Biological Functions Of miRNAs Research Articles

High-throughput screening identified miR-7-2-3p and miR-29c-3p as metastasis suppressors in gallbladder carcinoma.

Gallbladder carcinoma (GBC) is one of the most aggressive and lethal tumors, with extremely high metastatic activity and poor prognosis. Previously we have studied miRNAs that promote metastasis and progression of GBC, the aim of present study was to systematically elucidate the metastasis suppressor miRNAs in GBC. A novel designed high-throughput screening method that combined high content screening (HCS) and miRNA microarray analysis was conducted to filter out anti-metastatic miRNAs of GBC. Frozen samples were analyzed for the expression of goal miRNAs by real-time PCR. The biological functions of miRNAs were studied by transwell, immunoblot. Liver metastasis model via spleen injection was further examined in nude mice. Kaplan-Meier and Cox regression analyses were used to analyze the effect of goal miRNAs on overall survival. The target genes and interaction network of goal miRNAs were determined by whole transcriptome genome sequencing. Out of the miRNAs library, a series of prominent metastatic suppressor miRNA candidates were filtered out. Among them, miR-7-2-3p and miR-29c-3p were discovered downregulated in GBC, and upregulation of them could reverse epithelial-mesenchymal transition and decrease the metastasis ability of GBC cells in vitro and in vivo, which was dominated by the miRNA-mRNA-lncRNA co-expression network. And DCLK1 and SLC36A1 are the direct target genes of miR-7-2-3p and miR-29c-3p. Moreover, the deficiency of miR-7-2-3p and miR-29c-3p was closely associated with poor prognosis of GBC patients. Our findings indicate that miR-7-2-3p and miR-29c-3p play crucial roles in the pathogenesis and worse prognosis of GBCs, which may serve as prognosis biomarkers and promise potential therapeutic targets in the future.

Integrated mRNA and miRNA profile expression in livers of Jinhua and Landrace pigs.

ObjectiveTo explore the molecular mechanisms of fat metabolism and deposition in pigs, an experiment was conducted to identify hepatic mRNAs and miRNAs expression and determine the potential interaction of them in two phenotypically extreme pig breeds.MethodsmRNA and miRNA profiling of liver from 70-day Jinhua (JH) and Landrace (LD) pigs were performed using RNA sequencing. Blood samples were taken to detect results of serum biochemistry. Bioinformatics analysis were applied to construct differentially expressed miRNA-mRNA network.ResultsSerum total triiodothyronine and total thyroxine were significantly lower in Jinhua pigs, but the content of serum total cholesterol (TCH) and low-density lipoprotein cholesterol were strikingly higher. A total of 467 differentially expressed genes (DEGs) and 35 differentially expressed miRNAs (DE miRNAs) were identified between JH and LD groups. Gene ontology analysis suggested that DEGs were involved in oxidation-reduction, lipid biosynthetic and lipid metabolism process. Interaction network of DEGs and DE miRNAs were constructed, according to target prediction results.ConclusionWe generated transcriptome and miRNAome profiles of liver from JH and LD pig breeds which represent distinguishing phenotypes of growth and metabolism. The potential miRNA-mRNA interaction networks may provide a comprehensive understanding in the mechanism of lipid metabolism. These results serve as a basis for further investigation on biological functions of miRNAs in the porcine liver.

MicroRNA‑206 exerts anti‑oncogenic functions in esophageal squamous cell carcinoma by suppressing the c‑Met/AKT/mTOR pathway.

