Figure 4

The role of miR-16 targeting MAP7 in the regulation of proliferation, apoptosis and cell cycle progression. (A and B) Efficient overexpression or knockdown of MAP7 expression. For knockdown of MAP7, siRNA against MAP7 and a scrambled control siRNA were transfected into A549 cells. For overexpression of MAP7, MAP7 overexpressing plasmid and an empty plasmid were transfected into A549 cells. Cells were harvested at 24 h post-transfection. MAP7 mRNA and protein levels were assessed by relative quantification RT-PCR (A) and Western blotting (B). Left panel: quantitative analysis; right panel: representative image. (C) MTT cell viability assay at 12, 24, 36, and 48 h after transfection of A549 cells with equal doses of pre-miR-control, pre-miR-16, anti-miR-control or anti-miR-16. (D) MTT cell viability assay at 12, 24, 36, and 48 h after transfection of A549 cells with equal doses of control siRNA or siRNA against MAP7 or equal doses of empty plasmid or MAP7 overexpressing plasmid. (E) MTT cell viability assay at 12, 24, 36, and 48 h after transfection of A549 cells with equal doses of pre-miR-control, pre-miR-16, or pre-miR-16 along with the MAP7 overexpressing plasmid. (F) A549 cells transfected with equal doses of pre-miR-control, pre-miR-16, control siRNA or siRNA against MAP7 were labeled with FITC-Annexin V/PI, and serum deprivation-induced apoptosis was measured by flow cytometry. (G) Quantification of the apoptotic cells in panel F. (H) A549 cells were transfected with equal doses of scrambled ncRNA or pre-miR-16 or equal doses of control siRNA or siRNA against MAP7. Cell cycle profiles were analyzed using flow cytometry. Shown in the panel are histograms of cell numbers (y axis) against DNA content (x axis) determined by measuring fluorescence intensity. (I) Quantification of the percentages of cells in the G0/G1, S, and G2/M phases in panel (H) (mean ± SD; ** p < 0.01; *** p < 0.001).