQC) and often relies on the removal of the 5′ cap structure and their subsequent 5′ to 3′ degradation in cytoplasmic processing (P-)bodies. In parallel, many eukaryotes degrade exogenous and selected endogenous RNAs through post-transcriptional gene silencing (PTGS). In plants, PTGS depends on small interfering (si)RNAs produced after the conversion of single-stranded RNAs to double-stranded RNAs by the cellular RNA-dependent RNA polymerase 6 (RDR6) in cytoplasmic siRNA-bodies. PTGS and RQC compete for transgene-derived RNAs, but it is unknown whether this competition also occurs for endogenous transcripts. We show that the lethality of decapping mutants is suppressed by impairing RDR6 activity. We establish that upon decapping impairment hundreds of endogenous mRNAs give rise to a new class of rqc-siRNAs, that over-accumulate when RQC processes are impaired, a subset of which depending on RDR6 for their production. We observe that P- and siRNA-bodies often are dynamically juxtaposed, potentially allowing for cross-talk of the two machineries. Our results suggest that the decapping of endogenous RNA limits their entry into the PTGS pathway. We anticipate that the rqc-siRNAs identified in decapping mutants represent a subset of a larger ensemble of endogenous siRNAs. PMID:25694514

  1. miRNA engineering of CHO cells facilitates production of difficult-to-express proteins and increases success in cell line development.

    PubMed

    Fischer, Simon; Marquart, Kim F; Pieper, Lisa A; Fieder, Juergen; Gamer, Martin; Gorr, Ingo; Schulz, Patrick; Bradl, Harald

    2017-03-06

    In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult-to-express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi-specific affinity scaffolds (e.g. bispecific antibodies), cytokines or fusion proteins. Hence, the availability of robust and versatile Chinese hamster ovary (CHO) host cell factories is fundamental for high-yielding bioprocesses. MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve process performance of CHO manufacturing cell lines. However, there has not been any report demonstrating the impact of beneficial miRNAs on industrial cell line development yet. To address this question, we established novel CHO host cells constitutively expressing a pro-productive miRNA: miR-557. Novel host cells were tested in two independent cell line development campaigns using two different mAb candidates including a normal as well as a DTE antibody. Presence of miR-557 significantly enhanced each process step during cell line development in a product independent manner. Stable expression of miR-557 increased the probability to identify high-producing cell clones. Furthermore, production cell lines derived from miR-557 expressing host cells exhibited significantly increased final product yields in fed-batch cultivation processes without compromising product quality. Strikingly, cells co-expressing miR-557 and a DTE antibody achieved a twofold increase in product titer compared to clones co-expressing a negative control miRNA. Thus, host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial cell line development especially with regard to DTE proteins. This article is protected by copyright. All rights reserved.

  2. siRNA vs. shRNA: similarities and differences.

    PubMed

    Rao, Donald D; Vorhies, John S; Senzer, Neil; Nemunaitis, John

    2009-07-25

    RNA interference (RNAi) is a natural process through which expression of a targeted gene can be knocked down with high specificity and selectivity. Using available technology and bioinformatics investigators will soon be able to identify relevant bio molecular tumor network hubs as potential key targets for knockdown approaches. Methods of mediating the RNAi effect involve small interfering RNA (siRNA), short hairpin RNA (shRNA) and bi-functional shRNA. The simplicity of siRNA manufacturing and transient nature of the effect per dose are optimally suited for certain medical disorders (i.e. viral injections). However, using the endogenous processing machinery, optimized shRNA constructs allow for high potency and sustainable effects using low copy numbers resulting in less off-target effects, particularly if embedded in a miRNA scaffold. Bi-functional design may further enhance potency and safety of RNAi-based therapeutics. Remaining challenges include tumor selective delivery vehicles and more complete evaluation of the scope and scale of off-target effects. This review will compare siRNA, shRNA and bi-functional shRNA.

  3. Differential regulation of host mRNA translation during obligate pathogen-plant interactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus infection reprograms the plant messenger RNA (mRNA) transcriptome by activating or interfering with a variety of signaling pathways, but the effects on host mRNA translation have not been explored on a genome-wide scale. To address this issue, Arabidopsis thaliana mRNA transcripts were quantif...

  4. Highly Efficient and Safe Delivery of VEGF siRNA by Bioreducible Fluorinated Peptide Dendrimers for Cancer Therapy.

    PubMed

    Cai, Xiaojun; Zhu, Haofang; Zhang, Yanmei; Gu, Zhongwei

    2017-03-22

    RNA interference (RNAi) has a great promise in treating various acquired and hereditary diseases. However, it remains highly desirable to develop new delivery system to circumvent complex extra- and intracellular barriers for successful clinical translation. Here, we report on a versatile polymeric vector, bioreducible fluorinated peptide dendrimers (BFPD), for efficient and safe small interfering RNA (siRNA) delivery. In virtue of skillfully integrating all of the unique advantages of reversible cross-linking, fluorination, and peptide dendrimers, this novel vector can surmount almost all extra- and intracellular barriers associated with local siRNA delivery through highly improved physiological stability and serum resistance, significantly increased intratumoral enrichment, cellular internalization, successful facilitation of endosomal escape, and cytosolic siRNA release. BFPD polyplexes, carrying small interfering vascular endothelial growth factor (siVEGF), demonstrated excellent VEGF silencing efficacy (∼65%) and a strong capability for inhibiting HeLa cell proliferation. More importantly, these polyplexes showed superior performance in long-term enrichment in the tumor sites and had a high level of tumor growth inhibition. Furthermore, these polyplexes not only exhibited excellent in vivo antitumor efficacy but also demonstrated superior biocompatibility, compared with LPF2000, both in vivo and in vitro. These findings indicate that BFPD is an efficient and safe siRNA delivery system and has remarkable potential for RNAi-based cancer treatment.

  5. Decreased microRNA(miR)-145 and increased miR-224 expression in T cells from patients with systemic lupus erythematosus involved in lupus immunopathogenesis

    PubMed Central

    Lu, M-C; Lai, N-S; Chen, H-C; Yu, H-C; Huang, K-Y; Tung, C-H; Huang, H-B; Yu, C-L

    2013-01-01

    Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with abnormal T cell immune responses. We hypothesized that aberrant expression of microRNAs (miRNAs) in T cells may contribute to the pathogenesis of SLE. First, we analysed the expression profiles of 270 human miRNAs in T cells from five SLE patients and five healthy controls and then validated those potentially aberrant-expressed miRNAs using real-time polymerase chain reaction (PCR). Then, the expression of mRNAs regulated by these aberrant-expressed miRNAs was detected using real-time PCR. Finally, miRNA transfection into Jurkat T cells was conducted for confirming further the biological functions of these miRNAs. The initial analysis indicated that seven miRNAs, including miR-145, miR-224, miR-513-5p, miR-150, miR-516a-5p, miR-483-5p and miR-629, were found to be potentially abnormally expressed in SLE T cells. After validation, under-expressed miR-145 and over-expressed miR-224 were noted. We further found that STAT1 mRNA targeted by miR-145 was over-expressed and apoptosis inhibitory protein 5 (API5) mRNA targeted by miR-224 was under-expressed in SLE T cells. Transfection of Jurkat cells with miR-145 suppressed STAT1 and miR-224 transfection suppressed API5 protein expression. Over-expression of miR-224 facilitates activation-induced cell death in Jurkat cells. In the clinical setting, the increased transcript levels of STAT1 were associated significantly with lupus nephritis. In conclusion, we first demonstrated that miR-145 and miR-224 were expressed aberrantly in SLE T cells that modulated the protein expression of their target genes, STAT1 and API5, respectively. These miRNA aberrations accelerated T cell activation-induced cell death by suppressing API5 expression and associated with lupus nephritis by enhancing signal transducer and activator of transcription-1 (STAT)-1 expression in patients with SLE. PMID:23199328

  6. The dominant-negative inhibition of double-stranded RNA-dependent protein kinase PKR increases the efficacy of Rift Valley fever virus MP-12 vaccine.

    PubMed

    Lihoradova, Olga; Kalveram, Birte; Indran, Sabarish V; Lokugamage, Nandadeva; Juelich, Terry L; Hill, Terence E; Tseng, Chien-Te K; Gong, Bin; Fukushi, Shuetsu; Morikawa, Shigeru; Freiberg, Alexander N; Ikegami, Tetsuro

    2012-07-01

    Rift Valley fever virus (RVFV), belonging to the genus Phlebovirus, family Bunyaviridae, is endemic to sub-Saharan Africa and causes a high rate of abortion in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. MP-12 is the only RVFV strain excluded from the select-agent rule and handled at a biosafety level 2 (BSL2) laboratory. MP-12 encodes a functional major virulence factor, the NSs protein, which contributes to its residual virulence in pregnant ewes. We found that 100% of mice subcutaneously vaccinated with recombinant MP-12 (rMP12)-murine PKRN167 (mPKRN167), which encodes a dominant-negative form of mouse double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in place of NSs, were protected from wild-type (wt) RVFV challenge, while 72% of mice vaccinated with MP-12 were protected after challenge. rMP12-mPKRN167 induced alpha interferon (IFN-α) in sera, accumulated RVFV antigens in dendritic cells at the local draining lymph nodes, and developed high levels of neutralizing antibodies, while parental MP-12 induced neither IFN-α nor viral-antigen accumulation at the draining lymph node yet induced a high level of neutralizing antibodies. The present study suggests that the expression of a dominant-negative PKR increases the immunogenicity and efficacy of live-attenuated RVFV vaccine, which will lead to rational design of safe and highly immunogenic RVFV vaccines for livestock and humans.

  7. The Dominant-Negative Inhibition of Double-Stranded RNA-Dependent Protein Kinase PKR Increases the Efficacy of Rift Valley Fever Virus MP-12 Vaccine

    PubMed Central

    Lihoradova, Olga; Kalveram, Birte; Indran, Sabarish V.; Lokugamage, Nandadeva; Juelich, Terry L.; Hill, Terence E.; Tseng, Chien-Te K.; Gong, Bin; Fukushi, Shuetsu; Morikawa, Shigeru; Freiberg, Alexander N.

