Fig. 5. ET-1/ETAR axis downregulates miR-200b/c via ZEB1.
a Expression of ZEB1 and ETAR proteins in HEY cells transfected for 48 h with siRNA control (si-Ctr) or si-ETAR or treated with MAC and stimulated or not for 48 h with ET-1 analyzed by WB. β-actin is used as loading control. b Luciferase activity in HEY cells co-transfected for 48 h with si-Ctr, si-ETAR, and the ZEB1 promoter reporter plasmid and stimulated with ET-1 and/or MAC for 24 h.Values are the mean ± SD expressed as fold induction (n = 3; *p < 0.0001 vs. si-Ctr; **p < 0.0001 vs. ET-1 stimulated si-Ctr). c qRT-PCR for miR-200b/c expression in HEY cells transfected with si-Ctr or si-ZEB1 and stimulated with ET-1 for 72 h in the absence or in the presence of MAC. U6 is used to normalize. Values are the mean ± SD (n = 3; *p < 0.001 vs. unstimulated Ctr; **p < 0.001 vs. ET-1-stimulated Ctr). d Pri-miR-200b/c expression in cells transfected and treated as in c for 48 h is evaluated by qRT-PCR and normalized to cyclophilin-A. Values are the mean ± SD (n = 3; *, p < 0.01 vs. unstimulated Ctr; **p < 0.001 vs. ET-1-stimulated Ctr). e Schematic representation of the ZEB1 binding sites contained in the miR-200b/c promoter reporter plasmids. f Luciferase activity in HEY cells co-transfected for 48 h with si-Ctr, si-ETAR, or si-ZEB1 and the reporter plasmids for miR-200b/c promoters and stimulated or not with ET-1 and/or MAC for 24 h. Values are the mean ± SD expressed as fold induction (n = 3; *p < 0.001 vs. unstimulated Ctr; **p < 0.001 vs. ET-1-stimulated Ctr).