Figure 6.

Knockdown of miR-125b induces cell apoptosis independent on p53 expression. (A) Effect of UV radiation of p53 expression in U251 (p53 mutation) and U87 with p53 wild-type and p53 knock-down [U87(p53KD)] cells. (B) Downregulation of miR-125b activates p53 expression in p53 wild-type U87 cells but not in U251 and U87(p53 KD) cells. (C) Effect of 125b-AS on the proliferation of glioblastoma cell lines. 125b-AS was transfected into cells and the cell proliferation inhibitory rates were evaluated by MTT assay at 12, 24, 36, 48 and 72 h, respectively. The inhibitory rates of cells were calculated using the formula: the OD value in cells transfected with 125b-AS /to the OD value in cells transfected with NC-AS × 100%. (D) Observation of nuclear morphology. Cells were stained with Hoechst 33342 and nuclear morphology was observed under a fluorescence microscope. The white arrows showed the apoptosis cells with condensed, fragmented nuclei. (E) Quantities for the apoptosis cells with Annexin V-FITC/PI stain and Flow cytometry analysis. U251, U87 and U87(p53 KD) cells were transfected with NC-AS or 125b-AS. After incubation for 72 h, the cell apoptosis rates were detected by Annexin V-FITC/ PI stain and Flow cytometer analysis. The two-tail Student's t-test was employed to statistically analyse the results: **P<0.01. (F) Effect of 12b-AS on caspases expression in U251, U87 and U87(p53 KD) cells. Cells were treated with 125b-AS. After incubation for 72 h, cells were collected and the expression of caspase-9, cleaved caspase-9, caspase-3 and cleaved caspase-3 were detected by western blot. The expression of β-actin was used as a loading control.