Targeting EZH1 and EZH2 contributes to the suppression of fibrosis-associated genes by miR-214-3p in cardiac myofibroblasts

Figures

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  Figure 1. MicroRNA-214-3p (miR-214-3p) expression in the fibrotic myocardium of a mouse Ang-II infusion model A. The systolic pressure measurement. B. The ratio of heart weight/tibia length (HW/TL). The scale bar was 2 cm.C. Masson trichrome stainning. The scale bar was 100 μm. D. Expressions of Col1a1, Col3a1 and α-SMA in mouse myocardium by Western blot assay. E. Expressions of mir-1, -133a, -133b, 16, -21, -214-3p and -29b in mouse myocardium by RT-qPCR assay. Data are shown as mean ± sem, *p < 0.05, **p < 0.01, ***p < 0.001. N = 5-8.

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  Figure 2. Phenotype of myocardial fibrosis of a mouse Ang-II infusion model with enforced expression of miR-214-3p A. Representative sections of mouse myocardium with Masson trichrome staining. The scale bar is 100 μm. B. Expressions of Col1a1, Col3a1 and α-SMA in mouse myocardium by Western blot assay. Data are shown as mean ± sem, *p < 0.05, **p < 0.01, ***p < 0.001. N = 5-8.

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  Figure 3. Expression of miR-214-3p in Ang-II-induced myofibroblasts and its effect on expressions of Col1a1, Col3a1 and α-SMA in myofibroblasts A. Expressions of Col1a1, Col3a1 and α-SMA in Ang-II-induced myofibroblasts by Western blot assay. B. Expression of miR-214-3p in Ang-II-induced myofibroblasts by RT-qPCR assay. C. Expressions of Col1a1, Col3a1 and α-SMA in miR-214-3p-modified myofibroblasts by FIHC assay. The scale bar is 100 μm. Data are shown as mean ± sem, *p < 0.05, **p < 0.01. N = 3.

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  Figure 4. MicroRNA-214-3p (miR-214-3p) negatively modulates EZH1 and -2 expression A. Verification of EZH1 and -2 as a target gene of miR-214-3p by the dual luciferase reporter system. Predicted miR-214-3p seed matches to the sequence in the 3′UTR of EZH1 and -2 mRNA. The seed sequence of miR-214-3p is CAGCAGG, and the complementary nucleotide sequences are shown in red words. Data are shown as mean ± sem, *p < 0.05, **p < 0.01 vs pGl3-promoter vector control, N = 3. B. EZH1 and -2 expression in Ang-II-induced myofibroblasts by Western blot assay. C. Determination of miR-214-3p level in miR-214-3p-modified myofibroblasts by RT-qPCR assay. MRNA and protein expressions of EZH1 D. and EZH2 E. in miR-214-3p-modified myofibroblasts by RT-qPCR and Western blot assay, respectively. Data are shown as mean ± sem, *p < 0.05, **p < 0.01, ***p < 0.001. N = 3.

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  Figure 5. Expressions of Col1a1, Col3a1 and α-SMA in mouse myofibroblasts with transfection of miR-214-3p mimic, EZH1 siRNA, EZH2 siRNA and PPAR-γ siRNA, or overexpression of EZH1 and -2, respectively A. Protein expression of EZH1, Col1a1, Col3a1, α-SMA and PPAR-γ in myofibroblasts with transfection of miR-214-3p mimic, EZH1 siRNA, respectively. B. Expression of EZH1, Col1a1, Col3a1, α-SMA and PPAR-γ in myofibroblasts with enforced expression of EZH1 by Western blot assay. C. Protein expression of EZH2, Col1a1, Col3a1, α-SMA and PPAR-γ in myofibroblasts with transfection of miR-214-3p mimic, EZH2 siRNA, respectively. D. Expression of EZH2, Col1a1, Col3a1, α-SMA and PPAR-γ in myofibroblasts with enforced expression of EZH2 by Western blot assay. E. Protein expression of PPAR-γ, Col1a1, Col3a1 and α-SMA in myofibroblasts with transfection of PPAR-γ siRNA. Data are shown as mean ± sem, *p < 0.05, **p < 0.01 vs scramble control, N = 3 in A., C., E. *p < 0.05, **p < 0.01 vs rAd-GFP control, N = 3 in B., D.

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  Figure 6. Up-regulation of microRNA-214-3p (miR-214-3p) in mouse myofibroblasts through NF-kB pathway A. Location of NF-κB P65 by FIHC assay. The scale bar is 50 μm. B. Activation of NF-kB signaling in Ang-II-treated myofibroblasts in a time-course study. MiR-214-3p expression in Ang-II-induced myofibroblasts with knockdown of P65 C., or with pre-treatment with NF-kB inhibitor JSH23 and QNZ, respectively D., was assessed by RT-qPCR assay. Data are shown as mean α sem, *p < 0.05, **p < 0.01, ***p < 0.001. N = 3.

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  Figure 7. Schematic diagram of the mechanism whereby miR-214-3p exerts the anti-fibrotic effect in cardiac fibrosis MiR-214-3p is upregulated in Ang-II-induced myofibroblasts via NF-κB pathway. MiR-214-3p enhances PPAR-γ expression by targeting EZH1 and -2, resulting in the suppressing the expression of fibrosis related genes, including Col1a1 and Col3a1, in myofibroblasts.