MiR-3910 Promotes The Growth Of Hepatocellular Carcinoma Via Targeting MST1

ObjectiveTo study the expression pattern of miR-3910 in the hepatocellular carcinoma(HCC)and explore the functions and molecular mechanisms of miR-3910 in the progression of HCC.MethodsPart 1:The expression of miR-3910 in HCC and its adjacent tissues was detected by quantitative PCR(qPCR);and the transfection of kRasV12 in normal hepatocytes(L02)and HCC cells(Huh-7 and 7404)was performed,and the effects of kRasV12 on the expression of miR-3910 were evaluated.Part 2:The effects of miR-3910 expression on the growth of HCC cells were examined by MTT assay.The effects of miR-3910 expression on the migration of HCC cells were examined by Boyden chamber assay.The effects of miR-3910 expression on the anchorage-independent growth of HCC cells were examined by soft agar assay.The effects of miR-3910 expression on the metastasis of HCC cells were examined by the intrahepatic metastasis assay.Part 3:The three miRNA databases were used to predict the potential target gene of miR-3910.The expression level of miR-3910 and MST1 in HCC samples was compared,and the effect of the expression of miR-3910 on the functions of MST1 in apoptosis and colony formation of HCC cells was tested.ResultsPart 1:miR-3910 was up-regulated in HCC.1.The expression level of miR-3910 in HCC was higher than that in adjacent tissues.With U6 as the internal reference,the value of Cycle Threshold(Ct)between U6 and miR-3910 in the adjacent tissue was-18.7±0.4,and that in cancer tissues was-17.3±0.3.The difference between the average values of the two groups was significant(P<0.01).2.The lowest expression level of miR-3910 was observed in the normal hepatocytes(Chang and L02),and the expression of miR-3910 was increased in a series of HCC cell lines,especially Hep3B,HepG2,QGY and MHCC97.3.After the overexpression of Flag-kRasV12 in 7404 and Huh-7 cells,miR-3910 was up-regulated.The expression of miR-3910 in 7404 cells overexpressing FlagkRasV12 was up-regulated about 2-fold higher than the control cells,and that in Huh7 cells overexpressing Flag-kRasV12 was up-regulated about 6-fold higher than the control cells.Part 2:MiR-3910 promoted the malignant phenotypes of HCC cells.1.Overexpression of miR-3910 in 7404 and Huh-7 cells promoted the growth,migration and colony formation of HCC cells.The expression level of miR-3910 in the stable cell line was 6-12-fold higher than that in the control cell line.In the cell growth assay,the absorbance of miR-3910 overexpression group was about 1.5-fold higher than that of the control group.Statistical analysis showed that there was a significant difference in absorbance between the two groups.In the cell migration assay,the migratory cells in the overexpression group were 1.5-2.0-fold more than those in the control group.Moreover,compared with the control cells,7404 cells overexpressing miR-3910 formed more colonies on soft agar.2.The loss-function-of miR-3910 in 7404 and Huh-7 cells inhibited the growth,migration and colony formation of HCC cells.The loss-function-of miR-3910 inhibited the growth of 7404 and Huh-7 cells.The absorbance of the experimental group was about 60%～70%of that of the control group.Consistent with this,the loss-function-of miR-3910 resulted in a 50%decrease in the migration ability of 7404 and Huh-7 cells.In the colony formation assay,the loss-function-of miR-3910 significantly inhibited 7404 colonies on soft agar(P<0.01).3.In 7404,the loss-function-of miR-3910 weakened the metastasis ability of HCC cells in the liver of nude mice.There was no significant difference in liver size between the control group and the experimental group.Compared with the control cells,7404 cells could hardly form visible intrahepatic metastasis after the loss function of miR-3910.In the survival analysis,half of the mice injected with control cells died on the 40th day,and no mice in the experimental group died on the 40th day,which showed that the loss function of miR-3910 significantly prolonged the survival time of mice.Part 3:miR-3910 inhibited the expression of MST1 in HCC cells.1.The 3’-UTR region of MST1 matched the sequence of miR-3910.In 7404 and Huh-7 cells,miR-3910 down-regulated the protein level of MST1,while antimiR-3910 up-regulated the protein level of MST1.In HCC clinical samples,the mRNA abundance of miR-3910 reversely correlated with that of MST1.2.In 7404 and Huh-7 cells,miR-3910 activated YAP/TEAD reporter activity in a dose-dependent manner.Overexpression of miR-3910 up-regulated the protein levels of yap and downstream target genes(Cyr61 and cyclinE)in 7404 cells.However,overexpression of anti-miR-3910 down-regulated the protein levels of YAP and downstream target genes(Cyr61 and cyclinE).3.In annexin V/PI staining,overexpression of MST1 in 7404 cells promoted apoptosis,which could be reversed by overexpression of miR-3910.Similar phenomenon was observed in the colony formation analysis.Conclusions1.miR-3910 is up-regulated in HCC.kRasV12 can induce its expression.2.miR-3910 can promote the growth,migration and colony formation of HCC cells,and the loss-function-of miR-3910 can inhibit the growth,migration,colony formation and intrahepatic metastasis of HCC cells.3.miR-3910 can activate YAP transcription activity by targeting MST1.