Fig. 3.
P4 affects the miR-200-ZEB contractile axis via direct induction of ZEB1 expression. (A) Diagram depicting P4 injection of mice in late gestation. IL, laboring myometrium; NIL, nonlaboring control. (B) miR-200b/429 are only modestly down-regulated by exogenous P4 treatment during late gestation. (C) ZEB1 but not ZEB2 mRNA is significantly increased by P4 treatment (same samples as in B). Expression of each miRNA/mRNA was determined by qRT-PCR, normalized to U6/GAPDH, and depicted as the fold increase over vehicle-treated controls. Mean ± SEM values are shown (Student’s t test, *P < 0.05; n = 7 mice per group). Data are representative of three similar experiments. (D) ZEB1 protein in myometrial nuclear extracts is increased by P4 treatment. β-actin was used as a loading control. Densitometry analysis of blots comparing P4-treated mice with vehicle controls revealed that P4 causes a significant increase in ZEB1 protein (Student’s t test, P < 0.05; n = 7 mice per group). (E) ZEB1 but not ZEB2 mRNA levels are induced in T74D cells treated with 10−7 M P4 for 12 or 24 h. Expression of each gene was determined by qRT-PCR, normalized to h36B4, and expressed as the fold increase over vehicle-treated control cells. Data are the mean ± SD values from three replicate experiments (Student’s t test, *P < 0.05, **P < 0.01). (F) P4 acting via PR induces ZEB1 promoter activity. HEK293 cells were transiently transfected with a ZEB1-Luciferase reporter construct containing 978 bp of the 5′-flanking sequence from the hZEB1 gene and with empty expression vector (control) or with a CMV expression vector containing the wild-type mouse PR-B isoform or PR-B containing a mutation in the DNA-binding domain (mutPR-BDBD). The cells were cultured with or without P4 (10−7 M) for 24 h, and luciferase activity was assayed, normalized to β-gal, and expressed as the fold increase over vector-transfected control cells. Data are the mean ± SD values from three replicate experiments (Student’s t test, *P < 0.05). (G) Overexpression of ZEB1 causes induction of ZEB2 mRNA expression. Primary murine myometrial cells infected with recombinant adenoviruses expressing CMV/ZEB1 manifested induction of endogenous ZEB2 mRNA after 72 and 96 h. Expression of ZEB2 was determined by qRT-PCR, normalized to GAPDH, and expressed as the fold increase over ZEB2 expression in cells transduced with β-gal–expressing adenoviruses. Data are the mean ± SD values from two replicate experiments (Student’s t test, *P < 0.05).