METTL16 Regulates The Pulmonary Microvascular Injury Induced By PM2.5 Through M6A And Participates In The COPD Process

Objective:Chronic obstructive pulmonary disease(COPD)is a common,preventable and treatable disease characterized by persistent respiratory symptoms and airflow limitation caused by airway and alveolar abnormalities resulting from significant exposure to harmful particles or gases.The WHO predicts that COPD will become the third leading cause of death by 2030.Current treatment options for COPD are limited,only partially alleviating symptoms,and have not yet been shown to alter the progression of COPD disease.Therefore,there is an urgent need to develop disease mitigation therapies for COPD.The role of pulmonary vascular changes in the development of COPD in the last decade has attracted academic attention.During the development of COPD,some research groups have found that pulmonary vascular dysfunction occurs in COPD patients,such as pulmonary hypertension,changes in peripheral airway microvascular permeability index(AMPI),injury of pulmonary microvascular endothelial cells,and destruction of vascular bed.This study aims to clinical data from this study and animal model,this paper using molecular biology,cell biology and bioinformatics,PM2.5 as incentives,respectively in clinical samples,the animal in vivo and in vitro cell/molecular experiment explore endothelial injury involved in the COPD process,for the future response to PM2.5 induced pulmonary microvascular endothelial injury treatment of new ideas.Methods:Part Ⅰ:1.Plasma was obtained from COPD patients and healthy people.①Flow cytometry was performed by circulating endothelial microparticle EMPs,and the proportion of CD31+/CD42b-microparticles in AnnexinV+microparticles was measured by statistical analysis.② VEGF content in alveolar lavage fluid was identified by ELISA and angiogenesis indexes were detected.2.Through THE PM exposure device,our research group established a batch of rat models of COPD caused by biofuel exposure lasting for 6 months,n=40.Lung tissue sections,alveolar lavage fluid,serum and plasma samples were obtained from successfully modeled rats,and lung tissue RNA and protein samples were obtained through experimental treatment.① The successful establishment of A COPD model was determined by the analysis of HE staining in paraffin sections of lung tissue and alveolar septum,alveolar circumference and inflammation;②The number of microvessels was evaluated by immunofluorescence staining of CD31 in paraffin sections of lung tissue;③EMPs ratio in plasma apoptotic particles of COPD rats was identified by flow cytometry,and endothelial apoptosis index was detected;④ The vascular endothelial permeability index was identified by Evans blue in vivo staining;⑤VEGF content in alveolar lavage fluid was identified by ELISA,and angiogenesis indexes were detected.3.In this study,primary pulmonary microvascular endothelial cells(PMVEC)of rats were extracted and exposed to PM2.5.① Apoptosis was detected by flow cytometry;②Angiogenesis experiment was used to detect newborn indexes;③ FITC dextrose fluorescence leakage method was used to detect the permeability index.Part Ⅱ:1.In this study,the methylation level of the pulmonary tissue transcriptome of COPD rats was quantitatively detected to determine the differential expression of methylation.2.The qPCR and WB were used to detect transcriptome of COPD rat model lung tissue to screen methylated enzymes and binding proteins that may be involved in regulation.3.Through THE detection of PMVEC exposed to PM2.5 by qPCR and WB,the selected methylase METTL16 was verified.4.METTL16 in PMVEC was interfered with by lentivirus:①angiogenesis experiment was used to detect the neovascularization index;②The permeability index was detected by FITC dextrose fluorescence leakage method.Part Ⅲ:1.In this study,high-throughput sequencing RNA-SEQ and transcriptome methylation merip-seq of transcriptome in COPD rats were used to obtain genes with differences in RNA expression and RNA methylation expression,and the downstream target genes of methylase were predicted by generation of mrna.2.Through qPCR detection of PMVEC exposed to PM2.5,the screened downstream target genes were verified.Results:Part Ⅰ:1.In this study,the proportion of plasma endothelial circulating particles EMPs in annexinV-labeled apoptosis particles in COPD patients was much higher than that in healthy subjects,indicating that the degree of endothelial apoptosis in COPD patients was higher than that in healthy subjects;VEGF expression in alveolar lavage fluid was detected by ELISA.2.The results of HE staining showed that in the alveolar structure of the rats exposed to biofuel exposed COPD,the diameter and circumference of the alveoli of the rats were much larger than those of the rats exposed to clean air,and the alveoli were fused with each other,which proved the success of the establishment of the model of COPD rats.On the basis of THE COPD rat model,the number of pulmonary microvascular endothelial cells was evaluated by immunofluorescence staining marker CD31.The number of pulmonary microvascular endothelial cells in the PM2.5 exposed group was significantly reduced compared with that in the clean air group.Flow cytometry was used to identify the number of EMPs of apoptotic endothelial circulating particles in the PM2.5 exposed group was significantly higher than that in the clean air group.In vivo Evans blue staining showed increased vascular endothelial permeability in PM2.5 infusion group.VEGF was reduced by ELISA in the alveolar lavage fluid.These can prove that pulmonary microvascular endothelium is damaged under PM2.5 exposure.3.PM2.5 exposure was performed on the extracted primary pulmonary microvascular endothelial cells,and the angiogenesis experiment found that PMVEC regeneration ability of PM2.5 exposure was reduced.The apoptosis rate was increased by flow cytometry.FITC dextroglucose fluorescence leakage was used to detect the increased permeability of endothelial monolayer cells.Part Ⅱ:1.We quantitatively detected the methylation level of the pulmonary tissue transcriptome of COPD rats with the m6A quantitative kit,and found that the methylation ratio increased significantly.2.The expression levels of methylated enzymes and binding proteins that MAY be regulated by RNA in the lung tissues of COPD rats were screened by qPCR,in which METTL16 showed a significant trend of increase,and we believed that METTL16 methylation might be an important factor for pulmonary microvascular damage in THE process of COPD caused by PM2.5 exposure.3.Through the detection of PMVEC exposed to PM2.5 by qPCR and WB,it was verified that the selected methylase METTL16 showed a significant upward trend,which was consistent with the results of animal models.4.After interfering with METTL16 in PMVEC of pulmonary microvascular endothelial cells,it was found that apoptosis was reduced,newborn ability was enhanced,and endothelial permeability was restored.Part Ⅲ:1.In this study,the lung tissues of COPD rats were merip-seq and RNA-SEQ to screen the downstream target genes of methylase.The results showed that a total of 1342 m6A methylation peaks were distributed,and 1301 m6A-modified genes were found in both groups.Among them,there were 62 genes differentially expressed at m6A level and mRNA level.A total of 10 genes related to endothelial injury were selected through GO analysis.Sulf2 and the m6A methylation peak sequence of Cythl were consistent with the reported binding motif of METTL16,and were potential binding sites for METTL16.2.QPCR test for COPD rat lung tissue and qPCR/WB test for PM2.5 PMVEC exposure showed a decrease in the expression levels of Sulf2 and Cythl,consistent with the sequencing results.Conclusion:During THE COURSE of PM2.5 exposure in COPD,pulmonary microvascular endothelial cells apoptosis,loss of angiogenesis ability and barrier ability.Methylation of METTL16 is involved in the pulmonary microvascular injury of COPD caused by PM2.5 exposure.Sulf2 and Cythl are potential binding sites for METTL16.