Figure 3. miR-182 regulates PHD2 and FIH1 expression by directly targeting their 3′-UTR.

(a,b) The sequences of predicted and conserved miR-182 binding sites in PHD2 3′-UTR (a) and FIH1 3′-UTR (b). The binding site is conserved across species. The predicted binding sites for miR-96 and miR-183 are also shown. (c) Western blot analysis of PHD2 and FIH1 protein expression in DU145 and PC-3 cells transfected with miR-182 mimics, control oligos (NC), miR-182 antagomir (anti-182), or antagomir control (anti-NC) as indicated. (d,e) RT-PCR analysis of PHD2 and FIH1 expression in DU145 (d) and PC-3 (e) cells transfected with miR-182 mimics and control oligos (NC). (f) miR-182 significantly inhibits PHD2 and FIH1 3′-UTR reporter luciferase activity in 293T cells. Different amount of miR-182 mimics (4 nM, 10 nM) or control oligos (NC) were co-transfected with reporter plasmids as indicated. (g) miR-182 suppresses activity of the reporter containing the 3′-UTR of PHD2 or FIH1 in DU145 prostate cancer cells. (h) Luciferase reporter containing wild type or mutant 3′-UTR of PHD2 and FIH1 was co-transfected into DU145 cells with miR-182 or control oligos (NC). 48 hours after transfection, luciferase activity was measured. (i,j) Cellular hypoxia was induced in DU145 cells for different times as indicated. HIF1α, PHD2, and FIH1 protein levels were determined by western blot analysis (i). miR-182 expression was analyzed by RT-PCR (j, n = 3). Data are mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01. Full-length blots are presented in Supplementary Fig. S6.