A20 (TNFAIP3) alleviates viral myocarditis through ADAR1/miR-1a-3p-dependent regulation

Figures

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  Fig. 1. A20 is underexpressed in the myocardium of VMC mice. Notes: Male BABL/C mice were intraperitoneally injected with CVB3. Weight (A) and survival (B) of the mice; C H&E staining revealed the pathological changes in the myocardium; ELISA measured the serum levels of CK (D), CK-MB (E) and cTnI (F); qRT-PCR (G) and Western blotting (H) detected the expression of A20 in the myocardium. *P < 0.05; **P < 0.01; ***P < 0.001; VMC viral myocarditis, CVB3 Coxsackie virus B3, CK creatine kinase, CK-MB creatine kinase isoenzyme, cTnI cardiac troponin I

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  Fig. 2. A20 suppresses inflammation and apoptosis in VMC mice. Notes CVB3-infected mice were injected with LV-sh-A20, LV-sh-NC, LV-A20 or LV-NC in the tail vein. qRT-PCR (A) and western blotting (B) detected the expression of A20 in the myocardium; ELISA measured the serum levels of IL-6 (C), IL-18 (D) and TNF-α (E); F TUNEL staining assessed the cell apoptosis in the myocardium. *P < 0.05; **P < 0.01; ***P < 0.001; VMC viral myocarditis, CVB3 Coxsackie virus B3

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  Fig. 3. A20 suppresses inflammation and apoptosis of CVB3-infected cardiomyocytes. Notes qRT-PCR (A) and western blotting (B) detected the expression of A20 in primary neonatal rat cardiomyocytes and H9c2 cells. CVB3-infected primary neonatal rat cardiomyocytes and H9c2 cells were transfected with LV-sh-A20, LV-sh-NC, LV-A20 or LV-NC. qRT-PCR (C) and western blotting (D) detected the expression of A20 in the cells; ELISA measured the levels of IL-6 (E), IL-18 (F) and TNF-α (G) in the supernatant; H flow cytometry assessed the cell apoptosis. *P < 0.05; **P < 0.01; ***P < 0.001; CVB3 Coxsackie virus B3

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  Fig. 4. A20 is a target gene of miR-1a-3p. Notes qRT-PCR detected the expression of miR-1a-3p in the myocardium of VMC mice (A) and in CVB3-infected primary neonatal rat cardiomyocytes and H9c2 cells (B); C starBase predicted the binding sites between miR-1a-3p and A20; dual-luciferase reporter assay (D) and RIP assay (E) verified the binding between miR-1a-3p and A20. *P < 0.05; **P < 0.01; ***P < 0.001; VMC viral myocarditis, CVB3 Coxsackie virus B3

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  Fig. 5. miR-1a-3p promotes inflammation and apoptosis of CVB3-infected cardiomyocytes by targeting A20. Notes CVB3-infected primary neonatal rat cardiomyocytes and H9c2 cells were transfected with miR-1a-3p inhibitor, inhibitor NC, miR-1a-3p inhibitor + LV-sh-NC or miR-1a-3p inhibitor + LV-sh-A20. A qRT-PCR detected the expression of miR-1a-3p; qRT-PCR (B) and western blotting (C) detected the expression of A20; ELISA measured the levels of IL-6 (D), IL-18 (E) and TNF-α (F) in the supernatant; G flow cytometry assessed the cell apoptosis. *P < 0.05; **P < 0.01; ***P < 0.001; CVB3 Coxsackie virus B3

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  Fig. 6. ADAR1 promotes the expression of miR-1a-3p by binding to Dicer. Notes qRT-PCR and Western blotting detected the expression of ADAR1 in the myocardium of VMC mice (A, B), and in CVB3-infected primary neonatal rat cardiomyocytes and H9c2 cells (C, D); E Co-IP assay verified the binding between ADAR1 and Dicer; qRT-PCR (F) and Western blotting (G) detected the expressions of ADAR1, miR-1a-3p and A20 in primary neonatal rat cardiomyocytes and H9c2 cells that were transfected with LV-sh-NC, LV-sh-ADAR1, LV-NC or LV-ADAR1. *P < 0.05; **P < 0.01; ***P < 0.001; VMC viral myocarditis, CVB3 Coxsackie virus B3, Co-IP co-immunoprecipitation