The Mechanism Of Mettl16-mediated Sulf2 M~6A Modification In Pulmonary Microvascular Injury In PM2.5-induced COPD Rats

ObjectiveEpigenetic changes affect the development of various human diseases,classical epigenetic studies include DNA methylation,chromatin remodeling and histone modification.With the development of technology,various RNA modifications have attracted people’s attention and become a hot spot in epigenetic research.N6methyladenosine(m6A)is the most abundant RNA chemical modification of eukaryotic messenger RNA(mRNA),regulating the metabolism of mRNA.The previous study of the research group found that m6A methyltransferase Mettl16 was involved in lung microvascular injury in rats with chronic obstructive pulmonary disease(COPD)induced by atmospheric particulate matter(PM2.5),and the target gene Sulf2 of Mettl16 was predicted by RNA transcriptome sequencing and methylation sequencing of lung tissue of modeled rats,combined with bioinformatics,but the specific mechanism has yet to be elucidated.This study aims to explore the specific mechanism of Mettl16 and Sulf2 in PM2.5-induced pulmonary microvascular endothelial cell(PMVEC)damage in COPD,and provide a theoretical basis for the elucidation and treatment of COPD pulmonary microvascular injury.MethodsIn order to determine the relationship between Mettl16 and lung microvascular endothelial cells,the expression and localization of Mettl16 and CD31 were detected by immunofluorescence in lung tissue in a rat model with 6 months of biofuel(BM)exposure.In order to prove that the high expression of Mettl16 after PM2.5 exposure damages lung microvascular,lentivirus infection with Mettl16 overexpressing PMVEC cells was detected by FITC dextrose fluorescence leakage method,and angiogenesis was detected by angiogenesis experiment.In order to explore whether Sulf2 is involved in the pathological process of COPD,the expression of Sulf2 and CD31 was detected by immunofluorescence in lung tissue of rat model with BM exposure for 6 months.Western Blot technology measures the protein expression level of Sulf2 in its lung tissue;qPCR detects the RNA expression level of Sulf2 at different time points of PM2.5-treated PMVEC cells.In order to explore whether Sulf2 affects the function of lung microvascular endothelial cells,lentivirus infected PMVEC cells that overexpressed Sulf2 were detected by FITC dextrose fluorescence leakage method,and angiogenesis was detected by angiogenesis experiment.To determine whether Mettl16 regulates methylation levels in vascular endothelial cells,the m6A methylati on kit was used to measure the m6A level of PMVEC cells after knocking out Mettl16.To confirm whether Sulf2 is only regulated by the m6A methylase Mettl16,qPCR detects the RNA expression level of Sulf2 after knocking out Mettl3 or Mettl14.qPCR and Western Blot techniques detect RNA and protein expression levels of Sulf2 after knocking out Mettl16.In order to explore the specific mechanism of Mettl16 affecting Sulf2 expression,the binding of Mettl16 and Sulf2 was detected by double luciferase reporter gene experiment and RNA co-immunoprecipitation experiment.PMVEC cells after knocking out Mettl16 were treated with Actin-D to detect Sulf2 RNA stability.MeRIPqPCR detects the Sulf2 RNA m6A modification site in PMVEC cells after knocking out Mettl16.ResultsThe results of immunofluorescence experiments in lung tissue in rat model with BM exposure for 6 months showed that the endothelial cell marker CD31 decreased and the expression of Mettl16 increased after BM exposure.PMVEC cells overexpressing Mettl16 have impaired function;The results of immunofluorescence experiments and Western Blot experiments in rat models with BM exposure for 6 months showed that the expression of Sulf2 protein decreased after BM exposure.The results of qPCR experiments showed that the expression level of Sulf2 RNA in PMVEC cells decreased with the treatment time of PM2.5,and overexpression of Sulf2 improved the function of PMVEC cells.The results of m6A methylation test showed that the m6A level of PMVEC cells decreased after knockout Mettl16.The results of qPCR experiments showed that the RNA level of Sulf2 was not affected after PMVEC cells knocked out Mettl3 and Mettl14.The results of Western Blot experiment and qPCR experiment showed that the protein and RNA expression levels of Sulf2 in PMVEC cells that knocked out Mettl16 increased.The results of double luciferase reporter gene detection and RNA co-immunoprecipitation showed that Mettl16 and Sulf2 3’UTR region were bound to each other,and the RNA stability experiment showed that knocking out Mettl16 increased the stability of Sulf2 RNA.MeRIP-qPCR assay results showed that Mettl16 modified Sulf2 RNA 3’UTR m6A recognition sequence of one A base.ConclusionIn the process of PM2.5-induced COPD pulmonary microvascular injury,the expression level of Mettl16 protein in pulmonary microvascular endothelial cells increased,thereby enhancing the methylation modification of Sulf2 mRNA,affecting the stability of Sulf2 mRNA,downregulating the expression of Sulf2 protein,impairing the function of lung microvascular endothelial cells,leading to the occurrence of pulmonary microvascular injury.This study revealed the specific mechanism of m6A modification involved in COPD pulmonary microvascular injury,which laid a theoretical foundation for future drug development and clinical treatment.