Effects of lncRNA MIR31HG knockdown on adhesion,migration,invasion and EMT of hepatocellular carcinoma cells
Objective To investigate the effects of knocking down long non-coding RNA(lncRNA)MIR31HG on cell adhesion,migration,invasion and epithelial-mesenchymal transition(EMT)in hepatocellular carcinoma(HCC)and their mechanism.Methods Highly invasive HCC cell line MHCC97H,low invasive HCC cell line MHCC97L and normal human liver cell line LO2 were cultured in vitro,and the relative expression levels of lncRNA MIR31HG and miR-361 in each cell line were detected by RT-qPCR.HCC cells with high relative expression of lncRNA MIR31HG were selected for follow-up experiments.HCC cells were taken and randomly divided into the control group and knockdown group.Cells in the control group were transfected with NC siRNA,while cells in the knockdown group were transfected with MIR31HG siRNA.After 6-hour transfection,the medium was replaced with fresh medium and the culture continued for 48 h.RT-qPCR method was used to verify transfection efficiency.The transfected cells of the two groups were collected,and cell adhesion,migra-tion,and invasion abilities were detected by cell-matrix adhesion assay and Transwell chamber assay,respectively.The expression levels of EMT-related proteins such as E-cadherin,N-cadherin,and Fibronectin were detected by Western blot-ting.LncBase V.2 online prediction software was used to predict the targeting binding site of lncRNA MIR31HG and miR-361,and double luciferase reporter gene experiment was used to verify the targeted regulatory relationship between lncRNA MIR31HG and miR-361.Results The relative expression of lncRNA MIR31HG in MHCC97H and MHCC97L cells was higher than that in LO2 cells,and the relative expression of miR-361 was lower than that in LO2 cells(both P<0.05).The relative expression of lncRNA MIR31HG in MHCC97H cells was higher than that in MHCC97L cells,and the relative expression of miR-361 was lower than that in MHCC97L cells(both P<0.05).Therefore,MHCC97H cells were selected for follow-up experiments.The relative expression of lncRNA MIR31HG in the knockdown group was lower than that in the control group(P<0.05).The cell adhesion,invasion and migration abilities in the knockdown group were lower than those in the control group(all P<0.05).The expression of E-cadherin protein in knockdown group was higher than that in the con-trol group,and the expression of N-cadherin and Fibronectin protein was lower than that in the control group(both P<0.05).LncBase V.2 online prediction software predicted that there were target binding sites between the lncRNA MIR31HG and miR-361.Dual luciferase reporter gene assay confirmed that the luciferase activity of HCC cells transfected with MIR31HG WT + miR-361 mimics was lower than that of HCC cells transfected with MIR31HG WT + NC mimics(P<0.05).There was no significant difference in luciferase activity between HCC cells transfected with MIR31HG MUT + miR-361 mimics and those transfected with MIR31HG MUT + NC mimics(P>0.05).Conclusion The expression of lncRNA MIR31HG is high in HCC cells,and the knockdown of lncRNA MIR31HG expression can inhibit the adhesion,invasion,migration ability and EMT of HCC cells,which may be related to the targeted regulation of miR-361.
万方数据
维普