Increasing evidence suggests that the dysregulation of microRNAs (miRNAs) has an important role in the progression of human cancer, including ESCC. However, the exact functions and mechanisms of miRNAs in ESCC remain largely unclear. The aim of the present study was to investigate the expression and biological functions of miRNAs in ESCC and reveal the underlying molecular mechanisms. miRNA microarray and reverse transcription-quantitative polymerase chain reaction analyses were performed, which identified and confirmed that miR-206 was significantly downregulated in ESCC tissues and cell lines. Its low expression was associated with lymph node metastasis, advanced TNM stage and N classification, as well as poorer overall survival in patients with ESCC. CCK-8 and flow cytometry assays demonstrated that ectopic miR-206 expression inhibited ESCC cell proliferation and induced cell apoptosis. In addition, MET proto-oncogene, receptor tyrosine kinase (c-Met), a well-known oncogene, was a direct target of miR-206. An inverse correlation between the levels of miR-206 and c-Met mRNA in ESCC tissue samples was confirmed. Notably, c-Met overexpression inhibited the effects of miR-206 on the proliferation and apoptosis of ESCC cells. Additionally, it was confirmed that the tumor-suppressive functions of miR-206 may have contributed to the inactivation of the c-Met/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) signaling pathway. In conclusion, the findings of the present study suggested that miR-206 exerts its anti-cancer functions via the c-Met/AKT/mTOR signaling pathway, providing a novel candidate prognostic factor and a potential therapeutic target in ESCC.

MiR-454-3p and miR-374b-5p suppress migration and invasion of bladder cancer cells through targetting ZEB2.

Bladder cancer (BCa) threatens human health due to the high occurrence and mortality. Nowadays, more and more researchers focussed on the molecular mechanisms and biological functions of miRNAs in human cancers. The present study aims to study the biological role of miR-454-3p and miR-374b-5p in BCa. The expression levels of miR-454-3p and miR-374b-5p were detected in BCa tissues and cell lines by qRT-PCR analysis. Kaplan–Meier analysis revealed that the expression levels of miR-454-3p and miR-374b-5p were positively correlated with the overall survival (OS) rate of BCa patients. Gain-of-function assays were conducted to demonstrate the inhibitory effects of miR-454-3p and miR-374b-5p on the invasion, migration, and epithelial–mesenchymal transition (EMT) of BCa cells. Mechanically, ZEB2 was found to be a target of both miR-454-3p and miR-374b-5p. Rescue assays revealed that ZEB2 reversed the inhibitory effects of miR-454-3p and miR-374b-5p on the invasion and migration of BCa cell lines. In summary, miR-454-3p and miR-374b-5p negatively regulated invasion and migration of BCa cell lines via targetting ZEB2.

MiR-671-5p inhibits gastric cancer cell proliferation and promotes cell apoptosis by targeting URGCP.

Various studies have demonstrated that microRNA (miRNA) serves an important role in the development of gastric cancer. However, the expression level, clinical significance and the biological function of miRNA in gastric cancer remain largely unknown. The present study investigated the exact roles of miR-671-5p in gastric cancer, confirmed its target and explored its mechanism. Initially, the low expression levels of miR-671-5p in gastric cancer cells were confirmed by reverse transcription-quantitative polymerase chain reaction. TargetScan and MiRanda databases were utilized to forecast the target genes of miR-671-5p, and the prediction was verified by dual-luciferase reporter assay and western blot analysis. Cell Counting Kit-8 was used for cell proliferation detection. An annexin V-fluorescein isothiocyanate kit was used for cell apoptosis determination. Western blot analysis was adopted to measure the protein expression levels in different groups. The results of the present study revealed that there were lower expression levels of miR-671-5p in gastric cancer cells than in normal gastric cells. Upregulator of cell proliferation (URGCP) is a direct target of miR-671-5p and it may be negatively regulated by miR-671-5p. miR-671-5p mimics induced reduction of MKN28 cell proliferation. miR-671-5p mimics caused upregulation of MKN28 cell apoptosis. In addition, western blotting results indicated that the ratio of B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein was significantly decreased in the miR-671-5p mimic group compared with the negative control group (P<0.01). These results suggested that miR-671-5p had a protective role in gastric cancer through inhibiting gastric cancer cell proliferation and promoting cell apoptosis by targeting URGCP. Therefore, miR-671-5p may be an effective therapeutic target for gastric cancer.