    2012-01-01

    Rift Valley fever virus (RVFV), belonging to the genus Phlebovirus, family Bunyaviridae, is endemic to sub-Saharan Africa and causes a high rate of abortion in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. MP-12 is the only RVFV strain excluded from the select-agent rule and handled at a biosafety level 2 (BSL2) laboratory. MP-12 encodes a functional major virulence factor, the NSs protein, which contributes to its residual virulence in pregnant ewes. We found that 100% of mice subcutaneously vaccinated with recombinant MP-12 (rMP12)-murine PKRN167 (mPKRN167), which encodes a dominant-negative form of mouse double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in place of NSs, were protected from wild-type (wt) RVFV challenge, while 72% of mice vaccinated with MP-12 were protected after challenge. rMP12-mPKRN167 induced alpha interferon (IFN-α) in sera, accumulated RVFV antigens in dendritic cells at the local draining lymph nodes, and developed high levels of neutralizing antibodies, while parental MP-12 induced neither IFN-α nor viral-antigen accumulation at the draining lymph node yet induced a high level of neutralizing antibodies. The present study suggests that the expression of a dominant-negative PKR increases the immunogenicity and efficacy of live-attenuated RVFV vaccine, which will lead to rational design of safe and highly immunogenic RVFV vaccines for livestock and humans. PMID:22573861

  8. Mutations in VP2 and VP1 capsid proteins increase infectivity and mouse lethality of enterovirus 71 by virus binding and RNA accumulation enhancement.

    PubMed

    Huang, Sheng-Wen; Wang, Ya-Fang; Yu, Chun-Keung; Su, Ih-Jen; Wang, Jen-Ren

    2012-01-05

    Enterovirus 71 (EV71) is a major cause of hand-foot-and-mouth disease. EV71 infection occasionally associates with severe neurological sequelae such as brainstem encephalitis or poliovirus-like paralysis. We demonstrated that mouse-adapted strain increases infectivity, resulting in higher cytotoxicity of neuron cells and mortality to neonatal mice than a non-adapted strain. Results pointed to EV71 capsid region determining viral infectivity and mouse lethality. Mutant virus with lysine to methionine substitution at VP2(149) (VP2(149M)) or glutamine to glutamic acid substitution at VP1(145) (VP1(145E)) showed greater viral titers and apoptosis. Synergistic effect of VP2(149M) and VP1(145E) double mutations enhanced viral binding and RNA accumulation in infected Neuro-2a cells. The dual substitution mutants markedly reduced value of 50% lethal dose in neonatal mice infection, indicating they raised mouse lethality in vivo. In sum, VP2(149M) and VP1(145E) mutations cooperatively promote viral binding and RNA accumulation of EV71, contributing to viral infectivity in vitro and mouse lethality in vivo.

  9. Isorhapontigenin (ISO) inhibited cell transformation by inducing G0/G1 phase arrest via increasing MKP-1 mRNA Stability.

    PubMed

    Gao, Guangxun; Chen, Liang; Li, Jingxia; Zhang, Dongyun; Fang, Yong; Huang, Haishan; Chen, Xiequn; Huang, Chuanshu

    2014-05-15

    The cancer chemopreventive property of Chinese herb new isolate isorhapontigenin (ISO) and mechanisms underlying its activity have never been explored. Here we demonstrated that ISO treatment with various concentrations for 3 weeks could dramatically inhibit TPA/EGF-induced cell transformation of Cl41 cells in Soft Agar assay, whereas co-incubation of cells with ISO at the same concentrations could elicit G0/G1 cell-cycle arrest without redundant cytotoxic effects on non-transformed cells. Further studies showed that ISO treatment resulted in cyclin D1 downregulation in dose- and time-dependent manner. Our results indicated that ISO regulated cyclin D1 at transcription level via targeting JNK/C-Jun/AP-1 activation. Moreover, we found that ISO-inhibited JNK/C-Jun/AP-1 activation was mediated by both upregulation of MKP-1 expression through increasing its mRNA stability and deactivating MKK7. Most importantly, MKP-1 knockdown could attenuate ISO-mediated suppression of JNK/C-Jun activation and cyclin D1 expression, as well as G0/G1 cell cycle arrest and cell transformation inhibition, while ectopic expression of FLAG-cyclin D1 T286A mutant also reversed ISO-induced G0/G1 cell-cycle arrest and inhibition of cell transformation. Our results demonstrated that ISO is a promising chemopreventive agent via upregulating mkp-1 mRNA stability, which is distinct from its cancer therapeutic effect with downregulation of XIAP and cyclin D1 expression.

  10. Two-Pronged Approach to Overcome Spectroscopically Interfering Organic Compounds with Isotopic Water Analysis

    NASA Astrophysics Data System (ADS)

    Saad, Nabil; Hsiao, Gregor; Chapellet-Volpini, London; Vu, Danthu

    2013-04-01

    The ability to measure the stable isotopes of hydrogen (dD) and oxygen (d18O) has become much more accessible with the advent of Cavity Ring-Down Spectroscopy (CRDS) laser optical devices. These small and inexpensive analyzers have led to a significant increase in the acquisition of data from a variety of studies in the fields of groundwater, watershed, and other water source applications. However for some samples, such as those linked to fracking, mining, and other activities where higher than normal concentrations of organic materials are to be found, optical spectroscopy may require an adaptation from current methodologies in order to ensure data confidence. That is because CRDS is able to measure all the components within a spectral region - which will include the spectral characteristics of the isotopologues of water as well as the available features from interfering organic molecules. Although, at the first level, the information from the organic material provides spectral overlaps that can perturb the isotopic ratios, a more thorough review shows that these features are a source of information that will be inherently useful. This presentation will examine the approaches developed within the past year to allow for more accurate analyses of such samples by optical methods. The first approach uses an advanced spectroscopic model to flag the presence of organic material in the sample. Signals from known interfering compounds (i.e., alcohols, ketones, aldehydes, short-chain hydrocarbons, etc.) are incorporated into the overall fit of the measured spectra used to calculate the concentration of the individual isotopes. The second approach uses physical treatment of the sample to break down the organic molecules into non-interfering species. The vaporized liquid or solid sample travels through a cartridge packed with an oxidation catalyst. The interfering organic molecules will undergo high temperature oxidation using O2 present in the air carrier gas stream prior

  11. High yields and soluble expression of superoxide dismutases in Escherichia coli due to the HIV-1 Tat peptide via increases in mRNA transcription

    PubMed Central

    Sun, Yangdong; Ye, Qiao; Wu, Min; Wu, Yonghong; Zhang, Chenggang; Yan, Weiqun

    2016-01-01

    This study aimed to validate the high yield and soluble expression of proteins carrying the transactivator of transcription (Tat) peptide tag, and further explored the potential mechanism by which the Tat tag increases expression. Escherichia coli superoxide dismutase (SOD) proteins, including SodA, SodB and SodC, were selected for analysis. As expected, the yields and the solubility of Tat-tagged proteins were higher than those of Tat-free proteins, and similar results were observed for the total SOD enzyme activity. Bacterial cells that overexpressed Tat-tagged proteins exhibited increased anti-paraquat activity compared with those expressing Tat-free proteins that manifested as SodA>SodC>SodB. When compared with an MG1655 wild-type strain, the growth of a ΔSodA mutant strain was found to be inhibited after paraquat treatment; the growth of ΔSodB and ΔSodC mutant strains was also slightly inhibited. The mRNA transcript level of genes encoding Tat-tagged proteins was higher than that of genes encoding Tat-free proteins. Furthermore, the α-helix and turn of Tat-tagged proteins were higher than those of Tat-free proteins, but the β-sheet and random coil content was lower. These results indicated that the incorporation of the Tat core peptide as a significant basic membrane transduction peptide in fusion proteins could increase mRNA transcripts and promote the high yield and soluble expression of heterologous proteins in E. coli. PMID:27741225

  12. Chronic central infusion of ghrelin increases hypothalamic neuropeptide Y and Agouti-related protein mRNA levels and body weight in rats.

    PubMed

    Kamegai, J; Tamura, H; Shimizu, T; Ishii, S; Sugihara, H; Wakabayashi, I

    2001-11-01

    Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), was originally purified from the rat stomach. Like the synthetic growth hormone secretagogues (GHSs), ghrelin specifically releases growth hormone (GH) after intravenous administration. Also consistent with the central actions of GHSs, ghrelin-immunoreactive cells were shown to be located in the hypothalamic arcuate nucleus as well as the stomach. Recently, we showed that a single central administration of ghrelin increased food intake and hypothalamic agouti-related protein (AGRP) gene expression in rodents, and the orexigenic effect of this peptide seems to be independent of its GH-releasing activity. However, the effect of chronic infusion of ghrelin on food consumption and body weight and their possible mechanisms have not been elucidated. In this study, we determined the effects of chronic intracerebroventricular treatment with ghrelin on metabolic factors and on neuropeptide genes that are expressed in hypothalamic neurons that have been previously shown to express the GHS-R and to regulate food consumption. Chronic central administration of rat ghrelin (1 microg/rat every 12 h for 72 h) significantly increased food intake and body weight. However, it did not affect plasma insulin, glucose, leptin, or GH concentrations. We also found that chronic central administration of ghrelin increased both neuropeptide Y (NPY) mRNA levels (151.0 +/- 10.1% of saline-treated controls; P < 0.05) and AGRP mRNA levels (160.0 +/- 22.5% of saline-treated controls; P < 0.05) in the arcuate nucleus. Thus, the primary hypothalamic targets of ghrelin are NPY/AGRP-containing neurons, and ghrelin is a newly discovered orexigenic peptide in the brain and stomach.

  13. Enhancement of focusing properties by interfering spatial bending beams

    NASA Astrophysics Data System (ADS)

    Li, Hui; Xu, Yongzheng; Qu, Yu; Zhang, Bin; Wang, Li; Zhang, Zhongyue

    2016-12-01

    In this study, two slits were designed symmetrically on a metal film to excite surface plasmon polaritons (SPPs), and two groups of parallel dielectric rectangles were designed over a metal film to convert the SPPs into double mirror-symmetric spatial bending beams. The high-energy far-field focused beams were achieved by interfering double mirror-symmetric spatial bending beams using the finite-element method The focusing properties of the proposed structure are enhanced compared with the conventional metal grating structures. Furthermore, the effects of the structural parameters on the focusing properties were investigated Results show that the focusing properties of the proposed structure rely on the structural parameters of dielectric rectangles and on the distance between the dielectric rectangles and the metal film. The position of the focusing spot relies on the distance between two slits. These findings can be applied in the fields of biology imaging, nanolithography, optical data storage and photo-biomedical detection.

