Effects of miRNA-136 overexpression on S100 calcium binding protein A6 contents, proliferation and apoptosis in HCT116 cells

Abstract

Objective To investigate the effect of exogenous microRNA 136(mir136) on contents of S100 calcium binding protein A6(S100A6) and proliferation in human colon cancer cell line HCT116 through constructing a eukaryotic expression vector of mir136(pcDNA-mir136) and cationic liposome-mediated transfection.Methods Human genomic DNA was extracted from HEK293 cells and the DNA fragment containing mir136 precursor sequence was amplified by PCR and cloned into pcDNA to construct pcDNA-mir136.The plasmid was transfected into HCT116 cells via cationic liposome.The content of mir136 was determined by Real-time PCR,S100A6 protein was examined by Western blotting analysis,and apoptosis was detected by Flow Cytometry at 48 h after transfection.The proliferation ability of the tumor cells was examined by CCK-8 at 24 and 48 hours after transfection.Results The eukaryotic expression vector of mir136 was successfully constructed and transfected into HCT116 cells.Expression of mir136 was significantly higher in the transfected group than that in the untransfected group(P0.01) and the content of S100A6 protein was significantly lower than that of the untransfected group(P0.05) 48 h after transfection.Meanwhile,cell proliferation activity of the transfected group was significantly lower than that of the untransfected group(P0.05) and cell apoptosis was significantly increased compared with the untransfected group(P0.01).Conclusion Mir136 may inhibit proliferation and induce apoptosis of HCT116 cells by inhibiting the expression of S100A6 gene.