MicroRNA-132 and microRNA-212 mediate doxorubicin resistance by down-regulating the PTEN-AKT/NF-κB signaling pathway in breast cancer

Breast cancer is a serious health problem worldwide. Acquisition of multi-drug resistance (MDR) during the treatment of breast cancer is still considered a major clinical obstacle. Despite the biological functions of miRNAs becoming increasingly apparent, the function of miRNAs in regulating drug resistance of breast cancer remains under investigation. Quantitative real-time PCR (qRT-PCR) was used to quantify the expression of miR-132/-212 (miR-132 and miR-212) in doxorubicin (DOX)-resistant and -sensitive breast cancer tumors and cells. The function of miR-132/-212 in drug resistance was investigated in vitro (MTT assay, TUNEL assay, fluorescence, immunohistochemistry, luciferase reporter assay, Western blotting). We found that miR-132/-212 were commonly overexpressed in DOX-resistant breast cancer tumors and cells. Silenced miR-132/-212 expression induced DOX accumulation in MCF-7/ADR cells, while overexpression of miR-132/-212 led to breast cancer resistance protein (BCRP)-based DOX efflux in MCF-7 cells. Further study showed that up-regulation of miR-132/-212 in MCF-7/ADR cells suppressed the expression of PTEN, a target gene of miR-132/-212, which activated AKT phosphorylation and the NF-κB pathway and led to increased BCRP expression. Down-regulation of miR-132/-212 sensitized MCF-7/ADR cells to DOX. Mechanistic investigations suggested that miR-132/-212 enhancement was a result of NF-κB-mediated transactivation of the pri-miR-132/-212 gene. Taken together, our findings are among the first to demonstrate a novel aspect of the miR-132/-212-PTEN-AKT/NF-κB-BCRP pathway in the generation of breast cancer resistance and provides a potential method to reverse drug resistance.

MicroRNA-129-3p suppresses tumor growth by targeting E2F5 in glioblastoma.

Neuroma is the most common intracranial tumor. The mechanism of miRNA in glioma has gradually been understood. The purpose of this study was to investigate the role of MicroRNA-129-3p (miR-129-3p) in the pathogenesis of glioblastoma (GBM). Differential expression of miR-129-3p in samples was analyzed by bioinformatics. PCR was used to detect the expression of miR-129-3p in samples. CCK8 assay was used to detect the cell viability. Transfection of mimic and inhibitor altered the expression of miR-129-3p, and the biological function of miRNA was explored. Luciferase reporter gene was used to detect target genes of miRNA. E2F5 expression was inhibited by transfection of small interfering RNAs. Western blotting was used to detect protein expressions of cells. miR-129-3p was low-expressed in the tissue samples. By transfecting mimic and the inhibitor, we found that increasing the expression of miR-129-3p can inhibit the cell viability. In contrast, inhibition of miR-129-3p promoted cell growth. Luciferase reporter gene and Western blot results suggested that E2F5 can be used as the target gene of miR-129-3p. Knockdown the target gene of the miR-129-3p, E2F5, also inhibited proliferation of glioblastoma. miR-129-3p can inhibit the growth of glioblastoma by down-regulating the expression of E2F5. miR-129-3p can be a new target for the treatment of glioblastoma. Our research provides new ideas for the target therapy of glioma.

Circ-SHKBP1 Regulates the Angiogenesis of U87 Glioma-Exposed Endothelial Cells through miR-544a/FOXP1 and miR-379/FOXP2 Pathways.

Circular RNAs (circRNAs) are a type of endogenous non-coding RNAs, which have been considered to mediate diverse tumorigenesis including angiogenesis. The present study aims to elucidate the potential role and molecular mechanism of circ-SHKBP1 in regulating the angiogenesis of U87 glioma-exposed endothelial cells (GECs). The expression of circ-SHKBP1, but not linear SHKBP1, was significantly upregulated in GECs compared with astrocyte-exposed endothelial cells (AECs). circ-SHKBP1 knockdown inhibited the viability, migration, and tube formation of GECs dramatically. The expressions of miR-379/miR-544a were downregulated in GECs, and circ-SHKBP1 functionally targeted miR-544a/miR-379 in an RNA-induced silencing complex (RISC) manner. Dual-luciferase reporter assay demonstrated that forkhead box P1/P2 (FOXP1/FOXP2) were targets of miR-544a/miR-379. The expressions of FOXP1/FOXP2 were upregulated in GECs, and silencing of FOXP1/FOXP2 inhibited the viability, migration, and tube formation of GECs. Meanwhile, FOXP1/FOXP2 promoted angiogenic factor with G patch and FHA domains 1 (AGGF1) expression at the transcriptional level. Furthermore, knockdown of AGGF1 suppressed the viability, migration, and tube formation of GECs via phosphatidylinositol 3-kinase (PI3K)/AKT and extracellular signal-regulated kinase (ERK)1/2 pathways. Taken together, the present study demonstrated that circ-SHKBP1 regulated the angiogenesis of GECs through miR-544a/FOXP1 and miR-379/FOXP2 pathways, and these findings might provide a potential target and effective strategy for combined therapy of gliomas.

