Fig. 2.

Downregulation of HIF-1α by miR-18, −200c and − 549. a and b – miR-18 (a) and miR-549 (b) downregulate HIF-1α by targeting predicted binding sites in the 3′-UTR of HIF-1α. Luciferase activity in NC-transfected cells was set at 100%. ***p < 0.001. c – Luciferase activity decreased following co-transfection with miR-200c and wild-type 3′-UTR-luciferase plasmid. d – A549 cells were transfected with miRNA mimics and siRNA against HIF-1α (20 nM). The next day, cells were treated with CoCl₂ (200 μM). After 24 h, total RNA was isolated and subjected to quantitative RT-PCR. (−): no treatment with CoCl₂; (+): treatment with CoCl₂. ***p < 0.001. e – A549 cells were transfected and treated with CoCl₂ as indicated in (d). After treatment with CoCl₂, western blot was performed using total cell lysate. f – The transcriptional activity of HIF-1α was measured after co-transfection of cells with the HRE-luciferase plasmid, pGL4.75 plasmid and miRNA mimics. The luciferase activity was assayed 48 h after transfection. -, no treatment with CoCl₂; +, treatment with CoCl₂. ***p < 0.001. g and h – Downregulation of HIF-1α by miR-200c in hypoxia. The mRNA and protein levels of HIF-1α are downregulated by miR-200c in A549 (g) and NCI-H460 (h) lung carcinoma cells under hypoxic condition. N; normoxia, H; hypoxia. siR2210 was used to silence HIF-1α in the western blot and quantitative RT-PCR analyses. In quantitative RT-PCR, the mRNA level of HIF-1α obtained from NC-transfected cells in normoxia was set as 1. **p < 0.01, ***p < 0.001. i – Downregulation of downstream target genes by miR-200c in hypoxia. The mRNA level of downstream target genes in A549 cells was assessed using quantitative RT-PCR after transfection with miR-200c and siR2210. N; normoxia, H; hypoxia. The mRNA level of target genes from NC-transfected cells in normoxia was set as 1. *p < 0.05, **p < 0.01, ***p < 0.001