Figure 4

MiR-20a directly regulates Mcl-1 expression. (A) Wild-type and mutant of putative miR-20a target sequences of Mcl-1 3^′UTR. (B) MicroRNA luciferase reporter assay. Wild type and mutant miR-20a target sequences were fused with luciferase reporter and cotransfected with miR-20a precusor or control oligo into HEK293T cells. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity. MiR-20a significantly suppressed the luciferase activity of wild-type Mcl-1 3^′UTR (p = 0.027). (C) Effects of miR-20a overexpression on the level of cellular Mcl-1 in HepG2 and SMMC-7721 HCC cells without transfection or cells transfected with NC or miR-20a were analyzed by western blot. (D) Analysis of Mcl-1 and miR-20a expression in the same HCC tissue by IHC. Brown signal in IHC was considered as positive staining for Mcl-1. Scale bar = 200 μm.