Fig. 4.

Overexpression of miR-200b/429 mimics, ZEB1, and ZEB2 regulates contraction-associated genes and human myometrial cell contractility. (A) Transfection of hTERT-HM cells with miRmimics of miR-200b/429 causes a significant reduction in endogenous ZEB1 and ZEB2 within 24 h. (B) Primary murine myometrial cells were infected with recombinant adenoviruses expressing ZEB1, ZEB2, or β-gal (control); levels of miR-200b and miR-429 were analyzed by qRT-PCR 96 h posttransduction. (C) hTERT-HM cells were infected with recombinant adenoviruses expressing ZEB1, ZEB2, or β-gal (control); CXN-43 and OXTR mRNA levels were analyzed by qRT-PCR 48 h posttransduction. The levels of each mRNA/miRNA were normalized to h36B4/U6 and expressed as the fold change over control. Data are the mean ± SD values of three replicate experiments (Student’s t test, *P < 0.05, **P < 0.01). ChIP studies of endogenous ZEB1 binding to the promoters of its targets reveals robust binding to CXN-43 (D), OXTR (E), and the miR-200b-a-429 cluster (F). Binding of endogenous ZEB1 was determined by qPCR, normalized to input, and expressed as the fold increase over binding by IgG. Data shown are the mean ± SEM values of two replicate experiments (Student’s t test, *P < 0.05; n = 10 mice per group). Diagrammed above each graph are the locations of E-boxes within the gene promoter. (G) Overexpression of ZEB1 and ZEB2 in hTERT-HM cells inhibits oxytocin-mediated contraction. Cells were infected with recombinant adenoviruses expressing ZEB1, ZEB2, or β-gal, (control) and cultured for 48 h. These, as well as uninfected cells, were embedded into collagen gel matrices and treated with or without oxytocin (10 nM). Contraction of the gels was assessed after 24 h by measurement and calculation of mean gel area (mm²). A representative gel is shown below the data for each group. Data shown are the mean ± SD values from three replicate experiments (Student’s t test, *P < 0.05). (H) During pregnancy, P₄ and PR increase expression of ZEB1, which acts to suppress the miR-200 family as well as contraction-associated genes. Decreased expression of the miR-200 family relieves suppression of ZEB2 (as well as ZEB1), resulting in further down-regulation of contractile genes. Near term, a decrease in circulating P₄ and/or a decrease in PR function results in down-regulation of ZEB1 expression, and, in turn, up-regulation of the miR-200 family, further suppressing ZEB1 and ZEB2. This removes the brakes from contractile gene expression, resulting in increased uterine contractility and labor.