Fig. 2: HIF-1α-responsive miR-214-3p inhibits PlGF expression by targeting its mRNA 3′-UTR.

a–d HTR-8/SVneo cells were transfected with control (C), HIF1A siRNA (siHIF1A), control miRNA (C), or miR-214-3p mimics (M), followed by exposure to normoxia or hypoxia in the presence or absence of IDF-11774 for 24 h. The levels of miR-214-3p (n = 4, a), PlGF mRNA (n = 4, b), PlGF protein (n = 4, c), and secreted PlGF (n = 10, d) were determined using qRT‒PCR, western blotting, or ELISA. e Cells were transfected with control siRNA (siC), Twist1 siRNA (siTwist1), or pGL3-WT or -truncated Dnm3os promoter (see Supplementary Fig. 3i), followed by exposure to normoxia or hypoxia. Luciferase activity in the cell lysates was assayed using an enzyme assay kit and normalized to the signal under normoxic conditions (n = 4). f Cells were transfected with siRNA for control (C), HIF1A, or Twist1, followed by exposure to normoxia or hypoxia. MiR-214-3p levels were determined using qRT‒PCR (n = 4). g Cells were transfected with psiCHECK-2-WT or mutant PlGF 3′-UTRs (Mut-S1, Mut-S2, and Mut-S3 represent mutants at sites 1, 2, and 3, respectively; Supplementary Fig. 3k) in combination with control siRNA (C), Twist1 siRNA, control miRNA (C), or miR-214-3p mimics (M), followed by exposure to normoxia or hypoxia. Luciferase reporter activities were determined in cell lysates using an enzyme assay kit (n = 4). The data are presented as the mean ± SEM. Statistical significance was determined using one-way (a–d, f) or two-way ANOVA (e, g), followed by the Holm–Sidak′s multiple comparisons test.