Regulation of HIF-1α and VEGF by miR-20b Tunes Tumor Cells to Adapt to the Alteration of Oxygen Concentration (original) (raw)
Figure 5
Differential expression of miR-20b affects different aspects of tumor cells.
(A) miR-20b was required for H22 cell growth. H22 cells were transfected with miR-20b inhibitor or miR-20b inhibitor + HIF-1α siRNA or miR-20b or VHL siRNA or control oligonucleotide for 24 hr. Then the cells were seeded in 96-well plate (5×103 per well) for 48 h in normoxia. The proliferation assay was performed with MTT Cell Proliferation Kit (Roche Diagnostics, IN) according to the manufacturer's instructions. *, P<0.05, compared with control. (B) Downregulation of miR-20b enhanced the resistance to apotosis. H22 cells were transfected with miR-20b inhibitor or miR-20b inhibitor + HIF-1α siRNA for 24 hr. Then the cells (1×106) were irradiated by UVB (200 J/m2) or treated with mitomycin C (MMC, 10 µg/ml)for 12 h. The cells were stained with PE-Annexin V and 7-AAD for apoptotic analysis by flow cytometry. *, P<0.05, compared with control. (C) Analysis of the mRNA expressions of Bcl-2, Bcl-xL, Bax and Bad genes. H22 cells were transfected with miR-20b inhibitor or miR-20b inhibitor + HIF-1α siRNA for 72 hr. The cells were used for the analysis of gene expression by real time RT-PCR. *, P<0.05, compared with control. (D) Normoxic H22 cells were transfected with miR-20b for 24 hr. Then the cells were cultured under hypoxic condition for 12 h, and stained with PE-Annexin V and 7-AAD for apoptotic analysis by flow cytometry. *, P<0.05, compared with control.