Therapeutic targeting of non-coding RNAs in cancer.

The majority of the human genome encodes RNAs that do not code for proteins. These non-coding RNAs (ncRNAs) affect normal expression of the genes, including oncogenes and tumour suppressive genes, which make them a new class of targets for drug development in cancer. Although microRNAs (miRNAs) are the most studied regulatory ncRNAs to date, and miRNA-targeted therapeutics have already reached clinical development, including the mimics of the tumour suppressive miRNAs miR-34 and miR-16, which reached phase I clinical trials for the treatment of liver cancer and mesothelioma, the importance of long non-coding RNAs (lncRNAs) is increasingly being recognised. Here, we describe obstacles and advances in the development of ncRNA therapeutics and provide the comprehensive overview of the ncRNA chemistry and delivery technologies. Furthermore, we summarise recent knowledge on the biological functions of miRNAs and their involvement in carcinogenesis, and discuss the strategies of their therapeutic manipulation in cancer. We review also the emerging insights into the role of lncRNAs and their potential as targets for novel treatment paradigms. Finally, we provide the up-to-date summary of clinical trials involving miRNAs and future directions in the development of ncRNA therapeutics.

TUG1 promotes osteosarcoma tumorigenesis by upregulating EZH2 expression via miR-144-3p.

lncRNA-TUG1 (Taurine upregulated 1) is up regulated and highly correlated with poor prognosis and disease status in osteosarcoma. TUG1 knockdown inhibits osteosarcoma cell proliferation, migration and invasion, and promotes apoptosis. However, its mechanism of action has not been well addressed. Growing evidence documented that lncRNA works as competing endogenous (ce)RNAs to modulate the expression and biological functions of miRNA. As a putative combining target of TUG1, miR-144-3p has been associated with the progress of osteosarcoma. To verify whether TUG1 functions through regulating miR-144-3p, the expression levels of TUG1 and miR-144-3p in osteosarcoma tissues and cell lines were determined. TUG1 was upregulated in osteosarcoma tissues and cell lines, and negatively correlated with miR-144-3p. TUG1 knockdown induced miR-144-3p expression in MG63 and U2OS cell lines. Results from dual luciferase reporter assay, RNA-binding protein immuno precipitation (RIP) and applied biotin-avidin pull-down system confirmed TUG1 regulated miR-144-3p expression through direct binding. EZH2, a verified target of miR-144-3p was upregulated in osteosarcoma tissues and negatively correlated with miR-144-3p. EZH2 was negatively regulated by miR-144-3p and positively regulated by TUG1. Gain-and loss-of-function experiments were performed to analyze the role of TUG1, miR-144-3p and EZH2 in the migration and EMT of osteosarcoma cells. EZH2 overexpression partly abolished TUG1 knockdown or miR-144-3p overexpression induced inhibition of migration and EMT in osteosarcoma cells. In addition, TUG1 knockdown represses the activation of Wnt/β-catenin pathway, which was reversed by EZH2 over expression. The activator of Wnt/β-catenin pathway LiCl could partially block the TUG1-knockdown induced osteosarcoma cell migration and EMT inhibition. In conclusion, our results showed that TUG1 plays an important role in osteosarcoma development through miRNA-144-3p/EZH2/Wnt/β-catenin pathway.

