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           Search results for: MicroRNA Tools miRZip-331-3p anti-miR--331-3p microRNA construct   

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Transgene-Like Animal Models Using Intronic MicroRNAs.

Transgenic animal models are valuable tools for testing gene functions and drug mechanisms in vivo. They are also the best similitude for a human body for etiological and pathological research of diseases. All pharmaceutically developed medicines must be proven to be safe and effective in animals before approval by the Food and Drug Administration (FDA) to be used in clinical trials. To this end, the transgenic animal models of diseases serve as the front line of drug evaluation. However, there is currently no transgenic animal model for microRNA (miRNA)-related research. MiRNAs, small single-stranded regulatory RNAs capable of silencing intracellular gene transcripts (mRNAs) that contain either complete or partial complementarity to the miRNA, are useful for the design of new therapies against cancer polymorphism and viral mutation. Recently, varieties of natural miRNAs have been found to be derived from hairpin-like RNA precursors in almost all eukaryotes, including yeast (Schizosaccharomyces pombe), plant (Arabidopsis spp.), nematode (Caenorhabditis elegans), fly (Drosophila melanogaster), fish, mouse and human, involving intracellular defense against viral infections and regulation of certain gene expressions during development. To facilitate the miRNA research in vivo, we have developed a state-of-the-art transgenic strategy for silencing specific genes in zebrafish, chicken, and mouse, using intronic miRNAs. By the insertion of a hairpin-like pre-miRNA structure into the intron region of a gene, we have found that mature miRNAs were successfully transcribed by RNA polymerases type II (Pol-II), coexpressed with the encoding gene transcripts, and excised out of the encoding gene transcripts by intracellular RNA splicing and processing mechanisms. In conjunction with retroviral transfection, the designed hairpin-like pre-miRNA construct has also been placed in the intron regions of a cellular gene for tissue-specific expression, specifically regulated by the gene promoter of interest. Because the retroviral vectors are integrated into the genome of its host cells, we can select and propagate the most effective transgenic animals to form a stable model line for further research. Here, we have shown for the first time that transgene-like animal models were generated using the intronic miRNA expression system reported previously, which has been proven to be useful for studying miRNA function as well as the related gene regulation in vivo.

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Exploring the Potential Application of Short Non-Coding RNA-Based Genetic Circuits in Chinese Hamster Ovary Cells.

The majority of cell engineering for recombinant protein production to date has relied on traditional genetic engineering strategies, such as gene overexpression and gene knock-outs, to substantially improve the production capabilities of Chinese Hamster Ovary (CHO) cells. However, further improvements in cellular productivity or control over product quality is likely to require more sophisticated rational approaches to coordinate and balance cellular pathways. For these strategies to be implemented, novel molecular tools need to be developed to facilitate more refined control of gene expression. Multiple gene control strategies are developed over the last decades in the field of synthetic biology, including DNA and RNA-based systems, which allows tight and timely control over gene expression. microRNAs has received a lot of attention over the last decade in the CHO field and are used to engineer and improve CHO cells. In this review we focus on microRNA-based gene control systems and discuss their potential use as tools rather than targets in order to gain better control over gene expression.

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miR-CATCH Identifies Biologically Active miRNA Regulators of the Pro-Survival Gene XIAP, in Chinese Hamster Ovary Cells.

Genetic engineering of mammalian cells is of interest as a means to boost bio-therapeutic protein yield. X-linked inhibitor of apoptosis (XIAP) overexpression has previously been shown to enhance CHO cell growth and prolong culture longevity while additionally boosting productivity. The authors confirmed this across a range of recombinant products (SEAP, EPO, and IgG). However, stable overexpression of an engineering transgene competes for the cells translational machinery potentially compromising product titre. MicroRNAs are attractive genetic engineering candidates given their non-coding nature and ability to regulate multiple genes simultaneously, thereby relieving the translational burden associated with stable overexpression of a protein-encoding gene. The large number of potential targets of a single miRNA, falsely predicted in silico, presents difficulties in identifying those that could be useful engineering tools. The authors explored the identification of direct miRNA regulators of the pro-survival endogenous XIAP gene in CHO-K1 cells by using a miR-CATCH protocol. A biotin-tagged antisense DNA oligonucleotide for XIAP mRNA is designed and used to pull down and capture bound miRNAs. Two miRNAs are chosen out of the 14 miRNAs identified for further validation, miR-124-3p and miR-19b-3p. Transient transfection of mimics for both results in the diminished translation of endogenous CHO XIAP protein whereas their inhibition increases XIAP protein levels. A 3'UTR reporter assay confirms miR-124-3p to be a bona fide regulator of XIAP in CHO-K1 cells. This method demonstrates a useful approach to finding miRNA candidates for CHO cell engineering without competing for the cellular translational machinery.

