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. 2020 Nov 13;3:677. doi: 10.1038/s42003-020-01404-3

Fig. 4. ZEB1 regulates ETAR expression through the repression of miR-200b/c.

Fig. 4

a, b Expression of miR-200b/c and ETAR, ZEB1 proteins in HEY cells transfected for 72 h with siRNA control (si-Ctr) or si-ZEB1 as determined by qRT-PCR (a) and WB (b). U6 and β-actin are used to normalize miRNA and protein expression, respectively. Values are the mean ± SD (n = 3; *p < 0.0001 vs. Ctr). c, d Luciferase activity (c) in HEY cells co-transfected for 48 h with the indicated mimic-miRNAs, in the presence or in the absence of ZEB1 plasmid together with the ETAR 3′UTR reporter plasmid. qRT-PCR (d) of the miR-200b/c expression in cells transfected as above and normalized to U6. Values are the mean ± SD expressed as fold induction (n = 3; *p < 0.01 vs. Ctr; **p < 0.001 vs. ZEB1-transfected cells). e Expression of ETAR and ZEB1 proteins in lysates from cells treated as in c analyzed by WB. β-actin is used as loading control. f, g Luciferase activity (f) in HEY cells co-transfected for 48 h with the indicated anti-miRNAs, in the presence or absence of si-ZEB1 together with the ETAR 3′UTR reporter plasmid. qRT-PCR (g) of the miR-200b/c expression, in cells transfected as above and normalized to U6. Values are the mean ± SD expressed as fold induction (n = 3; *p < 0.01 vs. Ctr; **p < 0.001 vs. si-ZEB1-transfected cells). h Luciferase activity in HEY cells co-transfected for 48 h with si-Ctr or si-ZEB1 together with the reporter plasmid containing the 3’UTR region of ETAR (ETAR 3′UTR) or its triple mutant (mut/ΔETAR 3′UTR). Values are the mean ± SD expressed as fold induction (n = 3; *, p < 0.001 vs. ETAR 3′UTR-transfected Ctr cells). i Schematic model of the ETAR/ZEB1 and miR-200b/c feedback circuit.