Genome-wide identification of novel microRNAs from genome sequences using computational approach in the mudskipper (Boleophthalmus pectinirostris)

MicroRNAs (miRNAs), approximately 22 nucleotides (nt) long, are small, non-coding RNA molecules with important regulatory functions in gene expression. They are mostly conserved among the organisms and this conservation makes them a good source for the identification of novel miRNAs by computational genomic homology. The miRNA repertoire of a major aquaculture species, Boleophthalmus pectinirostris, has been unknown until recently. Currently, the B. pectinirostris whole-genome sequences have been completed, making it more convenient for us to focus on computational prediction for novel miRNA homologs. Following a range of strict filtering criteria, a total of 62 potential miRNAs were identified for the first time; they belong to 39 different miRNA families. All these miRNAs were observed in the stem portion of the stable stem–loop structures. The minimum free energy (MFE) of the predicted miRNAs ranged from −21.6 to −62.7 kcal/mol with an average of −39.2 kcal/mol. The A + U ranged from 32.5 to 69.1% with an average value of 52.2%. The phylogenetic analysis of predicted miRNAs revealed that miR-23a-3p, miR-184-3p, miR-214-5p, and miR-338-3p from B. pectinirostris are evolutionary highly conserved showing more similarity with other fish species. To verify the predicted miRNAs, selected miRNAs representing 16 of the 39 families were confirmed by stem–loop RT-PCR, indicating that the computational approach that we used to identify the miRNAs is a highly efficient and affordable alternative method. Taken together, these findings provide a reference point for further research on miRNAs identification in fish species, meanwhile, our study also will be useful for further insight into biological functions of miRNAs and improved understanding of genome in B. pectinirostris.

MiR-2425-5p targets RAD9A and MYOG to regulate the proliferation and differentiation of bovine skeletal muscle-derived satellite cells

Our group previously identified miR-2425-5p, a unique bovine miRNA; however, its biological function and regulation in muscle-derived satellite cells (MDSCs) remain unclear. Herein, stem-loop RT-PCR results showed that miR-2425-5p increased during MDSCs proliferation, but decreased during differentiation. Cell proliferation was examined using EdU assays, cyclin B1 (CCNB1) and proliferating cell nuclear antigen (PCNA) western blot (WB) and flow cytometry analysis. These results showed that miR-2425-5p mimics (miR-2425-M) enhanced MDSCs proliferation, whereas, miR-2425-5p inhibitor (miR-2425-I) had opposite effect. Conversely, cell differentiation studies by desmin (DES) immunofluorescence, myotubes formation, and myosin heavy chain 3 (MYH3) WB analyses revealed that miR-2425-M and miR-2425-I blocked and promoted MDSCs differentiation, respectively. Moreover, luciferase reporter, RT-PCR, and WB assays showed that miR-2425-5p directly targeted the 3′-UTR of RAD9 homolog A (RAD9A) and myogenin (MYOG) to regulate their expression. Rescue experiment showed RAD9A inhibited the proliferation of MDSCs through miR-2425-5p. In addition, we found that miR-2425-5p expression was regulated by its host gene NCK associated protein 5-like (NCKAP5L) rather than being transcribed independently as a separate small RNA. Collectively, these data indicate that miR-2425-5p is a novel regulator of bovine MDSCs proliferation and differentiation and provides further insight into the biological functions of miRNA in this species.

Target genes prediction and functional analysis of microRNAs differentially expressed in gastric cancer stem cells MKN-45.

Since mechanisms of microRNAs (miRNAs) in gastric cancer stem cells (CSCs) and their signaling pathways remain unknown, our aim was to predict the miRNA target genes that differentially expressed in gastric CSCs. Using miRanda, PicTar, and TargetScan algorithm, target genes of miRNAs differentially expressed in gastric CSCs versus parental cells were predicted. Afterward, signaling pathways and biological functions of miRNAs in gastric CSCs were analyzed by the Database for Annotation, Visualization and Integrated Discovery (DAVID) database and DIANA tools. Gene ontology (GO) tool indicated that most of miRNA target genes involved in regulation of cell cycle, apoptosis, cell migration, vasculogenesis, angiogenesis, etc. Some of miRNA target genes are connected to pivotal signaling pathways of the "stem cell genes," such as Notch, Wnt/β-catenin. Bioinformatics analysis such as DAVID database, GO biological process, GO molecular function, Kyoto encyclopedia of genes and genomes pathways, BioCarta pathway, Panther pathway, and Reactome pathway revealed that target genes of differentially expressed miRNAs in gastric CSCs were connected to pivotal biological pathways that involved in cell cycle regulation, stemness properties, and differentiation.

Role of miRNA and its potential as a novel diagnostic biomarker in drug-induced liver injury.