  14. Genome-scale methylation analysis of Parkinson's disease patients' brains reveals DNA hypomethylation and increased mRNA expression of cytochrome P450 2E1.

    PubMed

    Kaut, Oliver; Schmitt, Ina; Wüllner, Ullrich

    2012-02-01

    Multiple lines of evidence suggest a link between environmental toxins and Parkinson's disease (PD). Although numerous studies reported associations of genetic variants in de-toxifying enzymes, i.e. cytochrome genes, with PD. Epigenetic modifications of genes and subsequent altered expression may confer a yet unappreciated level of susceptibility. We present a genome-wide methylation analysis of PD with quantitative DNA methylation levels of 27.500 CpG sites representing 14.495 genes. We found decreased methylation of the cytochrome P450 2E1 gene and increased expression of CYP2E1 messenger RNA in PD patients' brains, suggesting that epigenetic variants of this cytochrome contribute to PD susceptibility.

  15. Metabolism studies of unformulated internally [3H]-labeled short interfering RNAs in mice.

    PubMed

    Christensen, Jesper; Litherland, Karine; Faller, Thomas; van de Kerkhof, Esther; Natt, François; Hunziker, Jürg; Krauser, Joel; Swart, Piet

    2013-06-01

    Absorption, distribution, metabolism, and excretion properties of two unformulated model short interfering RNA (siRNAs) were determined using a single internal [(3)H]-radiolabeling procedure, in which the full-length oligonucleotides were radiolabeled by Br/(3)H -exchange. Tissue distribution, excretion, and mass balance of radioactivity were investigated in male CD-1 mice after a single intravenous administration of the [(3)H]siRNAs, at a target dose level of 5 mg/kg. Quantitative whole-body autoradiography and liquid scintillation counting techniques were used to determine tissue distribution. Radiochromatogram profiles were determined in plasma, tissue extracts, and urine. Metabolites were separated by liquid chromatography and identified by radiodetection and high-resolution accurate mass spectrometry. In general, there was little difference in the distribution of total radiolabeled components after administration of the two unformulated [(3)H]siRNAs. The radioactivity was rapidly and widely distributed throughout the body and remained detectable in all tissues investigated at later time points (24 and 48 hours for [(3)H]MRP4 (multidrug resistance protein isoform 4) and [(3)H]SSB (Sjögren Syndrome antigen B) siRNA, respectively). After an initial rapid decrease, concentrations of total radiolabeled components in dried blood decreased at a much slower rate. A nearly complete mass balance was obtained for the [(3)H]SSB siRNA, and renal excretion was the main route of elimination (38%). The metabolism of the two model siRNAs was rapid and extensive. Five minutes after administration, no parent compound could be detected in plasma. Instead, radiolabeled nucleosides resulting from nuclease hydrolysis were observed. In the metabolism profiles obtained from various tissues, only radiolabeled nucleosides were found, suggesting that siRNAs are rapidly metabolized and that the distribution pattern of total radiolabeled components can be ascribed to small molecular

  16. Sodium bicarbonate ingestion augments the increase in PGC-1α mRNA expression during recovery from intense interval exercise in human skeletal muscle

    PubMed Central

    Percival, Michael E.; Martin, Brian J.; Gillen, Jenna B.; Skelly, Lauren E.; MacInnis, Martin J.; Green, Alex E.; Tarnopolsky, Mark A.

    2015-01-01

    We tested the hypothesis that ingestion of sodium bicarbonate (NaHCO3) prior to an acute session of high-intensity interval training (HIIT) would augment signaling cascades and gene expression linked to mitochondrial biogenesis in human skeletal muscle. On two occasions separated by ∼1 wk, nine men (mean ± SD: age 22 ± 2 yr, weight 78 ± 13 kg, V̇o2 peak 48 ± 8 ml·kg−1·min−1) performed 10 × 60-s cycling efforts at an intensity eliciting ∼90% of maximal heart rate (263 ± 40 W), interspersed with 60 s of recovery. In a double-blind, crossover manner, subjects ingested a total of 0.4 g/kg body weight NaHCO3 before exercise (BICARB) or an equimolar amount of a placebo, sodium chloride (PLAC). Venous blood bicarbonate and pH were elevated at all time points after ingestion (P < 0.05) in BICARB vs. PLAC. During exercise, muscle glycogen utilization (126 ± 47 vs. 53 ± 38 mmol/kg dry weight, P < 0.05) and blood lactate accumulation (12.8 ± 2.6 vs. 10.5 ± 2.8 mmol/liter, P < 0.05) were greater in BICARB vs. PLAC. The acute exercise-induced increase in the phosphorylation of acetyl-CoA carboxylase, a downstream marker of AMP-activated protein kinase activity, and p38 mitogen-activated protein kinase were similar between treatments (P > 0.05). However, the increase in PGC-1α mRNA expression after 3 h of recovery was higher in BICARB vs. PLAC (approximately sevenfold vs. fivefold compared with rest, P < 0.05). We conclude that NaHCO3 before HIIT alters the mRNA expression of this key regulatory protein associated with mitochondrial biogenesis. The elevated PGC-1α mRNA response provides a putative mechanism to explain the enhanced mitochondrial adaptation observed after chronic HIIT supplemented with NaHCO3 in rats. PMID:26384407

  17. Sodium bicarbonate ingestion augments the increase in PGC-1α mRNA expression during recovery from intense interval exercise in human skeletal muscle.

    PubMed

    Percival, Michael E; Martin, Brian J; Gillen, Jenna B; Skelly, Lauren E; MacInnis, Martin J; Green, Alex E; Tarnopolsky, Mark A; Gibala, Martin J

    2015-12-01

    We tested the hypothesis that ingestion of sodium bicarbonate (NaHCO3) prior to an acute session of high-intensity interval training (HIIT) would augment signaling cascades and gene expression linked to mitochondrial biogenesis in human skeletal muscle. On two occasions separated by ∼1 wk, nine men (mean ± SD: age 22 ± 2 yr, weight 78 ± 13 kg, V̇O(2 peak) 48 ± 8 ml·kg(-1)·min(-1)) performed 10 × 60-s cycling efforts at an intensity eliciting ∼90% of maximal heart rate (263 ± 40 W), interspersed with 60 s of recovery. In a double-blind, crossover manner, subjects ingested a total of 0.4 g/kg body weight NaHCO3 before exercise (BICARB) or an equimolar amount of a placebo, sodium chloride (PLAC). Venous blood bicarbonate and pH were elevated at all time points after ingestion (P < 0.05) in BICARB vs. PLAC. During exercise, muscle glycogen utilization (126 ± 47 vs. 53 ± 38 mmol/kg dry weight, P < 0.05) and blood lactate accumulation (12.8 ± 2.6 vs. 10.5 ± 2.8 mmol/liter, P < 0.05) were greater in BICARB vs. PLAC. The acute exercise-induced increase in the phosphorylation of acetyl-CoA carboxylase, a downstream marker of AMP-activated protein kinase activity, and p38 mitogen-activated protein kinase were similar between treatments (P > 0.05). However, the increase in PGC-1α mRNA expression after 3 h of recovery was higher in BICARB vs. PLAC (approximately sevenfold vs. fivefold compared with rest, P < 0.05). We conclude that NaHCO3 before HIIT alters the mRNA expression of this key regulatory protein associated with mitochondrial biogenesis. The elevated PGC-1α mRNA response provides a putative mechanism to explain the enhanced mitochondrial adaptation observed after chronic HIIT supplemented with NaHCO3 in rats.

  18. The nuclear splicing factor RNA binding motif 5 promotes caspase activation in human neuronal cells, and increases after traumatic brain injury in mice

    PubMed Central

    Jackson, Travis C; Du, Lina; Janesko-Feldman, Keri; Vagni, Vincent A; Dezfulian, Cameron; Poloyac, Samuel M; Jackson, Edwin K; Clark, Robert SB; Kochanek, Patrick M

    2015-01-01

    Splicing factors (SFs) coordinate nuclear intron/exon splicing of RNA. Splicing factor disturbances can cause cell death. RNA binding motif 5 (RBM5) and 10 (RBM10) promote apoptosis in cancer cells by activating detrimental alternative splicing of key death/survival genes. The role(s) of RBM5/10 in neurons has not been established. Here, we report that RBM5 knockdown in human neuronal cells decreases caspase activation by staurosporine. In contrast, RBM10 knockdown augments caspase activation. To determine whether brain injury alters RBM signaling, we measured RBM5/10 protein in mouse cortical/hippocampus homogenates after controlled cortical impact (CCI) traumatic brain injury (TBI) plus hemorrhagic shock (CCI+HS). The RBM5/10 staining was higher 48  to 72 hours after injury and appeared to be increased in neuronal nuclei of the hippocampus. We also measured levels of other nuclear SFs known to be essential for cellular viability and report that splicing factor 1 (SF1) but not splicing factor 3A (SF3A) decreased 4  to 72 hours after injury. Finally, we confirm that RBM5/10 regulate protein expression of several target genes including caspase-2, cellular FLICE-like inhibitory protein (c-FLIP), LETM1 Domain-Containing Protein 1 (LETMD1), and amyloid precursor-like protein 2 (APLP2) in neuronal cells. Knockdown of RBM5 appeared to increase expression of c-FLIP(s), LETMD1, and APLP2 but decrease caspase-2. PMID:25586139

  19. Therapeutic antidepressant potential of a conjugated siRNA silencing the serotonin transporter after intranasal administration.

