Supplementary Material for: miR-590-3p Is a Novel MicroRNA in Myocarditis by Targeting Nuclear Factor Kappa-B in vivo

Objective: Nuclear factor kappa-B (NF-κB)-induced inflammation leads to myocarditis and heart dysfunction. How microRNAs (miRNAs) contribute to this process is poorly defined. The aim of this study was to investigate whether miRNAs regulate NF-κB-induced inflammation in experimental autoimmune myocarditis (EAM) in vivo. Methods and Results: NF-κB and its related proinflammatory genes, including interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a), were activated in EAM. Profiling of NF-κB-related miRNAs revealed that miR-590-3p was strikingly reduced in EAM. We found IL-6-induced proinflammatory signaling via miR-590-3p reduction, p50 induction, NF-κB activation and IL-6/TNF-a expression. Moreover, a luciferase reporter assay demonstrated that miR-590-3p directly interacted with the 3' UTR (untranslated region) of the p50 subunit, and that miR-590-3p overexpression inhibited p50 expression. Finally, miR-590-3p transfection through adeno-associated virus significantly inhibited p50 expression, suppressed NF-κB activity and blocked IL-6/TNF-a expression in vivo, reducing the lesion area and improving cardiac function in EAM. Conclusion: miR-590-3p is a novel NF-κB-related miRNA that directly targets the p50 subunit. This may provide a novel strategy for the treatment of myocarditis.