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MicroRNA and target mRNA selection through invasion and cytotoxicity cell modeling and bioinformatics approaches in esophageal squamous cell carcinoma.

This study analyzed microRNA (miRNA) and mRNA expression profiles and investigated the biological characteristics of ESCC by using invasion and cytotoxicity cell models. miRNA profiles were evaluated through miRNA microarray. Transwell chamber and nedaplatin (NDP) were used to construct invasion and cytotoxicity cell models. Invasion Transwell and cytotoxicity assays were performed to examine the invasiveness and proliferation in the cell models. Functional miRNAs were selected from dysregulated miRNAs through qRT-PCR. Biometric Research Program (BRB)-array tools, Cytoscape plugins, and DAVID were utilized to find potential mRNAs targeted by these two miRNAs between ESCC and paired normal adjacent tissues. Our microarray obtained 11 dysregulated miRNAs expressed in three paired ESCC samples from Kazakhs (ethnicity in Northwestern China). qRT-PCR demonstrated the miRNA expression in the invasion and cytotoxicity cell models. miR‑652-5p and miR‑21‑5p exhibited a consistent expression level in the microarray and cell models. Bioinformatics revealed that the potential targets of PLD1, MSH2, STC1, and DSG1 might be involved in ESCC invasion and proliferation. Cell models with bioinformatics approaches may help distinguish functional genes. miR‑652-5p, miR‑21‑5p, and their potential target genes may participate in ESCC development and metastasis.

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New insights for therapeutic recombinant human miRNAs heterologous production: Rhodovolum sulfidophilum vs Escherichia coli.

RNA interference-based technologies have emerged as an attractive and effective therapeutic option with potential application in diverse human diseases. These tools rely on the development of efficient strategies to obtain homogeneous non-coding RNA samples with adequate integrity and purity, thus avoiding non-targeted gene-silencing and related side-effects that impair their application onto pre-clinical practice. These RNAs have been preferentially obtained by in vitro transcription using DNA templates or via chemical synthesis. As an alternative to overcome the limitations presented by these methods, in vivo recombinant production of RNA biomolecules has become the focus in RNA synthesis research. Therefore, using pre-miR-29b as a model, here it is evaluated the time-course profile of Escherichia coli and Rhodovolum sulfidophilum microfactories to produce this microRNA. As the presence of major host contaminants arising from the biosynthesis process may have important implications in the subsequent downstream processing, it is also evaluated the production of genomic DNA and host total proteins. Considering the rapidly growing interest on these innovative biopharmaceuticals, novel, more cost-effective, simple and easily scaled-up technologies are highly desirable. As microRNA recombinant expression fulfills those requirements, it may take the leading edge in the methodologies currently available to obtain microRNAs for clinical or structural studies.

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MicroRNAs 10a and 10b Regulate the Expression of Human Platelet Glycoprotein Ibα for Normal Megakaryopoiesis.

MicroRNAs are a class of small non-coding RNAs that bind to the three prime untranslated region (3'-UTR) of target mRNAs. They cause a cleavage or an inhibition of the translation of target mRNAs, thus regulating gene expression. Here, we employed three prediction tools to search for potential miRNA target sites in the 3'-UTR of the human platelet glycoprotein () gene. A luciferase reporter assay shows that miR-10a and -10b sites are functional. When miR-10a or -10b mimics were transfected into the GP Ibβ/GP IX-expressing cells, along with a DNA construct harboring both the coding and 3'-UTR sequences of the human gene, we found that they inhibit the transient expression of GP Ibα on the cell surface. When the miR-10a or -10b mimics were introduced into murine progenitor cells, upon megakaryocyte differentiation, we found that GP Ibα mRNA expression was markedly reduced, suggesting that a miRNA-induced mRNA degradation is at work. Thus, our study identifies GP Ibα as a novel target of miR-10a and -10b, suggesting that a drastic reduction in the levels of miR-10a and -10b in the late stage of megakaryopoiesis is required to allow the expression of human GP Ibα and the formation of the GP Ib-IX-V complex.

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WNT1 Gene from WNT Signaling Pathway Is a Direct Target of miR-122 in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is an invasive form of hepatic cancer arising from the accumulation of multiple genetic alterations. In this study, the causal role of disturbed canonical Wnt/β-catenin pathway was approved, and some of HCC-driven important gene candidates were determined. MicroRNAs (miRNAs), small non-coding RNAs, are the key regulators of important cancer genes, and their participation in tumorigenesis has been shown. By reviewing literature, WNT1 gene with functional significance was selected to approve miRNAs as new subjects for targeted therapy.For proper and fast miRNA detection and also confirmation of the role of bioinformatics in obtaining practical data, we benefited from different bioinformatics tools such as TargetScan, miRanda, and DIANA. In order to use an HCC model, we used HepG2 cell line. Luciferase assay was applied to assess the ability of the selected miRNAs in targeting WNT1 3'-UTR. To overexpress the selected miRNA in HepG2 cell line, viral construct was prepared. Quantitative real-time PCR was performed to evaluate selected miRNA and target gene expression levels. miR-122 was selected according to data concerning various bioinformatics tools.miR-122 was downregulated and WNT1 gene expression was upregulated in HepG2 cell line. After viral construct transduction, miR-122 expression was elevated and WNT1 expression was notably declined. Finally, we introduced WNT1 gene as one of the important genes in HCC, and also, we showed that miR-122 can regulate WNT1 gene expression.Moreover, our study determines the potential of bioinformatics analyses in providing accurate and reliable data for miRNA: messenger RNA (mRNA) prediction.

