miR-320 accelerates chronic heart failure with cardiac fibrosis through activation of the IL6/STAT3 axis

Figures

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  Figure 1. The expression of STAT3 in cardiomyocyte lines and miR-320 targeted-regulated STAT3. (A) The expression of STAT3 was low in standard samples (green) and high in case samples (red); P < 0.05. (B) Different expressed miRNA volcano map of standard group and case group in GEO dataset; (C) Venn diagram of predicted upstream, down-regulated, differently expressed miRNA for STAT3; (D) A heat map depicting miR-320 was higher in the cardiac fibrosis group, as compared with the controls; (D) GSEA pathway enrichment analysis results of STAT3; (E) Correlation analysis found that miR-320 was negatively correlated with KLF9 protein expression in cardiac tissues. (F) The relative expression levels of serum miR-320 were significantly elevated in patients with cardiac fibrosis. P < 0.05.

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  Figure 2. MiR-320 OE induced left ventricular (LV) hypertrophy and cardiac fibrosis. (A) Representative serial M-mode echocardiography in conscious sham and miR-320 OE and model mice measured before and after TAC; (B) Summary data of left ventricular internal dimension diastole (LVIDd), left ventricular internal dimension systole (LVIDs), left ventricular posterior wall dimensions diastole (LVPWd), left ventricular posterior wall dimensions systole (LVPWDs), and ejection fraction (E.F.). P < 0.05; miR-320 OE vs. model and sham vs. model.

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  Figure 3. MiR-320 OE accelerated cardiac function damage with significant cardiac fibrosis. (A) The histological assessment was performed to evaluate cardiac morphology between the model, miR-320 OE, and miR-320 OE + BP-1–102 samples. (B) Representative pictures of myocardial connective tissue Masson staining demonstrate cardiac fibrosis and fibrosis area. The quantitative analysis of fibrosis area/total areas, cardiocyte areas, and heart/weight ratio. P < 0.05.

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  Figure 4. MiR-320 OE increased the type I and III collagen synthesis; (Aa–d) Representative pictures of type I and type III collagen staining in the left ventricular tissues; (Ac–f) The expression levels of type I and type III collagen synthesis; P < 0.05; miR-320 OE vs. model; (Ba) Representative Western blots in the left ventricular tissues to show the collagenous fibers synthesis; (Bb) The quantitative analysis of type I and type III collagen; P < 0.05; miR-320 OE vs. model.

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  Figure 5. MiR-320 OE heightened the TGF-β, IL-6, t-STAT3, and p-STAT3 signaling and suppressed PTEN. (A) Representative Western blots in the left ventricular tissues; (B) The quantitative analysis of TGF-β, IL-6, p-STAT3, t-STAT3 and PTEN; P < 0.05; miR-320 OE vs. model.

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  Figure 6. PTEN was involved in miR-320 mimics-induced cardiac fibrosis. (A) Representative Western blots in fibroblasts with or without orexin B injection; (B) The quantitative analysis of TGF-β, IL-6, p-STAT3, and PTEN with or without orexin B injection; P < 0.05. (C) The proliferation of fibroblasts treated with miR-320 mimics, inhibitor or negative control (NC) was measured by CCK8 assay.

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  Figure 7. STAT3 was involved in miR-320 mimics-induced development of cardiac hypertrophy and fibrosis. (A) Western blots revealed that BP-1-102 significantly reversed the up-regulated STAT3, p-STAT3, type I and type III collagen, IL6, TGF-β, and down-regulated PTEN. (B) The quantitative analysis illustrated that inhibitor BP-1-102 exposure blunted the up-regulated type I and III collagen, IL6, TGF-β, p-STAT3, and STAT3 expression and enhanced the down-regulated PTEN expression in fibroblasts transfected with miR-320 mimic by BP-1-102. P < 0.05.