Expression Of MiR-186 And Its Role In Hepatocellular Carcinoma

Objective: To investigate the expression level of miR-186 in hepatocellular carcinoma and study its biological characteristics of HCC cell lines.Methods: qRT-PCR were performed to analyze the expression level of miR-186 in HCC tissue and adjacent non-tumor tissue.Simultaneously,qRT-PCR were performed to analyze the expression of miR-186 in four hepatoma cell(hepG2,Hep3 B,SMMC-7721,BEL-7402)and one normal hepatocyte cell(HL-7702).The eukaryotic expression vector of miR-186 was transfected into SMMC-7721 and BEL-7402 cells by using EndoFectionTM-Max,and then the effect of miR-186 on the cell proliferation,cell apoptosis,cell migration and invasion was detected by CCK-8 assay,flow cytometry technique,transwell migration and invasion assay.In addition,further verify the effect miR-186 on target genes by using qRT-PCR and Western blot.Results: The expression of miR-186(0.0342±0.0333)in the cancer tissues was significantly lower than that(0.0769±0.0559)in the adjacent tissues(p<0.001);meanwhile,it shows that the expression of miR-186 is related to tumor size and clinical TNM stage by analyzing the relationship between miR-186 expression and clinicopathological factors.The relative expression level of miR-186 in HCC cells was significantly lower than that in normal cell,especially in SMMC-7721 and BEL-7402(p<0.01 or p<0.001).CCK-8 assay showed that the cell proliferation rate of HCC cells transfected with miR-186 mimic was lower than that of HCC cells transfected with miR-186 mimic control,while the cell proliferation rate of HCC cells transfected with miR-186 inhibitor was higher than that of HCC cells transfected with miR-186 inhibitor control(P<0.05 or P<0.01).Flow cytometric analysis indicated that the cell apoptosis rate of HCC cells transfected with miR-186 mimic was higher than that of HCC cells transfected with miR-186 mimic control,while the cell apoptosis rate of HCC cells transfected with miR-186 inhibitor was lower than that of HCC cells transfected with miR-186 inhibitor control(P<0.05 or P<0.01)(P<0.05 or P<0.01).Transwell migration and invasion assay presents that the number othrough transwell chamber of HCC cells transfected with miR-186 mimic was lower than that that of HCC cells transfected with miR-186 mimic control,while the number othrough transwell chamber of HCC cells transfected with miR-186 inhibitor was higher than that that of HCC cells transfected with miR-186 inhibitor control(P<0.05 or P<0.01).Further,qRT-PCR and Western blot confirmed that the expression of mRNA and protein level of ROCK-1 in HCC cells transfected with miR-186 mimic was lower than that in HCC cells transfected with miR-186 mimic control.In contrast,the expression of mRNA and protein level of ROCK-1 in HCC cells transfected with miR-186 inhibitor was higher than that in HCC cells transfected with miR-186 inhibitor control.(P<0.05).Conclusion: miR-186 is down-regulated in HCC tissue and in HCC cells.miR-186 can inhibit the cell proliferation,migration and invasion and promote the cell apoptosis of HCC cells in vitro.It indicates that miR-186 acts as tumor suppressor and the effect is possibly by suppressing ROCK1 expression.