Relationship Between Methylat Ion Level Of CYP2S1 Promoter And Endothelial Cell Injury In High Altitude Pulmonary Edema

ObjectivesHigh-altitude pulmonary edema（HAPE）is the leading cause of nontraumatic death in alpine regions,and its morbidity and mortality quite high.However,HAPE has a complex pathogenesis and there is a lack of effective preventive measures.DNA methylation is involved in different pathways in hypoxic injury diseases including HAPE,and the DNA methylation also regulates the expression of genes in the body.CYP2S1 is highly expressed in lung tissue and is closely associated with lung injury.In order to further investigate the implications of DNA methylation of CYP2S1 in the development of HAPE,MassARRAY^? Epi TYPER^TM DNA methylation analysis was used in this study combined with in vitro biological function studies to analyze the differences and correlations between the methylation levels of CpG sites of CYP2S1 gene in HAPE patients and normal control population.The project also examined the alteration in the methylation levels of the promoter of CYP2S1 before and after demethylation with 5Aza under hypoxia conditions and its effects on the function of vascular endothelial cells,with a view to provide ideas and a theoretical basis for finding an ideal target for HAPE prevention.Methods1.There are 53 patients with HAPE and 53 normal controls were invited to collect peripheral blood samples.MassARRAY^? Epi TYPER^TM DNA methylation analysis technology was performed to identify variations in CpG sites in samples and the mean methylation level of the CYP2S1 gene.To assess the potential value of CYP2S1 methylation level in predicting HAPE,a ROC curve was plotted and the AUC was calculated.2.We simulated a high-altitude hypoxic environment,the rat pulmonary microvascular endothelial cells（PMVEC）were cultured in vitro and divided into normoxia and hypoxia groups.CYP2S1 was demethylated using 5Aza acting PMVEC.3.Methylation-specific PCR（MSP）primers were designed using the Meth primer software and the MSP kit was used to determine the methylation levels of the CYP2S1 promoter region in different states.4.CCK8 assay was used to screen the optimal hypoxia time and drug concentration,CYP2S1 mRNA and protein expression levels were measured using qRT-PCR and Western blot.Apoptosis rate and cell cycle changes were confirmed by flow cytometry.VEGF,MMP2 and MMP9 expression was determined by Western blot.Results1.CYP2S1 was methylated in both HAPE patients and normal controls.5 CpG sites showed significant differences in HAPE patients.The methylation level of 12 CpG sites in HAPE patients was higher than that in normal control group,1 CpG site could significantly increase the risk of HAPE,and 8 CpG sites had a protective effect on HAPE.The results of ROC curve showed that CYP2S1 methylation level had a high predictive value for HAPE.2.Hypoxia can induce hypermethylation of CYP2S1 promoter region.3.Under hypoxia,the proliferation,migration and invasiveness of PMVEC were obviously reduced,the expression of CYP2S1 and MMP9 were inhibited,and the cell cycle progression was blocked;the apoptosis rate of PMVEC and the protein expression levels of angiogenesis-related factors VEGF and MMP2 were increased.4.When CYP2S1 promoter region was hypermethylated,the proliferation,migration and invasion rates of PMVEC were obviously increased,and the expression of both CYP2S1 and MMP9 were promoted.The apoptosis rate of PMVEC and the protein expression levels of angiogenesis-related factors VEGF and MMP2 were significantly decreased.ConclusionsThis study found that the methylation level of CYP2S1 gene has a high predictive value for the pathogenesis of HAPE.Hypoxia can regulate the proliferation,apoptosis,migration,and invasive ability of PMVEC by mediating the methylation of the CYP2S1 promoter region.At the same time,it has different effects on the angiogenesis-related factors VEGF,MMP2 and MMP9,which are involved in PMVEC injury and angiogenesis.