Figure 6: miR-421 is a transcriptional target of E2F1.

(a) Mouse miR-421 promoter region contains a potential E2F1-binding site. (b) E2F1 activates miR-421 promoter activity. Cardiomyocytes were treated with the adenoviral β-gal or E2F1, the constructs of the empty vector (pGL-4.17), the wild type promoter or the promoter with mutations in the binding site (mutant), respectively. Luciferase activity was assayed. Data are shown as mean±s.e.m. of four independent experiments. Analysis was performed with one-way analysis of variance (ANOVA) followed by Tukey–Kramer post hoc analysis. *P<0.05. (c) E2F1 promotes miR-421 expression. Cardiomyocytes were infected with adenoviral β-gal or E2F1. E2F1 expression was analysed by immunoblot (left panel). Quantitative analysis of expression levels of E2F1 protein shown in lower panel. MiR-421 levels were analysed by qRT–PCR (right panel). Data are shown as mean±s.e.m. of four independent experiments. *P<0.05 versus untreated group in Student’s t-test. (d) MiR-421 levels in E2F1-deficient mice. E2F1-knockout mice (KO), wild-type littermates (WT). Data are shown as mean±s.e.m. of six independent experiments. *P<0.05 versus WT in Student’s t-test. (e) ChIP analysis of E2F1 binding to the promoter of miR-421. (f) Knockdown of E2F1 attenuates the increase of miR-421 promoter activity induced by H₂O₂. Cardiomyocytes were treated with the adenoviral E2F1-siRNA or E2F1-sc, the constructs of the empty vector (pGL-4.17), the wild type (WT) promoter, then were treated with H₂O₂. Luciferase activity was assayed. Data are shown as mean±s.e.m. of three independent experiments. Analysis was performed with one-way ANOVA followed by Tukey–Kramer post hoc analysis. *P<0.05.