    PubMed

    Ferrés-Coy, A; Galofré, M; Pilar-Cuéllar, F; Vidal, R; Paz, V; Ruiz-Bronchal, E; Campa, L; Pazos, Á; Caso, J R; Leza, J C; Alvarado, G; Montefeltro, A; Valdizán, E M; Artigas, F; Bortolozzi, A

    2016-03-01

    Major depression brings about a heavy socio-economic burden worldwide due to its high prevalence and the low efficacy of antidepressant drugs, mostly inhibiting the serotonin transporter (SERT). As a result, ~80% of patients show recurrent or chronic depression, resulting in a poor quality of life and increased suicide risk. RNA interference (RNAi) strategies have been preliminarily used to evoke antidepressant-like responses in experimental animals. However, the main limitation for the medical use of RNAi is the extreme difficulty to deliver oligonucleotides to selected neurons/systems in the mammalian brain. Here we show that the intranasal administration of a sertraline-conjugated small interfering RNA (C-SERT-siRNA) silenced SERT expression/function and evoked fast antidepressant-like responses in mice. After crossing the permeable olfactory epithelium, the sertraline-conjugated-siRNA was internalized and transported to serotonin cell bodies by deep Rab-7-associated endomembrane vesicles. Seven-day C-SERT-siRNA evoked similar or more marked responses than 28-day fluoxetine treatment. Hence, C-SERT-siRNA (i) downregulated 5-HT1A-autoreceptors and facilitated forebrain serotonin neurotransmission, (ii) accelerated the proliferation of neuronal precursors and (iii) increased hippocampal complexity and plasticity. Further, short-term C-SERT-siRNA reversed depressive-like behaviors in corticosterone-treated mice. The present results show the feasibility of evoking antidepressant-like responses by selectively targeting neuronal populations with appropriate siRNA strategies, opening a way for further translational studies.

  20. Time-course of SKF-81297-induced increase in glutamic acid decarboxylase 65 and 67 mRNA levels in striatonigral neurons and decrease in GABA(A) receptor alpha1 subunit mRNA levels in the substantia nigra, pars reticulata, in adult rats with a unilateral 6-hydroxydopamine lesion.

    PubMed

    Yamamoto, N; Soghomonian, J-J

    2008-06-26

    Striatal projection neurons use GABA as their neurotransmitter and express the rate-limiting synthesizing enzyme glutamic acid decarboxylase (GAD) and the vesicular GABA transporter vGAT. The chronic systemic administration of an agonist of dopamine D1/D5-preferring receptors is known to alter GAD mRNA levels in striatonigral neurons in intact and dopamine-depleted rats. In the present study, the effects of a single or subchronic systemic administration of the dopamine D1/D5-preferring receptor agonist SKF-81297 on GAD65, GAD67, PPD and vGAT mRNA levels in the striatum and GABA(A) receptor alpha1 subunit mRNA levels in the substantia nigra, pars reticulata, were measured in rats with a unilateral 6-hydroxydopamine (6-OHDA) lesion. After a single injection of SKF-81297, striatal GAD65 mRNA levels were significantly increased at 3 but not 72 h. In contrast, striatal GAD67 mRNA levels were increased and nigral alpha1 mRNA levels were decreased at 72 but not 3 h. Single cell analysis on double-labeled sections indicated that increased GAD or vGAT mRNA levels after acute SKF-81297 occurred in striatonigral neurons identified by their lack of preproenkephalin expression. Subchronic SKF-81297 induced significant increases in striatal GAD67, GAD65, preprodynorphin and vGAT mRNA levels and decreases in nigral alpha1 mRNA levels. In the striatum contralateral to the 6-OHDA lesion, subchronic but not acute SKF-81297 induced a significant increase in GAD65 mRNA levels. The other mRNA levels were not significantly altered. Finally, striatal GAD67 mRNA levels were negatively correlated with nigral alpha1 mRNA levels in the dopamine-depleted but not dopamine-intact side. The results suggest that different signaling pathways are involved in the modulation by dopamine D1/D5 receptors of GAD65 and GAD67 mRNA levels in striatonigral neurons. They also suggest that the down-regulation of nigral GABA(A) receptors is linked to the increase in striatal GAD67 mRNA levels in the dopamine

  1. Long non-coding RNA MALAT1 increases AKAP-9 expression by promoting SRPK1-catalyzed SRSF1 phosphorylation in colorectal cancer cells.

    PubMed

    Hu, Zhi-Yan; Wang, Xiao-Yan; Guo, Wen-Bin; Xie, Lin-Ying; Huang, Yu-Qi; Liu, Yan-Ping; Xiao, Li-Wei; Li, Sheng-Nan; Zhu, Hui-Fang; Li, Zu-Guo; Kan, Heping

    2016-03-08

    Our earlier findings indicate that the long non-coding RNA MALAT1 promotes colorectal cancer (CRC) cell proliferation, invasion and metastasis in vitro and in vivo by increasing expression of AKAP-9. In the present study, we investigated the molecular mechanism by which MALAT1 enhances AKAP9 expression in CRC SW480 cells. We found that MALAT1 interacts with both SRPK1 and SRSF1. MALAT1 increases AKAP-9 expression by promoting SRPK1-catalyzed SRSF1 phosphorylation. Following MALAT1 knockdown, overexpression of SRPK1 was sufficient to restore SRSF1 phosphorylation and AKAP-9 expression to a level that promoted cell proliferation, invasion and migration in vitro. Conversely, SRPK1 knockdown after overexpression of MALAT1 in SW480 cells diminished SRSF1 phosphorylation and AKAP-9 expression and suppressed cell proliferation, invasion and migration in vitro. These findings suggest MALAT1 increases AKAP-9 expression by promoting SRPK1-catalyzed SRSF1 phosphorylation in CRC cells. These results reveal a novel molecular mechanism by which MALAT1 regulates AKAP-9 expression in CRC cells.

  2. Sirolimus induces apoptosis and reverses multidrug resistance in human osteosarcoma cells in vitro via increasing microRNA-34b expression

    PubMed Central

    Zhou, Yan; Zhao, Rui-hua; Tseng, Kuo-Fu; Li, Kun-peng; Lu, Zhi-gang; Liu, Yuan; Han, Kun; Gan, Zhi-hua; Lin, Shu-chen; Hu, Hai-yan; Min, Da-liu

    2016-01-01

    Aim: Multi-drug resistance poses a critical bottleneck in chemotherapy. Given the up-regulation of mTOR pathway in many chemoresistant cancers, we examined whether sirolimus (rapamycin), a first generation mTOR inhibitor, might induce human osteosarcoma (OS) cell apoptosis and increase the sensitivity of OS cells to anticancer drugs in vitro. Methods: Human OS cell line MG63/ADM was treated with sirolimus alone or in combination with doxorubicin (ADM), gemcitabine (GEM) or methotrexate (MTX). Cell proliferation and apoptosis were detected using CCK-8 assay and flow cytometry, respectively. MiRNAs in the cells were analyzed with miRNA microarray. The targets of miR-34b were determined based on TargetScan analysis and luciferase reporter assays. The expression of relevant mRNA and proteins was measured using qRT-PCR and Western blotting. MiR-34, PAK1 and ABCB1 levels in 40 tissue samples of OS patients were analyzed using qRT-PCR and in situ hybridization assays. Results: Sirolimus (1–100 nmol/L) dose-dependently suppressed the cell proliferation (IC50=23.97 nmol/L) and induced apoptosis. Sirolimus (10 nmol/L) significantly sensitized the cells to anticancer drugs, leading to decreased IC50 values of ADM, GEM and MTX (from 25.48, 621.41 and 21.72 μmol/L to 4.93, 73.92 and 6.77 μmol/L, respectively). Treatment of with sirolimus increased miR-34b levels by a factor of 7.5 in the cells. Upregulation of miR-34b also induced apoptosis and increased the sensitivity of the cells to the anticancer drugs, whereas transfection with miR-34b-AMO, an inhibitor of miR-34b, reversed the anti-proliferation effect of sirolimus. Two key regulators of cell cycle, apoptosis and multiple drug resistance, PAK1 and ABCB1, were demonstrated to be the direct targets of miR-34b. In 40 tissue samples of OS patients, significantly higher miR-34 ISH score and lower PAK5 and ABCB1 scores were detected in the chemo-sensitive group. Conclusion: Sirolimus increases the sensitivity of human OS

  3. Biologically responsive carrier-mediated anti-angiogenesis shRNA delivery for tumor treatment

    PubMed Central

    Che, Junyi; Tao, Anqi; Chen, Shun; Li, Xiaoming; Zhao, Yi; Yuan, Weien

    2016-01-01

    Small interfering RNA (siRNA) has increased the hope for highly-efficient treatment of gene-related diseases. However, the stable and efficient delivery of therapeutic nucleic acids is a prerequisite for the successful clinical translation of RNA interfering therapy. To achieve this, we condensed the low molecular weight polyethyleneimine (PEI, Mw < 2000) with 2,6-pyridinedicarboxaldehyde (PDA) to synthesize a biologically responsive and degradable cationic polymer (abbreviated to PDAPEI) which was utilized as a gene vector for the delivery of a VEGF-A shRNA expression plasmid DNA (pDNA). The resulting electrostatic interaction between PDAPEI and pDNA led to the self-assembly of nanoscale polyplexes with suitable particle size and stable zeta potential. The PDAPEI/pDNA polyplexes demonstrated an outstanding gene transfection and silencing efficiency at 30 w/w ratio, as well as negligible cytotoxicity. Also, the designed polymer showed no stimulation to the innate immune system. Moreover, compared with PEI 25 KDa, the polyplexes accomplished comparatively better anti-angiogenesis efficacy, which resulted in the inhibition of tumor growth in subcutaneous tumor mice models. In conclusion, PDAPEI has great potential to be a gene delivery vector for cancer therapy. PMID:27759095

  4. Allergen-specific immune response suppresses interleukin 10 expression in B cells via increasing micro-RNA-17-92 cluster.