MicroRNAs (miRNA or miR) are the most abundant and stable class of small RNA. Unlike the typical RNA molecules present in the cell, they do not encode proteins but can control translation. and Hhence, they are found to play a major role in the regulation of cellular processes. miRNAs have been shown to differentially regulate various genes, and the expression levels of some miRNAs changes several fold in liver and serum, during drug- induced toxicity. This review summarises some of the latest findings about the biological functions of miRNA and its potential use as diagnostic biomarkers in drug- induced liver injury. The information presented in this article is taken from published literature, both original work and reviews on mechanisms of drug- induced liver injury, miRNA in liver pathophysiology, and studies exploring the use of miRNA as biomarker in drug- induced liver injury. Literature search was done using search engines:- PUBMED, Google scholar, and relevant journal sites. Recent research provides insight into the ability of miRNA to regulate various pathways in diseased and nondiseased states of liver. They also lay a foundation for development of diagnostic tests utilizing the potential of miRNAs that can not only be used for early detection of DILI but also to differentiate between different types of DILI. More studies on biological functions of miRNA and standardisation of protocol between research laboratories can lead to further advancement in this field. Considering the therapeutic and diagnostic potential of miRNA, the major challenge would be to integrate these findings to clinical settings where it can be used for the treatment of cases with DILI.

Advances in the effect of miRNA on regulating lung development

miRNAs are a group of 22~25 nucleotides endogenous non-coding RNAs, which regulate gene expression at the post-transcription level by cause the degradation or translational suppression of target mRNAs.In recent years, studies have demonstrated that miRNAs widely participate in cell differentiation, proliferation, organ development and lipid metabolism, and are closely related to the formation of many kinds of diseases.The biological function of miRNAs and their effects on regulating prenatal and postnatal lung development are reviewed in this paper. Key words: miRNA; Lung development; Microarray; Bronchopulmonary dysplasia

MicroRNA-193a-3p Reduces Intestinal Inflammation in Response to Microbiota via Down-regulation of Colonic PepT1

Intestinal inflammation is characterized by epithelial disruption, leading to the loss of barrier function, recruitment of immune cells, and host immune responses to gut microbiota. PepT1, a di/tripeptide transporter that uptakes bacterial products, is up-regulated in inflamed colon tissue, which implies its role in bacterium-associated intestinal inflammation. Although microRNA (miRNA)-mediated gene regulation has been found to be involved in various processes of inflammatory bowel disease (IBD), the biological function of miRNAs in the pathogenesis of IBD remains to be explored. In this study we detected miRNA expression patterns in colon tissues during colitis and investigated the mechanism underlying the regulation of colonic PepT1 by miRNAs. We observed an inverse correlation between PepT1 and miR-193a-3p in inflamed colon tissues with active ulcerative colitis, and we further demonstrated that miR-193a-3p reduced PepT1 expression and activity as a target gene and subsequently suppressed the NF-κB pathway. Intracolonic delivery of miR-193a-3p significantly ameliorated dextran sodium sulfate-induced colitis, whereas the overexpression of colonic PepT1 via PepT1 3'-untranslated region mutant lentivirus vector abolished the anti-inflammatory effect of miR-193a-3p. Furthermore, antibiotic treatment eliminated the difference in the dextran sodium sulfate-induced inflammation between the presence and absence of miR-193a-3p. These findings suggest that miR-193a-3p regulation of PepT1 mediates the uptake of bacterial products and is a potent mechanism during the colonic inflammation process. Overall, we believe miR-193a-3p may be a potent regulator of colonic PepT1 for maintaining intestinal homeostasis.

The biological functions of miRNAs: lessons from in vivo studies.

Despite their clear importance as a class of regulatory molecules, pinpointing the relevance of individual miRNAs has been challenging. Studies querying miRNA functions by overexpressing or silencing specific miRNAs have yielded data that are often at odds with those collected from loss-of-functions models. In addition, knockout studies suggest that many conserved miRNAs are dispensable for animal development or viability. In this review, we discuss these observations in the context of our current knowledge of miRNA biology and review the evidence implicating miRNA-mediated gene regulation in the mechanisms that ensure biological robustness.

