Potential Regulatory miRNAs Research Articles

Discovery of porcine miRNA-196a/b may influence porcine adipogenesis in longissimus dorsi muscle by miRNA sequencing.

Intramuscular fat (IMF) is one of the fat traits that has economic importance in the pork industry. Longissimus dorsi muscle contains IMF and is suitable for studying adipogenesis. To discover further potential regulatory miRNAs that may influence adipogenesis, we analyzed miRNA in the longissimus dorsi muscle of Yorkshire (YY, lean-type) and Chinese Wannanhua (WH, fatty) pigs using miRNA sequencing (miRNA-seq). From this dataset, we identified 598 unique miRNAs comprising 325 pre-miRNAs and 273 novel pre-miRNAs through comparison with known miRNAs in miRBase version 21. We found 42 miRNAs including nine up- and 33 down-regulated between the YY and WH pigs. Moreover, we found two miRNAs, miR-196a/b (miR-196a, miR-196b-5p), that had the highest level of expression in WH pigs, and miR-196a/b may influence porcine adipogenesis in longissimus dorsi muscle through an adipocytokine signaling pathway.

MicroRNA-127-5p regulates osteopontin expression and osteopontin-mediated proliferation of human chondrocytes.

The aim of this study was to determine the specific microRNA (miRNA) that regulates expression of osteopontin (OPN) in osteoarthritis (OA). The potential regulatory miRNAs for OPN messenger RNA (mRNA) were predicted by miRNA prediction programs. Among eight potential regulatory miRNAs, miR-220b, miR-513a-3p and miR-548n increased, while miR-181a, miR-181b, miR-181c, miR-181d and miR-127-5p decreased in OA patients. miRNA-127-5p mimics suppressed OPN production as well as the activity of a reporter construct containing the 3′-UTR of human OPN mRNA. In addition, mutation of miR-127-5p binding site in the 3′-UTR of OPN mRNA abolished miR-127-5p-mediated repression of reporter activity. Conversely, treatment with miR-127-5p inhibitor increased reporter activity and OPN production. Interestingly, miR-127-5p inhibited proliferation of chondrocytes through OPN. In conclusion, miRNA-127-5p is an important regulator of OPN in human chondrocytes and may contribute to the development of OA.

A combinative analysis of gene expression profiles and microRNA expression profiles identifies critical genes and microRNAs in oral lichen planus

ObjectiveOral lichen planus (OLP) is a chronic inflammatory disease but aetiology and pathogenesis has not fully elucidated. To gain insight into the mechanism of OLP, bioinformatic analysis was performed in this study. DesignGSE38616 and GSE38615 were downloaded from GEO, including 7 cases of OLP and 7 healthy controls. Differentially expressed genes (DEGs) and miRNAs (DEMs) between OLP and control were screened with package Limma of R. Potential regulatory miRNAs were screened via gene set enrichment analysis. A protein–protein interaction network was constructed for the DEGs. KEGG pathways for DEGs were revealed using Gene Set Analysis Toolkit V2. ResultsAfter DEGs and DEMs were obtained, potential regulatory miRNAs of the DEGs were revealed and only miR-362 was differentially expressed in OLP compared with DEMs. Four targets of miR-362 were SLIT-ROBO Rho GTPase activating protein 2 (SRGAP2), vesicle-associated membrane protein 4 (VAMP4), leucine rich repeat transmembrane neuronal 4 (LRRTM4) and lysine (K)-specific demethylase5C (KDM5C). Identified DEGs were significantly enriched in olfactory transduction and ribosome pathways. ConclusionmiR-362, targeting SRGAP2 and VAMP4, may be a potential risk miRNA to regulate OLP. The findings may provide potential biomarkers for diagnosis or treatment of the disease.

