Figure 4

HIF3α is regulated by miR-210-3p and miR-485-5p. (A) MiR-210-3p and miR-485-5p mimic overexpression reduce HIF3α protein induction under hypoxic conditions. The sarcoma cell lines SW872 and SK-UT-1 were transfected with mimics of miR-210-3p (m210), miR-485-5p (m485) and a scrambled negative control mimic (mneg). 48 h post-transfection cells were cultured under hypoxic conditions (1% O₂) for 24 h. Subsequently total protein lysates were prepared and analysed by Western blotting for HIF3α protein expression. β-Actin is used as a loading control. (B) Hypoxia responsive miRNAs target HIF3α 3’UTR. SW872 and SK-UT-1 cell lines were transfected with psiCHECK 2 constructs containing short and long 3’UTR fragments of HIF3α. 24 h later the cells were exposed to hypoxia for 24 h. Bars indicate average luciferase activity ± SD (n = 3) measured in hypoxic cell lysates relative to the luciferase activity in normoxic cell lysates which is arbitrarily set at 100. (C) MiR-210-3p and miR-485-5p regulate HIF3α. SW872 and SK-UT-1 cell lines were transfected with mimics of miR-210-3p (m210), miR-485-5p (m485) or a scrambled control mimic (mneg) followed after 24 h by a transfection with psiCHECK 2 constructs containing short and long fragments of the HIF3α 3’UTR. Bars indicate average luciferase activity ± SD (n = 3) measured in cell lysates after 24 h. (D) MiR-210-3p and miR-485-5p regulate HIF3α. SW872 and SK-UT-1 cell lines were transfected with mimics of miR-210-3p (m210), miR-485-5p (m485) or a scrambled control mimic (mneg) followed, after 24 h, by a transfection with a psiCHECK 2-HIF3α-short construct containing either wild-type (WT) or mutated (mut) miR-210-3p and miR-485-5p binding sites. Bars indicate average luciferase activity ± SD (n = 3) measured in cell lysates. Statistical significance was determined by two-sample t-tests: * = p < 0.05, ** = p < 0.005, *** = p < 0.0005.