    PubMed

    Geng, Xiao-Rui; Qiu, Shu-Qi; Yang, Li-Tao; Liu, Zhi-Qiang; Yang, Gui; Liu, Jiang-Qi; Zeng, Lu; Li, Xiao-Xi; Mo, Li-Hua; Liu, Zhi-Gang; Yang, Ping-Chang

    2016-08-01

    Interleukin (IL)-10-expressing B cells play a critical role in the immune homeostasis in the body; its regulation has not been fully understood. Micro-RNA (miR)-17-92 cluster has strong regulation in the immunity. This study tests a hypothesis that miR-17-92 cluster suppresses IL-10 expression in B cells. In this study, peripheral B cells were collected from patients with allergic rhinitis (AR). The B cells were treated with specific allergens, dust mite extracts, in the culture. The expressions of miR-17-92 cluster and IL-10 in the culture were assessed by real-time quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. The results showed that the levels of miR-19a, but not the rest of the 5 members (miR-17, miR-18a, miR-19b, miR-20a, and miR-92a), were significantly higher in peripheral B cells from AR patients as than in B cells from healthy participants. Exposure of B cells from AR patients to specific allergen, dust mite extracts, significantly increased the levels if miR-19a and suppressed the expression of IL-10 in B cells. The levels of histone deacetylase 11 and acetylated H3K9 were higher, and the RNA polymerase II and c-Maf (the IL-10 transcription factor) were lower, at the IL-10 promoter locus. In conclusion, miR-19a mediates the allergen-specific immune response-decreased IL-10 expression in B cells.

  5. Circular RNA Related to the Chondrocyte ECM Regulates MMP13 Expression by Functioning as a MiR-136 ‘Sponge’ in Human Cartilage Degradation

    PubMed Central

    Liu, Qiang; Zhang, Xin; Hu, Xiaoqing; Dai, Linghui; Fu, Xin; Zhang, Jiying; Ao, Yingfang

    2016-01-01

    Circular RNAs (circRNAs) are involved in the development of various diseases, but there is little knowledge of circRNAs in osteoarthritis (OA). The aim of study was to identify circRNA expression in articular cartilage and to explore the function of chondrocyte extracellular matrix (ECM)-related circRNAs (circRNA-CER) in cartilage. To identify circRNAs that are specifically expressed in cartilage, we compared the expression of circRNAs in OA cartilage with that in normal cartilage. Bioinformatics was employed to predict the interaction of circRNAs and mRNAs in cartilage. Loss-of-function and rescue experiments for circRNA-CER were performed in vitro. A total of 71 circRNAs were differentially expressed in OA and normal cartilage. CircRNA-CER expression increased with interleukin-1 and tumor necrosis factor levels in chondrocytes. Silencing of circRNA-CER using small interfering RNA suppressed MMP13 expression and increased ECM formation. CircRNA-CER could compete for miR-136 with MMP13. Our results demonstrated that circRNA-CER regulated MMP13 expression by functioning as a competing endogenous RNA (ceRNA) and participated in the process of chondrocyte ECM degradation. We propose that circRNA-CER could be used as a potential target in OA therapy. PMID:26931159

  6. Exposure to bis(maltolato)oxovanadium(IV) increases levels of hepcidin mRNA and impairs the homeostasis of iron but not that of manganese.

    PubMed

    Sánchez-González, Cristina; Rivas-García, Lorenzo; López-Chaves, Carlos; Rodríguez-Nogales, Alba; Algieri, Francesca; Gálvez, Julio; Gómez-Aracena, Jorge; Vera-Ramírez, Laura; Montes-Bayon, Maria; Sanz-Medel, Alfredo; Llopis, Juan

    2014-11-01

    The aim of this study was to examine whether alterations in iron homeostasis, caused by exposure to vanadium, are related to changes in the gene expression of hepatic hepcidin. Two groups of rats were examined: control and vanadium-exposed. Vanadium, as bis(maltolato)oxovanadium(IV) was supplied in the drinking water. The experiment had a duration of five weeks. Iron and manganese were measured in excreta, serum and tissues. Leptin, ferritin, IL-1β, IL-6, TNF-α, red blood cells, haemoglobin and haematocrit were determined. Protein carbonyl group levels and hepcidin gene expression were determined in the liver. In the vanadium-exposed rats, iron absorption, serum iron and leptin and all haematological parameters decreased. Levels of IL-6, TNF-α and ferritin in serum and of iron in the liver, spleen and heart increased. In the liver, levels of protein carbonyl groups and hepcidin mRNA were also higher in the vanadium-exposed group. Exposure to vanadium did not modify manganese homeostasis. The results obtained from this study provide the first evidence that bis(maltolato)oxovanadium(IV) produces an increase in the gene expression of the hepcidin, possibly caused by an inflammatory process. Both factors could be the cause of alterations in Fe homeostasis and the appearance of anaemia. However, Mn homeostasis was not affected.

  7. Gene silencing in non-model insects: Overcoming hurdles using symbiotic bacteria for trauma-free sustainable delivery of RNA interference: Sustained RNA interference in insects mediated by symbiotic bacteria: Applications as a genetic tool and as a biocide.

    PubMed

    Whitten, Miranda; Dyson, Paul

    2017-03-01

    Insight into animal biology and development provided by classical genetic analysis of the model organism Drosophila melanogaster was an incentive to develop advanced genetic tools for this insect. But genetic systems for the over one million other known insect species are largely undeveloped. With increasing information about insect genomes resulting from next generation sequencing, RNA interference is now the method of choice for reverse genetics, although it is constrained by the means of delivery of interfering RNA. A recent advance to ensure sustained delivery with minimal experimental intervention or trauma to the insect is to exploit commensal bacteria for symbiont-mediated RNA interference. This technology not only offers an efficient means for RNA interference in insects in laboratory conditions, but also has potential for use in the control of human disease vectors, agricultural pests and pathogens of beneficial insects.

  8. Cloning of a long HIV-1 readthrough transcript and detection of an increased level of early growth response protein-1 (Egr-1) mRNA in chronically infected U937 cells.

    PubMed

    Dron, M; Hameau, L; Benboudjema, L; Guymarho, J; Cajean-Feroldi, C; Rizza, P; Godard, C; Jasmin, C; Tovey, M G; Lang, M C

    1999-01-01

    To identify the pathways involved in HIV-1 modification of cellular gene expression, chronically infected U937 cells were screened by mRNA differential display. A chimeric transcript consisting of the 3' end of the LTR of a HIV-1 provirus, followed by 3.7 kb of cellular RNA was identified suggesting that long readthrough transcription might be one of the mechanisms by which gene expression could be modified in individual infected cells. Such a phenomenon may also be the first step towards the potential transduction of cellular sequences. Furthermore, the mRNA encoding for the transcription factor Egr-1 was detected as an over-represented transcript in infected cells. Northern blot analysis confirmed the increase of Egr-1 mRNA content in both HIV-1 infected promonocytic U937 cells and T cell lines such as Jurkat and CEM. Interestingly a similar increase of Egr-1 mRNA has previously been reported to occur in HTLV-1 and HTLV-2 infected T cell lines. Despite the consistent increase in the level of Egr-1 mRNA, the amount of the encoded protein did not appear to be modified in HIV-1 infected cells, suggesting an increased turn over of the protein in chronically infected cells.

  9. The hepatitis B virus X protein increases the cellular level of TATA-binding protein, which mediates transactivation of RNA polymerase III genes

    SciTech Connect

    Wang, Horng-Dar; Johnson, D.L.; Yuh, Chio-Hwa

    1995-12-01

    This report decribes the mechanism by which the hepatitis B virus X gene product induces RNA polymerase III genes. The RNA pol III transcription system serves as model for understanding the mechanism of X in the transactivation of cellular genes in both Drosophila and rat cell lines. 53 refs., 7 figs., 1 tab.

  10. Knockdown of Long Non-Coding RNA UCA1 Increases the Tamoxifen Sensitivity of Breast Cancer Cells through Inhibition of Wnt/β-Catenin Pathway

    PubMed Central

    Liu, Hongying; Wang, Gang; Yang, Lili; Qu, Jianjun; Yang, Zhihui; Zhou, Xiangyu

    2016-01-01

    Acquired resistance to tamoxifen remains a major obstacle in breast cancer (BC) treatment, since the underlying mechanism has not been fully elucidated. The long non-coding RNA (lncRNA) urothelial carcinoma-associated 1 (UCA1) has been recently shown to be dysregulated and plays important roles in progression of breast cancer. In the present study, we aimed to investigate the biological role and clinical significance of UCA1 in BC drug resistance. Hence, we used quantitative PCR assay to evaluate the UCA1 expression in tissues from patients with BC as well as established tamoxifen-resistant BC cell lines in vitro. We tested the viability, invasive ability and apoptosis rate in MCF-7 and T47D cells using MTT assay, transwell assay and flow cytometry assay, respectively. The influence of UCA1 on tumorigenesis was monitored by in vivo mice xenograft model. The activation of Wnt/β-catenin signaling pathway was evaluated by immunofluorescence assay, western blot assay and luciferase reporter assay, respectively. We found that the expression of UCA1 positively correlated with the pathological grade and mortality of breast cancer patients, moreover, expressions of UCA1 was increased significantly in the tamoxifen-resistant cell lines compared with the wild type parental cells. Ectopic expression of UCA1 promoted cell survival and resistance to tamoxifen treatment, whereas inhibition of UCA1 enhanced tamoxifen sensitivity of BC cells and induced more apoptotic cells. In addition, tamoxifen-resistant cells exhibited increased Wnt signaling activation as measured by the TOP/FOP Wnt luciferase reporter assay and β-catenin protein level compared with parental MCF-7 and T47D cells, respectively. In line with these data, UCA1 depletion attenuated the activity of Wnt/β-catenin pathway activation and the tumorigenicity of the tamoxifen-resistant BC cells. Taken together, our data highlights the pivotal role of UCA1-Wnt/β-catenin signaling pathway in the tamoxifen resistance in

  11. Small-interfering RNAs (siRNAs) as a promising tool for ocular therapy

    PubMed Central

    Guzman-Aranguez, A; Loma, P; Pintor, J

    2013-01-01

    RNA interference (RNAi) can be used to inhibit the expression of specific genes in vitro and in vivo, thereby providing an extremely useful tool for investigating gene function. Progress in the understanding of RNAi-based mechanisms has opened up new perspectives in therapeutics for the treatment of several diseases including ocular disorders. The eye is currently considered a good target for RNAi therapy mainly because it is a confined compartment and, therefore, enables local delivery of small-interfering RNAs (siRNAs) by topical instillation or direct injection. However, delivery strategies that protect the siRNAs from degradation and are suitable for long-term treatment would be help to improve the efficacy of RNAi-based therapies for ocular pathologies. siRNAs targeting critical molecules involved in the pathogenesis of glaucoma, retinitis pigmentosa and neovascular eye diseases (age-related macular degeneration, diabetic retinopathy and corneal neovascularization) have been tested in experimental animal models, and clinical trials have been conducted with some of them. This review provides an update on the progress of RNAi in ocular therapeutics, discussing the advantages and drawbacks of RNAi-based therapeutics compared to previous treatments. PMID:23937539

  12. Dowsing can be interfered with by radio frequency radiation.

    PubMed

    Huttunen, Paavo; Niinimaa, Ahti; Myllylä, Risto

    2012-04-01

    The soil radiation, watercourses and ores have been located for centuries by sensitive persons, dowsers. An ideomotoric explanation of the dowsing reaction, with no physical interaction, has been accepted. Our present re-analyses of some such results have shown, that there could be a physical phenomenon connecting the human reactions in field experiments, where the test subjects walked or were sitting in a slow-moving car, with the windows covered, and a dowsing rod in their hands was recorded. The correlations between the reaction points by test subjects in the moving car and the points by walking along the same path were highly significant. The correlation was not seen in all test locations. The distance between the test location and the radio tower, and the incidence angle of the transmitted radio wave, possibly had an effect on results. We hypothesize that the experiments carried out in the 20th century were interfered with by man-made radio frequency radiation, mainly FM radio and TV broadcasting, as test subjects' bodies absorbed the radio waves and unconscious hand movement reactions took place following the standing waves or intensity variations due to multipath propagation.