Upregulation of RHOA and NKIRAS1 genes in lung tumors is associated with loss of their methylation as well as with methylation of regulatory miRNA genes

Methylation of CpG-islands in promoter regions as well as interaction of miRNAs with messenger RNAs of target genes are related to multilayer mechanisms regulating gene expression. The goal of this study was to assess a possibility for miRNA gene methylation to influence indirectly activation of their target genes in lung tumors. By using a unified collection of samples of non-small cell lung cancer, it was demonstrated that elevated levels of mRNA for RHOA and NKIRAS1 genes were significantly (Spearman rank correlation, P < 10(-11)) associated both with loss of methylation in their CpG-islands and methylation in a number of miRNA genes, which, according to the miRWalk database, were predicted to possess regulatory functions. Novel potential regulatory miRNAs for RHOA (miR-9-1/-3, -34b/c, -129-2, -125b-1, -375, -1258) and NKIRAS1 (miR-34b/c, -129-2, -125b-1, -193a, -124a-1/-2/-3, -212, -132) genes in lung cancer were identified.

Circulating Mir-153 Participates in Occurrence of Acute Graft-Versus-Host Disease By Down-Regulating Indoleamine-2,3-Dioxygenase

Background Indoleamine-2,3-dioxygenase (IDO) is able to catalyze the first and rate-limiting step in the catabolism of tryptophan, which is the essential amino acid for T cell proliferation. Many previous studies have proved that IDO is able to play important roles in immunosuppression-associated with tumor immunity, autoimmunity and chronic infection. Since donor T cells will be activated, proliferate, differentiate and finally lead to tissue injury, growing research paid more attention to the immunoregulatory role of IDO in the process of aGVHD. Our previous work also demonstrated that plasma IDO level was correlated with the severity of aGVHD. Increased IDO expression would serve as a protective molecular during aGVHD. However, the exact regulatory mechanism of IDO is still unclear. MicroRNAs (miRNAs) are a class of small non-coding RNA that negatively regulate gene expression by translational repression or induction of mRNA degradation. Recently, circulating miRNAs have been reported to be the promising biomarkers for various kinds of diseases. We speculated that miRNA that might regulate those GVHD-related molecules/cytokines expression including IDO, and the circulating miRNA that directed at IDO would serve as new potential biomarker for aGVHD. This study is to find the potential regulatory miRNA of IDO and investigate the correlation between plasma miRNA expression and aGVHD.Methods The potential regulatory miRNA for IDO was identified through bioinformatics analysis using TargetScan and Miranda. The regulatory function of target miRNA on IDO protein and gene was confirmed by Western Blot analysis and luciferase assay. Human plasma samples from 70 patients who underwent allo-HSCT from Sep 2012 to Jun 2013 were prospectively collected at +7 day(d), +14d, +21d, +30d, +45d, +60d, +90d and the occurrence of aGVHD from Peking University Institute of Hematology. All patients provided informed consent. This study was approved by the institutional review board at the Peking University Institute of Hematology (Protocol number 2013-42). Real-time quantitative PCR (RQ-PCR) analysis was performed to examine the miRNA expression of plasma at different time points posttransplant. Receiver operating characteristic (ROC) analysis was employed to determine the optimal cut-off value for predict the occurrence of aGVHD.Results Through bioinformatics analysis, we found the predicted target sites in its 3’UTR binding miRNA-153. The intracellular IDO protein would be reduced when miRNA-153 was transfected into interferon-gamma-treated Hela cells. Surprisingly, the mRNA expression level of IDO was also affected in miRNA-153 transfected cells obviously (P<0.001). In addition, luciferase assay in 293T cells showed that miRNA-153 was able to bind directly to the promoter region of IDO gene and affect its transcription. Luciferase activity dropped around 44% when wild-type construct was co-transfected into 293T cells with miRNA-153 (P=0.009). However, when the potential binding sites were mutated, this phenomenon could not be observed. This indicates that the IDO 3’UTR is normally targeted by endogenous miR-153. Among the 70 patients who underwent allo-HSCT, 35 patients developed into aGVHD and 35 patients did not. A significant difference in miRNA-153 expression levels between the two groups was confirmed at +7d (P<0.001). Furthermore, we used ROC analysis to evaluate the diagnostic accuracy. Plasma levels of miRNA-153 at day 7 could predict an impending aGVHD with a sensitivity of 38.71% and a specificity of 100%, and the area under the curve (AUC) was 0.898 (95%CI: 0.821-0.974, P<0.0001).Conclusions Our study suggested that miRNA-153 might participate in occurrence of aGVHD by down regulating IDO and might be a putative target for novel aGVHD therapy. The plasma level of miRNA-153 at +7d after allo-HSCT would be a good biomarker for predicting the occurrence of aGVHD. DisclosuresNo relevant conflicts of interest to declare.

