miR-338-3p suppresses HIF-1α expression by directly targeting the <i>HIF1A</i> 3′UTR.

<p>(A) Predicted miR-338-3p target sequences in the <i>HIF1A</i> 3′UTR. (B) Western blot analysis of HIF-1α levels in NC- or miR-338-3p-transfected cells. β-actin was used as loading control. Cells were cultured under hypoxia at two days post-transfection with cells cultured under normoxia as reference, and 24 h later protein levels were analyzed. (C) Relative miR-338-3p levels in miR-338-3p mimic transfected HCC cells. Transcript levels were normalized to <i>U6</i> expression. n = 4. (D) Six nucleotides (red) of HIF 3′UTR were mutated to prevent binding with miR-338-3p. (E) Luciferase reporter assay of cells transfected with the wild type (wt) or mutant (mut) <i>HIF1A</i> 3′UTR luciferase reporter plasmid with increasing amounts (20 to 50 nM) of negative control miRNA (NC) or miR-338-3p in HCC cells two days post-transfection; n = 4. **p≤0.01 compared to NC group. Data are shown as mean ± SEM of three independent experiments.