Figure 3

Overexpression of miR-302b and knockdown of miR-302b-target gene EGFR decrease hepatoma cell growth and induce G1/S arrest in vitro. A- qRT-PCR analysis of miR-302b in SMMC-7721 cells transfected with miR-302b over-expression construct and miR-ctrl (left). qRT–PCR (upper portion) and western blot analysis (lower portion) were performed to determine the expression level of EGFR after transfection of siEGFR and siRNA-ctrl. (*P < 0.05, **P < 0.01, Student’s t-test) (right). B- The effects of miR-302b or siEGFR on SMMC-7721 cell proliferation were determined by MTT assay at 24 h, 48 h, and 72 h after transfecting of miR-302b over-expression construct or siEGFR, with miR-ctrl or siRNA-ctrl as the respective controls (*P < 0.05, **P <0.01, Student’s t-test). C- Representative results of colony formation of SMMC-7721 after transfection of miR-302b re-expression or siEGFR. D- Cell cycles determined in SMMC-7721 cells after transfection of miR-302b over-expression construct or siEGFR at 24 h, 48 h, 72 h, with miR-ctrl or siRNA-ctrl as the respective controls. Histogram indicates the percentage of cells at G0–G1, S, and G2–M cell-cycle phases (*P < 0.05, Student’s t-test). E- Expression of Ki-67 was verified by immunofluorescence staining after transfecting with miR-ctrl, miR-302b, siRNA-ctrl, or siEGFR. Merged pictures are overlays of both Ki-67 red signals and nuclear staining by DAPI (blue).