MicroRNAs regulate methionine adenosyltransferase 1A expression in hepatocellular carcinoma

MicroRNAs (miRNAs) and methionine adenosyltransferase 1A (MAT1A) are dysregulated in hepatocellular carcinoma (HCC), and reduced MAT1A expression correlates with worse HCC prognosis. Expression of miR-664, miR-485-3p, and miR-495, potential regulatory miRNAs of MAT1A, is increased in HCC. Knockdown of these miRNAs individually in Hep3B and HepG2 cells induced MAT1A expression, reduced growth, and increased apoptosis, while combined knockdown exerted additional effects on all parameters. Subcutaneous and intraparenchymal injection of Hep3B cells stably overexpressing each of this trio of miRNAs promoted tumorigenesis and metastasis in mice. Treatment with miRNA-664 (miR-664), miR-485-3p, and miR-495 siRNAs reduced tumor growth, invasion, and metastasis in an orthotopic liver cancer model. Blocking MAT1A induction significantly reduced the antitumorigenic effect of miR-495 siRNA, whereas maintaining MAT1A expression prevented miRNA-mediated enhancement of growth and metastasis. Knockdown of these miRNAs increased total and nuclear level of MAT1A protein, global CpG methylation, lin-28 homolog B (Caenorhabditis elegans) (LIN28B) promoter methylation, and reduced LIN28B expression. The opposite occurred with forced expression of these miRNAs. In conclusion, upregulation of miR-664, miR-485-3p, and miR-495 contributes to lower MAT1A expression in HCC, and enhanced tumorigenesis may provide potential targets for HCC therapy.

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

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

Solexa Sequencing Identification of Conserved and Novel microRNAs in Backfat of Large White and Chinese Meishan Pigs

The domestic pig (Sus scrofa), an important species in animal production industry, is a right model for studying adipogenesis and fat deposition. In order to expand the repertoire of porcine miRNAs and further explore potential regulatory miRNAs which have influence on adipogenesis, high-throughput Solexa sequencing approach was adopted to identify miRNAs in backfat of Large White (lean type pig) and Meishan pigs (Chinese indigenous fatty pig). We identified 215 unique miRNAs comprising 75 known pre-miRNAs, of which 49 miRNA*s were first identified in our study, 73 miRNAs were overlapped in both libraries, and 140 were novelly predicted miRNAs, and 215 unique miRNAs were collectively corresponding to 235 independent genomic loci. Furthermore, we analyzed the sequence variations, seed edits and phylogenetic development of the miRNAs. 17 miRNAs were widely conserved from vertebrates to invertebrates, suggesting that these miRNAs may serve as potential evolutional biomarkers. 9 conserved miRNAs with significantly differential expressions were determined. The expression of miR-215, miR-135, miR-224 and miR-146b was higher in Large White pigs, opposite to the patterns shown by miR-1a, miR-133a, miR-122, miR-204 and miR-183. Almost all novel miRNAs could be considered pig-specific except ssc-miR-1343, miR-2320, miR-2326, miR-2411 and miR-2483 which had homologs in Bos taurus, among which ssc-miR-1343, miR-2320, miR-2411 and miR-2483 were validated in backfat tissue by stem-loop qPCR. Our results displayed a high level of concordance between the qPCR and Solexa sequencing method in 9 of 10 miRNAs comparisons except for miR-1a. Moreover, we found 2 miRNAs, miR-135 and miR-183, may exert impacts on porcine backfat development through WNT signaling pathway. In conclusion, our research develops porcine miRNAs and should be beneficial to study the adipogenesis and fat deposition of different pig breeds based on miRNAs.

