Figure 9
Effects of ANP/NPRA signaling and NPRA genotype on candidate microRNA levels in embryonic ventricles. (A–C) Expression analysis of miR-1a (A), miR-133 (B) and miR-27b (C) was performed on total microRNA isolated from E11.5 ventricular cell cultures treated with or without ANP (1 μg/ml) and NPRA inhibitor, A71915 (1 µM). MiR-1 and miR-133 target HCN4 mRNA and miR-27b regulates Cx40 mRNA levels. For all three miRNAs analyzed, addition of ANP significantly reduced expression of microRNAs, whereas addition of A71915 increased expression of microRNAs. N = 7 independent experiments. *P < 0.005 Vs. Cont, #P < 0.005 Vs. ANP or ANP + A71915, One-way ANOVA with Tukey’s post hoc test. (D–F) Expression analysis of miR-1a (D), miR-133 (E) and miR-208a (F) was also performed on total microRNA isolated from E14.5 ventricles of NPRA wild type (WT), heterozygous (HET) and knockout (KO) mice. Compared to WT mice, NPRA KO mice demonstrated upregulation of miR-1a (D) and miR-133 (E) and downregulation of miR-208a (F). There were no significant differences in miR-27b expression between genotypes. For all panels, microRNA expression levels were normalized using U6 levels via ∆∆CT method. Each bar represents mean ± SEM. N = 4 independent experiments. *P < 0.05 Vs. WT and HET, One-way ANOVA with Tukey’s post hoc test.
