Figure 4.

Suppression of TIMP2 by hypoxia was mediated by HIF‐1α/miR‐210/HIF‐3α regulatory circuit in human HCCs. (A) Hypoxic suppression of TIMP2 was abolished upon HIF‐1α knockdown, but not HIF‐2α knockdown. mRNA levels of TIMP2 in shNTC, shHIF‐1α, and shHIF‐2α MHCC‐97L clones were determined by qPCR and normalized to HPRT (P < 0.050, t test; mean ± SD; n = 3). (B) miR‐210 inhibition suppressed the transcriptional activity of HIF‐1α. Luciferase reporter assay demonstrated that miR‐210 inhibition suppressed the activity of HRE‐luciferase reporter construct under hypoxia (P < 0.001, t test; mean ± SD). (C) qPCR analysis of TIMP2 levels in SMMC‐7721 cells, subject to transfection with negative control (NC) LNA inhibitor or miR‐210 LNA inhibitor and incubated under normoxia (20% O₂) or hypoxia (0.1% O₂; P < 0.010, t test). (D) In silico analysis (i) RNA hybrid and (ii) TargetScan 5.2 revealed a putative miR‐210 target sequence in the 3′UTR of HIF‐3α. This fragment was fused with the pmiRGLO vector to examine the binding by luciferase reporter assay. The mutated seed sequence is underlined. (iii) miRNA luciferase reporter assay. pmiRGLO luciferase reporter fused with two copies of wild‐type miR‐210 target sequences were transfected into BEL‐7402 cells after transfection with NC precursor or miR‐210 precursor. miR‐210 precursor significant suppressed luciferase activity of wild‐type 3′UTR of HIF‐3α (P < 0.001, t test; mean ± SD; n = 5). (E) Luciferase reporter assay demonstrated that ectopic expression of HIF‐3α ORF suppressed activity of HRE‐luciferase reporter construct under hypoxia (P < 0.001, t test; mean ± SD; n = 5). (F) Ectopic expression of HIF‐3α ORF suppressed hypoxic induction of mR‐210. Transcript levels of miR‐210 in SMMC7721 stably expressing the empty vector GFP or HIF‐3α ORF are shown. U6 was used as a housekeeping gene for data normalization (P < 0.010, t test; mean ± SD; n = 3). Abbreviations: GFP, green fluorescent protein; ORF, open reading frame; RLU, relative light units.