Evaluation of a novel functional single-nucleotide polymorphism (rs35010275 G>C) in MIR196A2 promoter region as a risk factor of gastric cancer in a Chinese population.

Single-nucleotide polymorphisms (SNPs) in microRNAs (miRNAs) have been suggested to influence the occurrence and progression of cancer through altering the expression and biological function of miRNAs. The aim of this study was to investigate whether the potential functional SNPs in MIR196A2 promoter had effect on the susceptibility to gastric cancer (GC) in a Chinese population.We conducted a 2-stage case-control study (753 cases and 854 controls in testing set; 940 cases and 1061 controls in validation set) to evaluate the association between 2 potential functional SNPs in MIR196A2 promoter (rs12304647 A>C and rs35010275 G>C) and GC risk. The luciferase reporter assay and electrophoretic mobility shift assay were used to examine the functionality of the important polymorphism.We found that the rs35010275 C allele was significantly associated with the decreased risk of GC (adjusted odds ratio = 0.85, 95% confidence interval = 0.77-0.94) in the combined case-control studies. The miR-196a expression levels in GC tissues were significantly higher than that in corresponding adjacent normal tissues (P < 0.001). Besides, each allele of rs35010275 displayed completely opposite effects to influence the transcription activity of MIR196A2 promoter via recruiting different transcription factors or complexes.The functional rs35010275 G>C polymorphism in MIR196A2 promoter was significantly associated with miR-196a expression and influenced the genetic susceptibility to GC.

Dysregulated miR-124 and miR-200 expression contribute to cholangiocyte proliferation in the cholestatic liver by targeting IL-6/STAT3 signalling

Cholestatic liver disease is associated with dysregulated expression of microRNAs (miRNAs). However, it remains unknown whether miRNAs are involved in the cholestasis-induced proliferation of cholangiocytes. In this study, we tested the hypothesis that miRNAs modulate cholangiocyte proliferation through effects on the IL-6 pathway, a known regulator of cholangiocyte proliferation. Expression of IL-6, Foxa2, and phosphorylated signal transducer activator of transcription 3 (STAT3) was investigated in patients with biliary atresia (BA) and in rats subjected to bile duct ligation (BDL). miRNA expression was determined in BA patients and BDL rats, with miRNA array and quantitative real-time PCR. Biological functions of miRNAs were studied using immunoblot, immunohistochemical and proliferation assays. Luciferase reporter assays and Western blots were performed to identify miRNA targets. Hepatic interleukin-6 (IL-6) expression was significantly elevated in BA patients and BDL rats, while the expression of miR-124 was dramatically decreased in comparison to controls. Moreover, mRNA levels of STAT3 and IL-6 receptor (IL-6R) were inversely correlated with those of miR-124. Ectopic expression of miR-124 inhibited IL-6-mediated cholangiocyte proliferation in vitro and cholangiocyte hyperplasia in vivo, through a mechanism involving direct targeting of the 3'-untranslated region of STAT3 and IL-6R. We further demonstrated that miR-200 family members were significantly upregulated in cholestasis and inhibited FOXA2 expression in cholangiocytes, which further enhanced the expression of IL-6. Our findings suggest that downregulation of miR-124 and upregulation of miR-200 collaboratively promote bile duct proliferation through the IL-6/STAT3 pathway.

Computational identification of microRNAs and their targets in perennial Ryegrass (Lolium perenne).

MicroRNAs (miRNAs) are small non-coding RNA molecules of 22 nucleotides in length that have been characterized as regulators of messenger RNA (mRNA) regulating a number of developmental processes in plants and animals by silencing genes using multiple mechanisms. miRNAs have been extensively studied in various plant species; however, few information are available about miRNAs in perennial ryegrass, animal feed, and industrial raw materials. In this study, the 12 potential perennial ryegrass miRNAs were identified for the first time by computational approach. Using the newly identified miRNA sequences, the perennial ryegrass mRNA database was further used for BLAST search and detected 33 potential targets of miRNAs. Prediction of potential miRNA target genes revealed their functions involved in various important plant biological processes. Our result should be useful for further investigation into the biological functions of miRNAs in perennial ryegrass. The selected miRNAs representing four families were verified by RT-PCR experiment, indicating that the prediction method that we used to identify the miRNAs was effective.