MiR-29a Suppresses The Growth And Metastasis Of Hepatocellular Carcinoma Via Targeting IFITM3

Objective: In this study,we investigated the expression of IFITM3 and miR-29 a in HCC and their effects on the biological behavior of HCC cells,as well as the association between IFITM3 and miR-29 a.Methods: The protein expression of IFITM3 in 52 HCC tissues and corresponding adjacent nontumor tissues were examined using immunohistochemistry(IHC)and western blotting analysis.To explore the expression of IFITM3 and miR-29 a in HCC tissues,qRT-PCR was performed for 52 HCC tissues and adjacent nontumor tissues,and then we analyzed the correlation between IFITM3 and miR-29 a expression levels and their correlation with the prognosis of HCC patients along with clinicopathologic characteristics.IFITM3 small interfering RNA(siRNA)fragments were constructed and transfected into HCCLM3 cells,and after transfection,the changes in mRNA and protein expression of IFITM3,migration and invasion ability,proliferative capacity,as well as apoptosis rate of the HCCLM3 cells were examined by qRT-PCR and Western blotting,cell wound-healing assay and Transwell,5-ethynyl-2’-deoxyuridine(EdU)proliferation assay,and flow cytometry,respectively.Then,miR-29 a mimics were also constructed and transfected into HCCLM3 cells.After transfection,the changes in the expression of miR-29 a and IFITM3 protein,migration and invasion ability,proliferative capacity,and apoptosis rate of the HCCLM3 cells were examined by qRT-PCR,cell wound healing assay and Transwell,EdU proliferation assay,and flow cytometry,respectively.The dual luciferase reporter gene assay was performed to verify whether IFITM3 was the direct target gene of miR-29 a.Results: IFITM3 is upregulated and miR-29 a downregulated in HCC tissues and that they are associated with poor prognosis of HCC patients,tumor size,tumor multifocal,and venous invasion.The expression of IFITM3 in HCC tissues was negatively correlated with miR-29 a expression.Knockdown of IFITM3 inhibited migration,invasion,proliferation and promoted apoptosis of HCC cells.After upregulated the expression of miR-29 a,the expression of IFITM3 in HCC cells was decreased firstly,and then the ability of migration,invasion and proliferation for HCC cells was declined,but apoptosis rate was increased.Subsequently,dual luciferase reporter gene assay revealed that the miR-29 a mimics could reduce the luciferase activities of the wild-type IFITM3 reporter vector(IFITM3-WT)rather than the mutant-type IFITM3 reporter vector(IFITM3-MUT),suggesting that IFITM3 is the target gene of miR-29 a.Conclusion: Our findings demonstrate that the migration,invasion,proliferation of HCC cells can be inhibited and apoptosis can be induced by miR-29 a via targeting IFITM3,and IFITM3 is the target gene of miR-29 a.Thus,our study indicates that miR-29 a and IFITM3 play critical roles in the development and progression of HCC,suggesting that mi R-29 a and IFITM3 may be novel potential therapeutic targets for patients with HCC.