Fig. 3.

Identification of Sufu as the target gene of miR-214-3p during TNFα-induced iSMC differentiation from AdSPCs. a, b Sufu is closely modulated during iSMC differentiation from AdSPCs. c, d miR-214-3p over-expression in iSMCs inhibits Sufu gene expression. e, f Upregulation of Sufu by miR-214-3p inhibition. AdSPCs were differentiated into iSMCs for 4 days, then transfected with miR-214-3p inhibitor or negative control (miR ctrl) and cultured for 48 h in the same culture medium. Total RNAs and proteins were harvested and subjected to RT-qPCR (e) and Western blot (f) analysis, respectively. g The binding site is required for miR-214-3p-mediated Sufu gene repression. The potential wild-type binding site (WT) for miR-214-3p within Sufu gene 3′UTR and its mutant (BS^mut) are depicted in this illustration (left). miR-214-3p mimics or negative control (miR ctrl) was co-transfected into iSMCs with wild-type Sufu 3′UTR reporter (WT) or miR-214-3p binding site mutants [miR-214BS^mut], respectively. Luciferase activity assay was measured at 48 h post-transfection. The data presented here are representative (left panels in b, d and f) or mean ± S.E.M. of five (a, c, e and right panels in b, d and f, n = 5) or eight (g, n = 8) independent experiments. *P < 0.05 (vs vehicle/miR ctrl); ^#P < 0.05 (TGFβ1/TNFα vs TGFβ1 or TNFα/miR-214 vs TNFα/miR ctrl). (One-way ANOVA with a post hoc test of Tukey’s analysis for a–d; unpaired t test for e–g). miR-214 indicates miR-214-3p