  13. Frizzled2 signaling regulates growth of high-risk neuroblastomas by interfering with β-catenin-dependent and β-catenin-independent signaling pathways

    PubMed Central

    Zins, Karin; Schäfer, Romana; Paulus, Patrick; Dobler, Silvia; Fakhari, Nazak; Sioud, Mouldy; Aharinejad, Seyedhossein; Abraham, Dietmar

    2016-01-01

    Frizzled2 (FZD2) is a receptor for Wnts and may activate both canonical and non-canonical Wnt signaling pathways in cancer. However, no studies have reported an association between FZD2 signaling and high-risk NB so far. Here we report that FZD2 signaling pathways are critical to NB growth in MYCN-single copy SK-N-AS and MYCN-amplified SK-N-DZ high-risk NB cells. We demonstrate that stimulation of FZD2 by Wnt3a and Wnt5a regulates β-catenin-dependent and –independent Wnt signaling factors. FZD2 blockade suppressed β-catenin-dependent signaling activity and increased phosphorylation of PKC, AKT and ERK in vitro, consistent with upregulation of β-catenin-independent signaling activity. Finally, FZD2 small interfering RNA knockdown suppressed tumor growth in murine NB xenograft models associated with suppressed β-catenin-dependent signaling and a less vascularized phenotype in both NB xenografts. Together, our study suggests a role for FZD2 in high-risk NB cell growth and provides a potential candidate for therapeutic inhibition in FZD2-expressing NB patients. PMID:27323822

  14. Construction of RNA nanocages by re-engineering the packaging RNA of Phi29 bacteriophage

    NASA Astrophysics Data System (ADS)

    Hao, Chenhui; Li, Xiang; Tian, Cheng; Jiang, Wen; Wang, Guansong; Mao, Chengde

    2014-05-01

    RNA nanotechnology promises rational design of RNA nanostructures with wide array of structural diversities and functionalities. Such nanostructures could be used in applications such as small interfering RNA delivery and organization of in vivo chemical reactions. Though having impressive development in recent years, RNA nanotechnology is still quite limited and its programmability and complexity could not rival the degree of its closely related cousin: DNA nanotechnology. Novel strategies are needed for programmed RNA self-assembly. Here, we have assembled RNA nanocages by re-engineering a natural, biological RNA motif: the packaging RNA of phi29 bacteriophage. The resulting RNA nanostructures have been thoroughly characterized by gel electrophoresis, cryogenic electron microscopy imaging and dynamic light scattering.

  15. Dynamic macrophage polarization-specific miRNA patterns reveal increased soluble VEGF receptor 1 by miR-125a-5p inhibition.

    PubMed

    Melton, David W; Lei, XiuFen; Gelfond, Jonathan A L; Shireman, Paula K

    2016-05-01

    Dynamic, epigenetic mechanisms can regulate macrophage phenotypes following exposure to different stimulating conditions and environments. However, temporal patterns of microRNAs (miRNAs or miRs) across multiple macrophage polarization phenotypes have not been defined. We determined miRNA expression in bone marrow-derived murine macrophages over multiple time points (0.5, 1, 3, 24 h) following exposure to cytokines and/or LPS. We hypothesized that dynamic changes in miRNAs regulate macrophage phenotypes. Changes in macrophage polarization markers were detected as early as 0.5 and as late as 24 h; however, robust responses for most markers occurred within 3 h. In parallel, many polarization-specific miRNAs were also changed by 3 h and expressed divergent patterns between M1 and M2a conditions, with increased expression in M1 (miR-155, 199a-3p, 214-3p, 455-3p, and 125a) or M2a (miR-511 and 449a). Specifically, miR-125a-5p exhibited divergent patterns: increased at 12-24 h in M1 macrophages and decreasing trend in M2a. VEGF in the culture media of macrophages was dependent upon the polarization state, with greatly diminished VEGF in M2a compared with M1 macrophage culture media despite similar VEGF in cell lysates. Inhibition of miR-125a-5p in media-only controls (MO) and M1 macrophages greatly increased expression and secretion of soluble VEGF receptor-1 (sVEGFR1) leading to diminished VEGF in the culture media, partially converting MO and M1 into an M2a phenotype. Thus, the divergent expression patterns of polarization-specific miRNAs led to the identification and demonstrated the regulation of a specific macrophage polarization phenotype, sVEGFR1 by inhibition of miR-125a-5p.

  16. MicroRNA-191, by promoting the EMT and increasing CSC-like properties, is involved in neoplastic and metastatic properties of transformed human bronchial epithelial cells.

    PubMed

    Xu, Wenchao; Ji, Jie; Xu, Yuan; Liu, Yawei; Shi, Le; Liu, Yi; Lu, Xiaolin; Zhao, Yue; Luo, Fei; Wang, Bairu; Jiang, Rongrong; Zhang, Jianping; Liu, Qizhan

    2015-06-01

    Lung cancer is the leading cause of cancer mortality worldwide. A common interest in lung cancer research is the identification of biomarkers for early diagnosis and accurate prognosis. There is increasing evidence that microRNAs (miRNAs) are involved in lung cancer. To explore new biomarkers of chemical exposure in risk assessment of chemical carcinogenesis and lung cancer, we analyzed miRNA expression profiles of human bronchial epithelial (HBE) cells malignantly transformed by arsenite. High-throughput microarray analysis showed that 51 miRNAs were differentially expressed in transformed HBE cells relative to normal HBE cells. In particular, miR-191 was up-regulated in transformed cells. In HBE cells, arsenite induced increases of miR-191 and WT1 levels, decreased BASP1 expression, and activated the Wnt/β-catenin pathway, effects that were blocked by miR-191 knockdown. In addition, a luciferase reporter assay indicated that BASP1 is a direct target of miR-191. By inhibiting the expression of BASP1, miR-191 increased the expression of WT1 to promote activation of Wnt/β-catenin pathway. In transformed cells, inhibition of miR-191 expression blocked the epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC)-like properties of cells and decreased their migratory capacity and neoplastic properties. Thus, these results demonstrate that miR-191 modulates the EMT and the CSC-like properties of transformed cells and indicate that it is an onco-miR involved in the neoplastic and metastatic properties of transformed cells.

  17. Nanoparticles for targeted delivery of therapeutics and small interfering RNAs in hepatocellular carcinoma.

    PubMed

    Varshosaz, Jaleh; Farzan, Maryam

    2015-11-14

    Hepatocellular carcinoma (HCC) is the 5(th) most common malignancy which is responsible for more than half million annual mortalities; also, it is the third leading cause of cancer related death. Unfavorable systemic side-effects of chemotherapeutic agents and susceptibility to the degradation of small interfering RNAs (siRNAs), which can knock down a specific gene involved in the disease, have hampered their clinical application. So, it could be beneficial to develop an efficient carrier for the stabilization and specific delivery of drugs and siRNA to cells. Targeted nanoparticles have gained considerable attention as an efficient drug and gene delivery system, which is due to their capability in achieving the highest accumulation of cytotoxic agents in tumor tissue, modifiable drug pharmacokinetic- and bio-distribution, improved effectiveness of treatment, and limited side-effects. Recent studies have shed more light on the advantages of novel drug loaded carrier systems vs free drugs. Most of the animal studies have reported improvement in treatment efficacy and survival rate using novel carrier systems. Targeted delivery may be achieved passively or actively. In passive targeting, no ligand as homing device is used, while targeting is achieved by incorporating the therapeutic agent into a macromolecule or nanoparticle that passively reaches the target organ. However, in active targeting, the therapeutic agent or carrier system is conjugated to a tissue or cell-specific receptor which is over-expressed in a special malignancy using a ligand called a homing device. This review covers a broad spectrum of targeted nanoparticles as therapeutic and non-viral siRNA delivery systems, which are developed for enhanced cellular uptake and targeted gene silencing in vitro and in vivo and their characteristics and opportunities for the clinical applications of drugs and therapeutic siRNA are discussed in this article. Asialoglycoprotein receptors, low-density lipoprotein

  18. Nanoparticles for targeted delivery of therapeutics and small interfering RNAs in hepatocellular carcinoma

    PubMed Central

    Varshosaz, Jaleh; Farzan, Maryam

    2015-01-01

    Hepatocellular carcinoma (HCC) is the 5th most common malignancy which is responsible for more than half million annual mortalities; also, it is the third leading cause of cancer related death. Unfavorable systemic side-effects of chemotherapeutic agents and susceptibility to the degradation of small interfering RNAs (siRNAs), which can knock down a specific gene involved in the disease, have hampered their clinical application. So, it could be beneficial to develop an efficient carrier for the stabilization and specific delivery of drugs and siRNA to cells. Targeted nanoparticles have gained considerable attention as an efficient drug and gene delivery system, which is due to their capability in achieving the highest accumulation of cytotoxic agents in tumor tissue, modifiable drug pharmacokinetic- and bio-distribution, improved effectiveness of treatment, and limited side-effects. Recent studies have shed more light on the advantages of novel drug loaded carrier systems vs free drugs. Most of the animal studies have reported improvement in treatment efficacy and survival rate using novel carrier systems. Targeted delivery may be achieved passively or actively. In passive targeting, no ligand as homing device is used, while targeting is achieved by incorporating the therapeutic agent into a macromolecule or nanoparticle that passively reaches the target organ. However, in active targeting, the therapeutic agent or carrier system is conjugated to a tissue or cell-specific receptor which is over-expressed in a special malignancy using a ligand called a homing device. This review covers a broad spectrum of targeted nanoparticles as therapeutic and non-viral siRNA delivery systems, which are developed for enhanced cellular uptake and targeted gene silencing in vitro and in vivo and their characteristics and opportunities for the clinical applications of drugs and therapeutic siRNA are discussed in this article. Asialoglycoprotein receptors, low-density lipoprotein