Analysis of Regulatory miRNAs of Histone Demethylase JMJD3 In MDS CD34+ Hematoprogenitor Cells

AbstractAbstract 609 Introduction:Myelodysplastic syndromes (MDS) represent a very complex group of myeloid leukemias with diverse genetic and epigenetic characteristics. We have identified JMJD3, a histone demethylase, aberrantly upregulated in bone marrow CD34+ hematopoietic progenitor cells of MDS (Blood 2009). JMJD3 is a jmjc domain containing histone demethylase that removes methyl groups from Lys-27 of histone H3, and is also known to positively regulate the methylation on Lys-4 of histone H3. This results in activation of upstream regulators of NF-kB signaling, suggesting that JMJD3 plays a crucial role in the pathogenesis of MDS. Therefore, insight on the regulation of JMJD3 expression in MDS is of importance. Aberrant expression of miRNAs, including loss of expression, which leads to up-regulation of the expression of its target genes, has been identified in various types of malignancies including MDS. We hypothesized that alteration of regulatory miRNAs could be involved in the upregulation of JMJD3 in MDS CD34+ cells. Identification of JMJD3 regulatory miRNAs may help further dissect the molecular mechanisms underlying the pathogenesis of MDS. Methods and Results:To identify regulatory miRNAs targeting JMJD3, we performed an analysis using microRNA Genomics Resource (miRGen) and identified 40 candidate miRNAs that potentially interact with the 3′ untranslate region (3′-UTR) of JMJD3 RNA. We then characterized the interactions of these candidate miRNAs to JMJD3 by using a luciferase assay system by cloning the 3′-UTR region of JMJD3 gene into a luciferase reporter construct. By co-transfecting each of the 27 available miRNAs from the 40 candidates together with the luciferase reporter construct into human megakaryocytic blast cell line Meg-O1, we identified 7 mirRNAs (has-mir -29a, -29b, -29c, -99b, -101, -377, and -767-5p) that negatively regulate JMJD3 3′UTR associated luciferase activity. We then examined the expression for these 7 key potential regulatory miRNAs of JMJD3 in a cohort of 36 patient bone marrow CD34+ cells by microRNA Taqman probe based Q-PCR analysis. The median age of these patients was 67 years (32 to 85); 42% had low or INT-1 disease; 21% were diploid and 28% had an alteration of chromosome 5 or 7. Analysis showed that 16 of the 36 MDS CD34+ cell samples examined showed lost expression of mirRNA hsa-mir767-5p, and 7 had down-regulated expression of the same microRNA. For these 23 MDS samples with repressed mir-767-5p expression, 11 of them (~48%) had upregulated expression of JMJD3, as demonstrated by Q-PCR analysis Consistently, in miRNA transfected Meg-O1 cells, Q-PCR analysis indicates that over-expression of mir767-5p is accompanied with decrease of JMJD3 RNA by 68% based on Q-PCR analysis using 3 different probes for JMJD3. Of interest, 4 of the 5 patients with monosomy of chromosome 7 had downregulation of mir767-5p. Conclusion and Future Direction:These results support the hypothesis that mir767-5p is a key regulatory miRNA targeting JMJD3 in hematopoietic progenitor cells. Since one important mechanism for miRNA on target gene expression is through inhibiting the translation from mRNA to protein, we are now developing an antibody against endogenous JMJD3, with which we will be able to further correlate the level of mir767-5p and the expression of JMJD3 protein in both cell lines and primary MDS samples. Furthermore, correlation between alteration of mir767-5p with clinical, cytogenetic and molecular features in MDS patients will be characterized in a larger cohort of patients. These will further determine the role of mir767-5p for the regulation of JMJD3 and its role during the pathogenesis of MDS. By pursuing the studies described above, a potential link between two key epigenetic regulatory components, histone methylation regulator (JMJD3) and microRNAs, involved in pathogenesis of MDS, will be established. Disclosures:No relevant conflicts of interest to declare.