Figure 3.

Retn controls cardiac remodelling through regulation of miR148b-3p and Gadd45α. (A) Volcano plots showing differentially expressed genes (P < 0.05) in the hearts of the RKO-TAC vs. WT-TAC mice. Blue reflects downregulated and red upregulated genes. The dash line denotes significance level at P = 0.05. (B) Heat map of differentially expressed genes, red indicating gene-upregulated and blue indicating gene-downregulated. (C) Gadd45α mRNA and protein expression in groups of mice indicated (n = 3–4). (D) Western blot and real-time PCR analysis of the expression of Gadd45α in TAC-induced RKO mice hearts following AA9-empty or AAV9-mRetn overexpression (n = 3–4). (E) Bioinformatics analysis identified the Gadd45α gene as a putative target of miR148b-3p. Direct interaction was confirmed using dual-luciferase reporter assays (n = 3). (F) Real-time PCR analysis of the expression levels of miR148b-3p in sham and TAC-induced WT or RKO mice hearts for 10 weeks (n = 4). (G) Real-time PCR analysis of the expression levels of miR148b-3p in AAV9-empty or AAV9-mRetn overexpressing TAC-induced RKO mice hearts (n = 5). (H, I) Real-time PCR analysis of the expression levels of miR148b-3p and Gadd45α following adenovirus-encoding resistin (Ad.Retn) or Ad.β-Gal infection of cardiomyocytes (MOI 100) isolated from RKO mice (n = 3). (J) Western blot analysis and quantification (left panel), and real-time PCR verification (right panel) of Retn expression in cardiomyocytes (from H and I) following Ad.β-gal or Ad.Retn infection (MOI 100) (n = 3). Protein levels were normalized to GAPDH, a loading control; mRNA levels were normalized to 18S. N, each animal is shown as an individual point whereas horizontal lines represent median values. Values are shown as mean± S.E.M. with each experiment performed in biological and technical replicates as indicated. Significance was evaluated by Student’s t-test or one-way ANOVA with Tukey’s post-hoc test. P values <0.05 were considered significant.