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Applying 3D-FRAP microscopy to analyse gap junction-dependent shuttling of small antisense RNAs between cardiomyocytes.

Small antisense RNAs like miRNA and siRNA are of crucial importance in cardiac physiology, pathology and, moreover, can be applied as therapeutic agents for the treatment of cardiovascular diseases. Identification of novel strategies for miRNA/siRNA therapy requires a comprehensive understanding of the underlying mechanisms. Emerging data suggest that small RNAs are transferred between cells via gap junctions and provoke gene regulatory effects in the recipient cell. To elucidate the role of miRNA/siRNA as signalling molecules, suitable tools are required that will allow the analysis of these small RNAs at the cellular level. In the present study, we applied 3 dimensional fluorescence recovery after photo bleaching microscopy (3D-FRAP) to visualise and quantify the gap junctional exchange of small RNAs between neonatal cardiomyocytes in real time. Cardiomyocytes were transfected with labelled miRNA and subjected to FRAP microscopy. Interestingly, we observed recovery rates of 21% already after 13min, indicating strong intercellular shuttling of miRNA, which was significantly reduced when connexin43 was knocked down. Flow cytometry analysis confirmed our FRAP results. Furthermore, using an EGFP/siRNA reporter construct we demonstrated that the intercellular transfer does not affect proper functioning of small RNAs, leading to marker gene silencing in the recipient cell. Our results show that 3D-FRAP microscopy is a straightforward, non-invasive live cell imaging technique to evaluate the GJ-dependent shuttling of small RNAs with high spatio-temporal resolution. Moreover, the data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation where small RNAs act as signalling molecules within the intercellular network.

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Exosome-derived microRNAs contribute to prostate cancer chemoresistance.

Certain microRNAs (miRNAs) play a key role in cancer cell chemoresistance. However, the pleiotropic functions of exosome-derived miRNAs on developing chemoresistance remain unknown. In the present study, we aimed to construct potential networks of miRNAs, which derived from the exosome of chemoresistant prostate cancer (PCa) cells, with their known target genes using miRNA expression profiling and bioinformatic tools. Global miRNA expression profiles were measured by microarray. Twelve miRNAs were initially selected and validated by qRT-PCR. Known targets of deregulated miRNAs were utilized using DIANA-TarBase database v6.0. The incorporation of deregulated miRNAs and target genes into KEGG pathways were utilized using DIANA-mirPath software. To construct potential miRNA regulatory networks, the overlapping parts of miRNAs and their targer genes from the selected KEGG pathway 'PCa progression (hsa05215)' were visualized by Cytoscape software. We identified 29 deregulated miRNAs, including 19 upregulated and 10 downregulated, in exosome samples derived from two kinds of paclitaxel resistance PCa cells (PC3-TXR and DU145-TXR) compared with their parental cells (PC3 and DU145). The enrichment results of deregulated miRNAs and known target genes showed that a few pathways were correlated with several critical cell signaling pathways. We found that hub hsa-miR3176, -141-3p, -5004-5p, -16-5p, -3915, -488‑3p, -23c, -3673 and -3654 were potential targets to hub gene androgen receptor (AR) and phosphatase and tensin homolog (PTEN). Hub gene T-cell factors/lymphoid enhancer-binding factors 4 (TCF4) target genes were mainly regulated by hub hsa-miR-32-5, -141-3p, -606, -381 and -429. These results may provide a linkage between PCa chemoresistance and exosome regulatory networks and thus lead us to propose that AR, PTEN and TCF4 genes may be the important genes which are regulated by exosome miRNAs in chemoresistance cancer cells.

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Identifying High-Risk Stage II Colon Cancer Patients: A Three-MicroRNA-Based Score as a Prognostic Biomarker.

The potential benefit of adjuvant chemotherapy in surgically resected patients with stage II colorectal cancer is controversial. The current guidelines, which are based solely on clinical factors, have limited usefulness, and a clear need exists for biomarkers to supplement the clinical information. MicroRNAs (miRNAs) have previously been shown to be useful cancer biomarkers. In the present study, we assessed the usefulness of a miRNA score to help identify the subset of high-risk patients likely to benefit from adjuvant chemotherapy.

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