Screening Of Key Regulators For Carotid Atherosclerosis Based On DNA Methylation And RNA Sequencing

Objective:Stroke has high morbidity,high disability rate and high mortality rate,which brings huge economic and psychological burden to individuals,families and society,and seriously endangers human health.About 80% of cerebral apoplexy is ischemic stroke,and about half of ischemic stroke patients have ipsilateral extracranial carotid artery stenosis,atherosclerotic carotid artery stenosis is the main pathological basis.Carotid atherosclerosis is the main cause of most serious cerebrovascular events.The pathogenesis of carotid atherosclerosis has been well understood in recent decades,but the pathogenesis of carotid atherosclerosis has not been elucidated,and the ideal intervention is still lacking.Current studies suggest that carotid atherosclerosis is a complex multifactorial disease in which environmental,genetic and epigenetic changes may be closely related to its occurrence and development.In the study of the mechanism of atherosclerosis,there have been many reports on the genetic susceptibility of atherosclerosis,but there are still different opinions.In recent years,due to the progress of sequencing technology and the broadening of research field,people have come to realize that epigenetic mechanism may play an important role in the formation of atherosclerosis.Although there have been some exploratory studies in this field,systematic studies based on differences in expression at the genome-wide methylation and transcriptome levels,as well as studies on the genes involved in the pathogenesis and progression of atherosclerosis and their roles,There are few reports at present.Therefore,in this project,we intend to study carotid atherosclerotic plaque patients by self-matched and case-control sampling methods,through the whole genome methyl ation and transcriptome expression difference sequencing strategy.From the point of view of the interaction between methylation and gene expression,the specific roles and pathawys of related genes in the progression of atherosclerosis were clarified,and the epigenetic mechanism of atherosclerotic plaque formation was discussed.In order to explain the pathogenesis of atherosclerosis from one side,this will have important clinical significance and scientific value.Methods:A total of 25 carotid plaques,classified into unstable and stable groups were conescutively collected.DNA methylation,RNA sequencing and immunohistochemistry were performed to identify potential regulators of AS.Isolation of CD14+ macrophages from patients’ plaques,si RNA transfection,cytokine quantification,cell proliferation and cell cycle analysis were performed to explore the function of signaling lymphocytic activation molecule family receptor 7(SLAMF7)in AS.Results:We identified 174 up-regulated genes with hypo-methylation in the promoter,and 86down-regulated genes with hyper-methylation in the promoter,in AS vs.healthy controls.Among them,high expression of SLAMF7 was examined in carotid plaque vs.intact tissue,in advanced plaque vs.early atherosclerotic tissue,and SLAMF7 protein expressed significantly higher in the unstable plaques than that in the stable plaques,especially enriched in the CD68-positive macrophage area.si RNA knockdown of SLAMF7 in the CD14+macrophages induced a suppressed secretion of proinflammatory cytokines,and further inhibited proliferation of vascular smooth muscle cells(VSMCs).Conclusions:By using a systematic approach in integrating genome-wide DNA methylation,gene expression,pathological phenotypic data,and functional study on primary-cultured cells,we identified SLAMF7 as a key regulator of carotid AS disease progression.These data provide emerging evidence that SLAMF7 could be a target of potential therapeutic intervention in carotid AS.