The Study On The Mechanism Of MiR-615-5p Regulates The Metastasis Of Hepatoma Cells

[object ive]MicroRNAs(miRNAs)are about 22-nucleotide,non-coding RNAs that are thought to regulate gene expression through sequence-specific base pairing with target mRNAs.MiRNAs play important roles in cell growth,proliferation,migration,invasion and adhesion.Recent reports indicates that many miRNAs function as oncogenes or tumor suppressors and are involved in cancer initiation and progression by regulating their target genes negatively.Meanwhile,more and more studies have shown that the down-regulation of several miRNAs is tightly linked to epigenetic mechanisms such as histone modification and DNA methylation.Hepatocellular carcinoma(HCC)is one of the five commonest solid tumor worldwide and the third leading cause of cancer-induced death after gastric cancer and esophageal cancer.Recent evidence show that changed expression or activity of specific genes are associated with the pathogenesis of HCC,including miRNA.We investigated the influence of miR-615-5p and its direct target gene on biological behavior of hepatocellular carcinoma cells and further investigated the functions and molecular mechanisms of miR-615-5p on hepatocellular carcinogenesis.[Methods]Real-time PCR and BSP analysis were used to detect that the CpG island is whether significantly methylated in the HCC cell lines tested.Next,through enforced or blocked expression of miR-615-5p in QGY-7703 cells,we detected the changes of cell phenotypes with in vitro scratch assay and vasculogenic mimicry assay(VM).And then,we established the HCC metastasis model by injecting the cells transfected with ASO-miR-615-5p to study the effect of miR-615-5p on metastasis of HCC in vivo.Then,the target gene was knocked down using RNA interference and changes of cell phenotypes were detected with in vitro scratch assay and vasculogenic minicry assay(VM).Subsequently,we further validated whether miR-615-5p directly regulated its target gene and whether miR-615-5p exerted its function through it.Finally,we analysied some related molecules with EMT and cell-matrix adhesion to further explore the possible regulation mechanism of miR-615-5p and its target gene in HCC metastasis and adhesion.[Results]BSP analysis indicate that compared with the control group,the methylation level of miR-615-5p promoter region is down-regulated in QGY-7703 treated with 5-Aza-CdR.After overexpression of miR-615-5p,the migration and vessel formation ability of hepatocellular carcinoma cell were enhanced.And downexpression of miR-615-5p got the opposite results.The intrahepatic metastasis nodules were significantly increased by ASO-miR-615-5p in vivo.Knockdown of Rab24 could inhibited the ability of migration and vessel formation.The results of western blot also found that enhanced expression of miR-615-5p or blocked expression of Rab24 could up-regulate the epithelial marker E-Cadherin whereas down-regulate the mesenchymal markers,Vimentin and ICAM-1 expression.In addition,up-regulation of miR-615-5p and down-regulation of Rab24 could reduce the expression of ?1-integrin which mediates cell-matrix adhesion.[Conclusions]Expression levels of miR-615-5p were regulated by DNA methylation of the promoter region in QGY-7703 cells.MiR-615-5p may function as a tumor suppressor that inhibited the the migration and vessel formation ability of hepatocellular carcinoma cells through negative regulation of Rab24.MiR-615-5p can suppress EMT process to affect cell mobility and suppress cell-matrix adhesion by regulating ?1-integrin in vitro.In conclusion,we demonstrated that miR-615-5p can inhibit migration and vessel formation ability of hepatocellular carcinoma cells through the downregulation of Rab24 and all of these will provide new understanding into the function and mechanisms of miR-615-5p in the metastasis of HCC and new theoretical clues for HCC.