The Role Of MiR-941 And Its Regulation Mechanism In Hepatoma Cells

Objective:Hepatocellular carcinoma(HCC)is one of the most common cancers in many parts of the world,The occurrence and development of genes is involving in the completion of the multi-step complex process.MicroRNAs(miRNAs)are a class of small non-coding RNAs that include 20-25nt nucleotides,which post-transcriptionally regulate gene expression.Recent evidence indicates that many miRNAs play important roles on cell proliferation,migration and invasion.Abnormal expression of miRNAs in tumor development are closely related human disease.We investigated the expression of miR-941 in hepatocellular carcinoma and its influence on biological behavior of hepatocellular carcinoma cells.We also identified its direct target gene and investigated its influence on biological behavior of hepatocellular carcinoma cells.Meanwhile,we identify that the silencing of miRNAs is tightly linked to the epigenetic mechanisms such as DNA methylation.To epigenetically silenced miRNAs is also illuminate in our study.Methods:Real-time PCR was used to detect the different expression of miR-941 in human hepatocellular carcinoma and adjacent normal tissues,and we also detect the different expression in six HCC cells and normal hepatocyte.With the f-unctions of miR-941 were enhanced or decreased in hepatocellular carcinoma cell lines QGY-7703 and HepG2,the changes of cell phenotypes were determined by MTT assay,colony formation assay,transwell migration and invasion assay.Subsequently,the candidate target gene of miR-941 was predicted by the bioinformatics analysis and was validated by EGFP reporter experiment.Furthermore,we used real-time PCR and immunofluorescence to detect the target gene on the mRNA level and protein level in hepatocellular carcinoma cells.then,the function of target gene was inhibited by RNA interference and the changes of cell phenotypes were detected with the above assays.Finally,we predicted the CpG island upstream of miR-941,and carried out BSP analysis to detect the methylation in the HCC cell lines,whice may have the relationship with gene silence.Results:Compared with normal hepatocellular tissues and cells,miR-941 is down-expressed in both of the hepatocellular carcinoma tissuesand cells.We found that overespression of miR-941 suppressed the proliferation activity,migration and invasion capability of QGY-7703 and HepG2 cells.Subsequently,the EGFP reporter experiment,Real-time PCR and immunofluorescence confirmed that miR-941 could directly bind to mRNA 3’UTR of a candidate target gene KDM6B and negatively regulated its expression.meanwhile,we found that proliferation activity,migration and invasion capability of QGY-7703 and HepG2 cells were reduced after knockdown of KDM6B.In contrast,overexpression of KDM6B can rescue the changes of cell phenotypes caused by ectopic expression of miR-941.Finally,we carried out BSP analysis and found that the CpG island upstream of miR-941 is significantly methylated in the HCC cell lines,whice induced the silence of gene.Conclusions:These results demonstrated that miR-941 is methylated and down-expressioned in hepatocellular carcinoma cells and tissues.miR-941 may function as a tumor suppressor that suppressed the proliferation activity,migration and invasion capability of hepatocellular carcinoma cells through negative regulation of KDM6B.