MiR-21 Induced Angiogenesis through AKT and ERK Activation and HIF-1α Expression
Figure 6
HIF-1α expression is required in miR-21 inducing VEGF expression and tumor angiogenesis.
(A) DU145 cells were transfected with pre-miR-21. After 24 h incubation, cells were infected with adenovirus carrying GFP (Ad-SCR) or siRNA against HIF-1α (Ad-siHIF-1α) at 20 MOI for 24 h. The total proteins were analyzed by immunoblotting. (B–C) The expression levels of VEGF and GAPDH mRNA were analyzed by RT-PCR (B), and by real-time RT-PCR (C). (D) After DU145 cells were transfected with pre-miR-21 for 24 h, the cells were infected with Ad-GFP or Ad-siHIF-1α at 20 MOI for 24 h. Then the cells were used to perform angiogenesis assay as described above. The picture showed the representative plugs from Ad-GFP and Ad-siHIF-1α treatment groups. Scale, 2 mm. (E) The number of blood vessels were counted from replicate experiments, and normalized to that of the Ad-siSCR group as relative angiogenesis. The data are mean ± SD from 6 replicate experiments. ##, indicates the significant difference when compared to that of the Ad-siSCR group (p<0.01).