Silencing of METTL3 attenuates cardiac fibrosis induced by myocardial infarction via inhibiting activation of cardiac fibroblasts

OBJECTIVE METTL3（N6-adenosinemethyltransferase 70 ku subunit）, a component of methyltransferase complex, participates in multiple biological processes associated with mammalian development and disease progression. However, the role of METTL3 in cardiac fibrosis is still unknown. METHODS Transforming growth factor（TGF）-β;(20 μg·m L;) were used to induce fibrotic phenotypes. Overexpressing plasmid or METTL3 siRNA were used to regulate METTL3 expression in cardiac fibroblasts. Lentivirus vector carrying si METTL3 gene were injected into healthy male C57 BL/6 mice by aortic clipping. Two weeks after injection, myocardial infarction（MI） was established by ligation of left anterior descending branch. Cardiac fibrosis was detected by Masson’s trichrome staining. Cardiac function was evaluated by echocardiography. Western blotting, Ed U fluorescence staining, quantitative reverse transcription-polymerase chain reaction were used to investigate the role of METTL3 in cardiac fibrogenesis. RNA sequencing（RNA-seq） and m6A sequencing（m6A-seq） were used to screen out the expression level and m6A level of collagen-related factors and fibrosis-promoting factors in fibroblasts after knockdown of METTL3. m6A me RIP-Seq was used to verify this result. Dot blot were used to analyze m6A methylation levels in cells effected by METTL3.RESULTS Compared to normal control group, the expression level of METTL3 was increased in cardiac fibrotic tissue of mice with chronic MI and cultured CFs treated with TGF-β（P<0.01）. Enforced expression of METTL3 remarkably increased the expression of collagensⅠ and Ⅲ in CFs at m RNA and protein expression levels（P<0.01, P<0.05）. In addition, overexpression of METTL3 significantly increased CFs proliferation（P<0.01） and collagens accumulation（P<0.01） and the activation of CFs. While silence of METTL3 existed the opposite effects（P<0.05）. In vivo, silence of METTL3 significantly alleviated cardiac fibrosis in MI mice（P<0.01） and improved cardiac function compared with non-treated MI mice（P<0.01）. Dot bot revealed a global increase of m6A levels in METTL3-treated cells and a significant decrease in si METTL3-treated cells. Transcriptome and N6-methyladenosine（m6A） profiling analyses revealed that the m6A profiles and cellular stability of genes related to cardiac fibrosis, especially the expression and m6A level of collagen-related genes, were altered after silence of METTL3（P<0.05）. CONCLUSION METTL3-mediated m6A modification is critical for the development of cardiac fibrosis, and might be a molecular target for manipulating fibrosis and the associated cardiac disease.