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. 2016 Jun 3;17(8):790–798. doi: 10.1080/15384047.2016.1195040

Figure 3.

Figure 3.

miR-147a upregulates HIF-1α via targeting HIF- 3α. (A) miR-147a directly targets the 3′UTR of HIF-3α. Predicted binding sequences between miR-147a and seed matches in HIF-3α 3′UTR. Luciferase reporter assay of wild type (WT) or mutated (MUT) HIF-3α 3′UTR vector co-transfected with NC, miR-147a respectively in normoxia (21%O2). Data shown are mean ± SD of 3 independent experiments, *P < 0.05. (B) Western blot analysis of HIF-3α and ACTB proteins in HeLa cells transfected with NC or miR-147a in normoxia (21%O2). Relative HIF-3α/ACTB ratios were determined by Image J densitometric analysis. Data shown are mean ± SD of 3 independent experiments, *P < 0.05. (C) Western blot analysis of HIF-3α and ACTB proteins in HeLa cells transfected with ant-miR-147a or anti-NC in hypoxia (1% O2). Relative HIF-3α/ACTB ratios were determined by Image J densitometric analysis. Data shown are mean ± SD of 3 independent experiments, **P < 0.01. (D) Overexpression of miR-147a doesn't effect the mRNA level of HIF-3α. HeLa cells were transfected with NC, miR-147a. Cells were harvested at 48h in normoxia (21%O2) for qRT-PCR. N.S., not significant. (E) HIF-1α accumulates when HIF-3α expression is inhibited. HeLa cells transfected with NC or 2 designed HIF-3α siRNAs in hypoxia (1% O2). Western blotting was performed to analyze the protein levels of HIF-1α and ACTB. (F) Relative HIF-1α / ACTB ratios from (E) were determined by Image J densitometric analysis. Data shown are mean ± SD of 3 independent experiments, *P < 0.05, student 2-tailed t test. (G) miR-147a stabilized HIF-1α through HIF-3α. HeLa cells transfected with HIF-3α siRNA firstly and then introduced miR-147a into the cells in hypoxia (1% O2). Western blot analysis of HIF-1α and ACTB proteins.