Figure 3

miR-744 directly targets and regulates c-Myc expression in HCC cells. (A) Wild-type (WT) and mutant (Mut) of putative miR-744 targeting sequences in c-Myc mRNA. Mutant sequences were shown in bold type. (B) Analysis of luciferase activity. HEK-293 cells were cotransfected with miR-744 mimics or control oligo nucleotides, pRL-TK and firefly luciferase reporter plasmid containing putative miR-744 targeting sequences of c-Myc. pRL-TK was cotransfected as an internal control to correct the differences in both transfection and harvest efficiencies. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity. The normalized luciferase activity control oligo nucleotides was set as relative luciferase activity 1 respectively. (C) Effects of miR-744 mimics on the endogenous c-Myc mRNA levels. HepG2 and SMMC-7721 cells were cotransfected with miR-744 mimics or negative control oligonucleotides. Forty-eight hours after transfection, cells were isolated, the expression of c-Myc was analyzed by qRT-PCR. (D) Effects of miR-744 on the endogenous c-Myc protein levels. Forty-eight hours after transfection with the miR-744 mimics or negative control oligonucleotides, HepG2 and SMMC-7721 cells were analyzed by Western blotting, with β-actin as an internal control. The value up each lane indicates the c-Myc expression in oligonucleotides-transfected cells compared with mock-transfected (with only transfection reagent) cells. The data were subjected to Student's t-test. *p < 0.05, **p < 0.01. NC, negative control oligonucleotides.