  19. PEITC-mediated inhibition of mRNA translation is associated with both inhibition of mTORC1 and increased eIF2α phosphorylation in established cell lines and primary human leukemia cells

    PubMed Central

    Yeomans, Alison; Lemm, Elizabeth; Wilmore, Sarah; Cavell, Breeze E.; Valle-Argos, Beatriz; Krysov, Sergey; Hidalgo, Marina Sanchez; Leonard, Elodie; Willis, Anne E.; Forconi, Francesco; Stevenson, Freda K.; Steele, Andrew J.; Coldwell, Mark J.; Packham, Graham

    2016-01-01

    Increased mRNA translation drives carcinogenesis and is an attractive target for the development of new anti-cancer drugs. In this work, we investigated effects of phenethylisothiocyanate (PEITC), a phytochemical with chemopreventive and anti-cancer activity, on mRNA translation. PEITC rapidly inhibited global mRNA translation in human breast cancer-derived MCF7 cells and mouse embryonic fibroblasts (MEFs). In addition to the known inhibitory effects of PEITC on mTORC1 activity, we demonstrate that PEITC increased eIF2α phosphorylation. PEITC also increased formation of stress granules which are typically associated with eIF2α phosphorylation and accumulation of translationally stalled mRNAs. Analysis of genetically modified MEFs demonstrated that optimal inhibition of global mRNA translation by PEITC was dependent on eIF2α phosphorylation, but not mTORC1 inhibition. We extended this study into primary leukemic B cells derived from patients with chronic lymphocytic leukaemia (CLL). CLL cells were stimulated in vitro with anti-IgM to mimic binding of antigen, a major driver of this leukemia. In CLL cells, PEITC increased eIF2α phosphorylation, inhibited anti-IgM-induced mTORC1 activation and decreased both basal and anti-IgM-induced global mRNA translation. PEITC also inhibited transcription and translation of MYC mRNA and accumulation of the MYC oncoprotein, in anti-IgM-stimulated cells. Moreover, treatment of CLL cells with PEITC and the BTK kinase inhibitor ibrutinib decreased anti-IgM-induced translation and induced cell death to a greater extent than either agent alone. Therefore, PEITC can inhibit both global and mRNA specific translation (including MYC) via effects on multiple regulatory pathways. Inhibition of mRNA translation may contribute to the chemopreventive and anti-cancer effects of PEITC. PMID:27579538

  20. Nanograting formation on metals in air with interfering femtosecond laser pulses

    SciTech Connect

    Miyazaki, Kenzo E-mail: kmiyazaki@wind.ocn.ne.jp; Miyaji, Godai; Inoue, Toshishige

    2015-08-17

    It is demonstrated that a homogeneous nanograting having the groove period much smaller than the laser wavelength (∼800 nm) can be fabricated on metals in air through ablation induced by interfering femtosecond laser pulses (100 fs at a repetition rate of 10 Hz). Morphological changes on stainless steel and Ti surfaces, observed with an increase in superimposed shots of the laser pulses at a low fluence, have shown that the nanograting is developed through bonding structure change at the interference fringes, plasmonic near-field ablation to create parallel grooves on the fringe, and subsequent excitation of surface plasmon polaritons to regulate the groove intervals at 1/3 or 1/4 of the fringe period over the whole irradiated area. Calculation for a model target having a thin oxide layer on the metal substrate reproduces well the observed groove periods and explains the mechanism for the nanograting formation.

  1. Retraction Statement: 'MicroRNA-218 increases cellular sensitivity to Rapamycin via targeting Rictor in cervical cancer' by Li J, Wang J, Wang Y, Qiu H.

    PubMed

    2017-02-01

    The above article from APMIS, published online on 24 April 2015 in Wiley Online Library (wileyonlinelibrary.com) and in Volume 123, pp. 562-570, has been retracted by agreement between the authors, the journal Editors in Chief, Professors Bodil Norrild, Ben Vainer and Elisabeth Ralfkiaer, and John Wiley & Sons Ltd. The article has been retracted due to errors in the reported results. In this study, the authors used HeLa and SiHa cell lines to investigate the biological roles of miR-218. However, subsequently it emerged that the two cell lines were contaminated in the laboratory by other unknown cell lines. When repeating the experiments, it was found that the functions of miR-218 were not as significant as had been previously reported, especially its effects on rapamycin sensitivity. Reference Li J, Li X, Wang J, Wang Y, Qiu H. MicroRNA-218 increases cellular sensitivity to Rapamycin via targeting Rictor in cervical cancer. APMIS 2015; 123:562-570. doi: 10.1111/apm.12387.

  2. Administration of Panobinostat Is Associated with Increased IL-17A mRNA in the Intestinal Epithelium of HIV-1 Patients

    PubMed Central

    Bjerg Christensen, Ane; Dige, Anders; Vad-Nielsen, Johan; Brinkmann, Christel R.; Bendix, Mia; Østergaard, Lars; Tolstrup, Martin; Søgaard, Ole S.; Rasmussen, Thomas A.; Randel Nyengaard, Jens; Agnholt, Jørgen

    2015-01-01

    Intestinal CD4+ T cell depletion is rapid and profound during early HIV-1 infection. This leads to a compromised mucosal barrier that prompts chronic systemic inflammation. The preferential loss of intestinal T helper 17 (Th17) cells in HIV-1 disease is a driver of the damage within the mucosal barrier and of disease progression. Thus, understanding the effects of new therapeutic strategies in the intestines has high priority. Histone deacetylase (HDAC) inhibitors (e.g., panobinostat) are actively under investigation as potential latency reversing agents in HIV eradication studies. These drugs have broad effects that go beyond reactivating virus, including modulation of immune pathways. We examined colonic biopsies from ART suppressed HIV-1 infected individuals (clinicaltrials.gov: NCT01680094) for the effects of panobinostat on intestinal T cell activation and on inflammatory cytokine production. We compared biopsy samples that were collected before and during oral panobinostat treatment and observed that panobinostat had a clear biological impact in this anatomical compartment. Specifically, we observed a decrease in CD69+ intestinal lamina propria T cell frequency and increased IL-17A mRNA expression in the intestinal epithelium. These results suggest that panobinostat therapy may influence the restoration of mucosal barrier function in these patients. PMID:26696749

  3. Nematode endogenous small RNA pathways

    PubMed Central

    Hoogstrate, Suzanne W; Volkers, Rita JM; Sterken, Mark G; Kammenga, Jan E; Snoek, L Basten

    2014-01-01

    The discovery of small RNA silencing pathways has greatly extended our knowledge of gene regulation. Small RNAs have been presumed to play a role in every field of biology because they affect many biological processes via regulation of gene expression and chromatin remodeling. Most well-known examples of affected processes are development, fertility, and maintenance of genome stability. Here we review the role of the three main endogenous small RNA silencing pathways in Caenorhabditis elegans: microRNAs, endogenous small interfering RNAs, and PIWI-interacting RNAs. After providing an entry-level overview on how these pathways function, we discuss research on other nematode species providing insight into the evolution of these small RNA pathways. In understanding the differences between the endogenous small RNA pathways and their evolution, a more comprehensive picture is formed of the functions and effects of small RNAs. PMID:25340013

  4. Molecular mechanisms of RNA interference.

    PubMed

    Wilson, Ross C; Doudna, Jennifer A

    2013-01-01

    Small RNA molecules regulate eukaryotic gene expression during development and in response to stresses including viral infection. Specialized ribonucleases and RNA-binding proteins govern the production and action of small regulatory RNAs. After initial processing in the nucleus by Drosha, precursor microRNAs (pre-miRNAs) are transported to the cytoplasm, where Dicer cleavage generates mature microRNAs (miRNAs) and short interfering RNAs (siRNAs). These double-stranded products assemble with Argonaute proteins such that one strand is preferentially selected and used to guide sequence-specific silencing of complementary target mRNAs by endonucleolytic cleavage or translational repression. Molecular structures of Dicer and Argonaute proteins, and of RNA-bound complexes, have offered exciting insights into the mechanisms operating at the heart of RNA-silencing pathways.

  5. RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond.

    PubMed

    Castel, Stephane E; Martienssen, Robert A

    2013-02-01

    A growing number of functions are emerging for RNA interference (RNAi) in the nucleus, in addition to well-characterized roles in post-transcriptional gene silencing in the cytoplasm. Epigenetic modifications directed by small RNAs have been shown to cause transcriptional repression in plants, fungi and animals. Additionally, increasing evidence indicates that RNAi regulates transcription through interaction with transcriptional machinery. Nuclear small RNAs include small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs) and are implicated in nuclear processes such as transposon regulation, heterochromatin formation, developmental gene regulation and genome stability.

  6. Increased MicroRNA-34b and -34c Predominantly Expressed in Stromal Tissues Is Associated with Poor Prognosis in Human Colon Cancer

    PubMed Central

    Okayama, Hirokazu; Inamura, Kentaro; Anami, Katsuhiro; Nguyen, Giang H.; Horikawa, Izumi; Hawkes, Jason E.; Bowman, Elise D.; Leung, Suet Yi; Harris, Curtis C.

