ALKBH5‐mediated m6A demethylation of pri‐miR‐199a‐5p exacerbates myocardial ischemia/reperfusion injury by regulating TRAF3‐mediated pyroptosis

Abstract

AbstractMyocardial ischemia‒reperfusion injury (MI/RI) is closely related to pyroptosis. alkB homolog 5 (ALKBH5) is abnormally expressed in the MI/RI models. However, the detailed molecular mechanism of ALKBH5 in MI/RI has not been elucidated. In this study, rats and H9C2 cells served as experimental subjects and received MI/R induction and H/R induction, respectively. The abundance of the targeted molecules was evaluated using RT‐qPCR, Western blotting, immunohistochemistry, immunofluorescence, and enzyme‐linked immunosorbent assay. The heart functions of the rats were evaluated using echocardiography, and heart injury was evaluated. Cell viability and pyroptosis were determined using cell counting Kit‐8 and flow cytometry, respectively. Total m6A modification was measured using a commercial kit, and pri‐miR‐199a‐5p m6A modification was detected by Me‐RNA immunoprecipitation (RIP) assay. The interactions among the molecules were validated using RIP and luciferase experiments. ALKBH5 was abnormally highly expressed in H/R‐induced H9C2 cells and MI/RI rats. ALKBH5 silencing improved injury and inhibited pyroptosis. ALKBH5 reduced pri‐miR‐199a‐5p m6A methylation to block miR‐199a‐5p maturation and inhibit its expression. TNF receptor‐associated Factor 3 (TRAF3) is a downstream gene of miR‐199a‐5p. Furthermore, in H/R‐induced H9C2 cells, the miR‐199a‐5p inhibitor‐mediated promotion of pyroptosis was reversed by ALKBH5 silencing, and the TRAF3 overexpression‐mediated promotion of pyroptosis was offset by miR‐199a‐5p upregulation. ALKBH5 silencing inhibited pri‐miR‐199a‐5p expression and enhanced pri‐miR‐199a‐5p m6A modification to promote miR‐199a‐5p maturation and enhance its expression, thereby suppressing pyroptosis to alleviate MI/RI through decreasing TRAF3 expression.