    2015-01-01

    The microRNA-34 family (miR-34a, -34b and -34c) have been reported to be tumor suppressor microRNAs (miRNAs) that are regulated by the TP53 and DNA hypermethylation. However, the expression, regulation, and prognostic value of the miR-34 family have not been systematically studied in colon cancer. To elucidate the roles of miR-34 family in colon carcinogenesis, miR-34a/b/c were measured in tumors and adjacent noncancerous tissues from 159 American and 113 Chinese colon cancer patients using quantitative RT-PCR, and we examined associations between miR-34a/b/c expression with TNM staging, cancer-specific mortality, TP53 mutation status and Affymetrix microarray data. All miR-34 family members were significantly increased in colon tumors, counter to the proposed tumor suppressor role for these miRNAs. Increased miR-34b/c were observed in more advanced tumors in two independent cohorts and increased expression of miR-34b/c was associated with poor cancer-specific mortality. While the expression of miR-34 family was not associated with TP53 mutation status, TP53 transcriptional activity was associated with miR-34a/b/c expression that is consistent with the proposed regulation of miR-34a/b/c by TP53. To examine where the miR-34 family is expressed, the expression of miR-34 family was compared between epitheliums and stromal tissues using laser microdissection technique. The expression of miR-34b/c was increased significantly in stromal tissues, especially in cancer stroma, compared with epithelial tissue. In conclusion, increased miR-34b/c predominantly expressed in stromal tissues is associated with poor prognosis in colon cancer. MiR-34 may contribute to cancer-stromal interaction associated with colon cancer progression. PMID:25894979

  7. MicroRNA-19b is a potential biomarker of increased myocardial collagen cross-linking in patients with aortic stenosis and heart failure

    PubMed Central

    Beaumont, Javier; López, Begoña; Ravassa, Susana; Hermida, Nerea; José, Gorka San; Gallego, Idoia; Valencia, Félix; Gómez-Doblas, Juan José; de Teresa, Eduardo; Díez, Javier; González, Arantxa

    2017-01-01

    This study analyzed the potential associations of 7 myocardial fibrosis-related microRNAs with the quality of the collagen network (e.g., the degree of collagen fibril cross-linking or CCL) and the enzyme lysyl oxidase (LOX) responsible for CCL in 28 patients with severe aortic stenosis (AS) of whom 46% had a diagnosis of chronic heart failure (HF). MicroRNA expression was analyzed in myocardial and blood samples. From the studied microRNAs only miR-19b presented a direct correlation (p < 0.05) between serum and myocardium. Compared to controls both myocardial and serum miR-19b were reduced (p < 0.01) in AS patients. In addition, miR-19b was reduced in the myocardium (p < 0.01) and serum (p < 0.05) of patients with HF compared to patients without HF. Myocardial and serum miR-19b were inversely correlated (p < 0.05) with LOX, CCL and LV stiffness in AS patients. In in vitro studies miR-19b inhibition increased (p < 0.05) connective tissue growth factor protein and LOX protein expression in human fibroblasts. In conclusion, decreased miR-19b may be involved in myocardial LOX up-regulation and excessive CCL, and consequently increased LV stiffness in AS patients, namely in those with HF. Serum miR-19b can be a biomarker of these alterations of the myocardial collagen network in AS patients, particularly in patients with HF. PMID:28091585

  8. Antagonism of microRNA-99a promotes cell invasion and down-regulates E-cadherin expression in pancreatic cancer cells by regulating mammalian target of rapamycin.

    PubMed

    Li, Dan; Li, Xiaohan; Cao, Wei; Qi, Yafei; Yang, Xianghong

    2014-06-01

    MicroRNA-99a (miRNA-99a), a potential tumor suppressor, has been implicated in tumorigenesis of many human malignancies. However, the role of miRNA-99a in pancreatic cancer remains unclear. In the present study, we transfected miRNA-99a antagonism into human pancreatic cancer AsPC-1 cells to inhibit miRNA-99a expression and investigated its influence on cell migration and invasion as well as the underlying possible mechanisms. We found that miRNA-99a antagonism significantly increased proliferation, migration and invasion abilities of AsPC-1 cells, which was accompanied by increased expression of mesenchymal phenotype cell biomarkers (N-cadherin, Vimentin, and α-SMA), and decreased expression of epithelial phenotype cell biomarker (E-cadherin). Interestingly, small interfering RNA (siRNA)-mediated knockdown of mammalian target of rapamycin (mTOR) remarkably restored miRNA-99a antagonism-induced down-regulation of E-cadherin. In conclusion, our data suggest that miRNA-99a is involved in pancreatic cancer migration and invasion by regulating mTOR, and may provide a target for effective therapies against pancreatic cancer.

  9. A novel long noncoding RNA LINC01133 is upregulated in lung squamous cell cancer and predicts survival.

    PubMed

    Zhang, Jing; Zhu, Ning; Chen, Xiaodong

    2015-09-01

    Lung adenocarcinoma (LAD) and lung squamous cell cancer (LSCC) are two major histological types of non-small cell lung cancer. LSCC differs greatly from LAD in many aspects. Accumulating evidence has shown that long noncoding RNA (lncRNA) plays an important role in the process of carcinogenesis and tumor progression. Expression of lncRNA is highly tissue-specific and could be biomarkers for cancer diagnosis and prognosis. Here, we identified differentially expressed lncRNA between LSCC and LAD by data mining of Affymetrix HG-U133 Plus 2.0 microarray. A set of 1646 differentially expressed lncRNA transcripts were identified. Among these lncRNAs, a novel lncRNA, LINC01133, showed the largest fold change among large intergenic noncoding RNAs. Quantitative real-time polymerase chain reaction (PCR) assay confirmed that LINC01133 was upregulated in LSCC (increasing fold 6.4, P < 0.01) but not in the LAD samples. LSCC patients with higher expression level of LINC01133 had shorter survival time (hazard ratio = 2.383; 95 % confidence interval 1.023-5.547, P = 0.044). Wound-healing and transwell assays demonstrated that silence of LINC01133 by small interfering RNA (siRNA) inhibited invasion ability of LSCC cell line. Thus, a set of lncRNA was differentially expressed between LAD and LSCC and could serve as potential biomarkers.

  10. Lipid Nanoparticle-mediated siRNA Transfer Against PCTAIRE1/PCTK1/Cdk16 Inhibits In Vivo Cancer Growth

    PubMed Central

    Yanagi, Teruki; Tachikawa, Kiyoshi; Wilkie-Grantham, Rachel; Hishiki, Asami; Nagai, Ko; Toyonaga, Ellen; Chivukula, Pad; Matsuzawa, Shu-ichi

    2016-01-01

    PCTAIRE1/CDK16/PCTK1 plays critical roles in cancer cell proliferation and antiapoptosis. To advance our previously published in vitro results with PCTAIRE1 silencing, we examined the in vivo therapeutic potential of this approach by using small interfering RNA (siRNA) encapsulated by lipid nanoparticles. Therapy experiments of PCTAIRE1 siRNA were performed using human HCT116 colorectal cancer cells and human A2058 melanoma cells. A single dose of PCTAIRE1 siRNA-lipid nanoparticles was found to be highly effective in reducing in vivo PCTAIRE1 expression for up to 4 days as assayed by immunoblotting. Therapy experiments were started 4 days after subcutaneous injection of cancer cells. Treatment with PCTAIRE1 siRNA-lipid nanoparticles (0.5 mg/kg RNA, twice a week) reduced tumor volume and weight significantly compared with the scramble-control group. Histopathological analysis (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) showed increased apoptosis of tumor cells treated with PCTAIRE1-siRNA. Overall, our results demonstrate that siRNA treatment targeting PCTAIRE1 is effective in vivo, suggesting that PCTAIRE1 siRNA-lipid nanoparticles might be a novel therapeutic approach against cancer cells. PMID:27351680

  11. RNA Interference

    MedlinePlus

    ... NIGMS Home > Science Education > RNA Interference Fact Sheet RNA Interference Fact Sheet Tagline (Optional) Middle/Main Content Area What is RNA interference? RNA interference (RNAi) is a natural process ...

  12. Life-long norepinephrine transporter (NET) knock-out leads to the increase in the NET mRNA in brain regions rich in norepinephrine terminals.

    PubMed

    Solich, Joanna; Kolasa, Magdalena; Kusmider, Maciej; Pabian, Paulina; Faron-Gorecka, Agata; Zurawek, Dariusz; Szafran-Pilch, Kinga; Kedracka-Krok, Sylwia; Jankowska, Urszula; Swiderska, Bianka; Dziedzicka-Wasylewska, Marta

    2015-08-01

    These studies aimed to identify the genes differentially expressed in the frontal cortex of mice bearing a life-long norepinephrine transporter knock-out (NET-KO) and wild-type animals (WT). Differences in gene expression in the mouse frontal cortex were studied using a whole-genome microarray approach. Using an alternative approach, i.e. RT-PCR (reverse transcription polymerase chain reaction) with primers complementary to various exons of the NET gene, as well as TaqMan arrays, the level of mRNA encoding the NET in other brain regions of the NET-KO mice was also examined. The analyses revealed a group of 92 transcripts (27 genes) that differentiated the NET-KO mice from the WT mice. Surprisingly, the studies have shown that the mRNA encoding NET accumulated in the brain regions rich in norepinephrine nerve endings in the NET-KO mice. Because there is no other source of NET mRNA besides the noradrenergic terminals in the brain regions studied, these results might speak in favor of the presence of mRNA in axon terminals. RNA-Binding Protein Immunoprecipitation approach indicated that mRNA encoding NET was detected in the Ago2 protein/mRNA complex. In addition, the amount of Ago2 protein in the frontal cortex was significantly higher in NET-KO mice as compared with that of the WT animals. These results are important for further characterization of the NET-KO mice, which - besides other merits - might serve as a good model to study the fate of truncated mRNA in neurons.

  13. DNA repair investigations using siRNA.

    PubMed

    Miller, Holly; Grollman, Arthur P

    2003-06-11

    Small interfering RNA (siRNA) is a revolutionary tool for the experimental modulation of gene expression, in many cases making redundant the need for specific gene mutations and allowing examination of the effect of modulating essential genes. It has now been shown that siRNA phenotypes resulting from stable transfection with short hairpin RNA (shRNA) can be transmitted through the mouse germ line and Rosenquist and his colleagues have used shRNA, which is processed in vivo to siRNA, to create germline transgenic mice in which a target DNA repair gene has been silenced. Here, Holly Miller and Arthur P. Grollman give the background of these discoveries, provide an overview of current uses, and look at future applications of this research.