Identification Of Key Genes And Potential Mechanisms In The Progression Of Coronary Atherosclerosis Using Bioinformatics Analysis

Objective: Screening gene chips in public databases to obtain sequencing data at different stages of atherosclerosis;separately analyze and screen out key genes and potential mechanisms of atherosclerosis formation and progression,and further explore the infiltration of inflammatory cells in hardened plaque at different stages of atherosclerosis.Method: Set inclusion and exclusion criteria,search and screen the gene chips in the GEO(Gene Expression Omnibus)database and ArrayExpress database to obtain mRNA transcription sequencing data at different stages of atherosclerosis.According to the included chip data,the experimental groups were divided into normal control group,atherosclerosis group;early atherosclerosis group,and advanced atherosclerosis group.The limma R software package was used to compare and analyze the differential expression of genes between the normal control group and atherosclerosis formation group to obtain the differential genes.The R language package was used to visualize the differential genes,and the corresponding heatmap and volcano map were drawn.Use the DAVID data analysis platform(https://david-d.ncifcrf.gov/summary.jsp)to perform GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)analysis on differential genes to explore the signal pathways for gene enrichment,Cell function and biological processes.The STRING database(https://string-db.org)was used to analyze the interaction between the corresponding proteins of the different genes,and Cytoscape software was used to make a network diagram of protein interactions,and the pivot nodes were further analyzed as key genes.Use the R language package related to WGCNA(Weighted gene co-expression network analysis)to perform weighted co-expression network analysis on the differential genes,subdivide them into different functional modules,and combine GO analysis to explore the specific biological functions of genes in each module.According to CIBERSORT’s official website(https://cibersort.stanford.edu)immune cell single cell sequencing data,the Rlanguage software package was used to compare and calculate the infiltration of immune cells in each sample.The limma R software package was also used to comparatively analyze the differential genes between the early atherosclerosis group and the advanced atherosclerosis group,and repeat all the above analysis steps.Results: Two data chips GSE43292 and GSE28829 were included in the screening.The atherosclerosis formation group contained 32 carotid atherosclerotic plaque samples,and the normal control group contained 32 samples of normal vascular tissues adjacent to the plaque.The early group contains 13 early carotid atherosclerotic plaque tissues,and the advanced atherosclerosis group contains 16 advanced carotid atherosclerotic plaque tissue samples.There were 75 up-regulated genes and 57 down-regulated genes in the atherosclerosis formation group compared with the normal group;there were 154 up-regulated genes and 23 down-regulated genes in the advanced plaque compared with the early plaque.The differential genes in the normal control group and the atherosclerosis group are mainly enriched in biological processes such as the classic pathway of complement activation and the humoral immune response mediated by circulating immunoglobulin;and the differential genes in the early and advanced stages of atherosclerosis are mainly involved Leukocyte migration,regulation of inflammatory response,regulation of immune effect process,positive regulation of cytokine production,and receptormediated endocytosis.PPI(protein-protein interaction)analysis showed that the key differential genes in the normal group and atherosclerosis formation group are MMP9,ITGAX,CD163,CXCL10 and other genes,and the key differential genes in the early and advanced stages of atherosclerosis are TYROBP,FCGR2 B,CSF1R,ITGB2.WGCNA analysis found that the differential genes in atherosclerosis and normal groups were mainly related to pathways related to complement activation,humoral immunity,protein activation,inflammation regulation,and phagocytosis.The differential genes in the early and advanced stages of atherosclerosis are mainly involved in the biological reactions related to complement activation,humoral immunity,phagocytosis,blood particles,peptidoglycan,phosphatidylcholine,and ammonium ions.Compared with the normal control group,the expression abundance of initial B cells,CD8 T cells,regulatory T cells(Tregs),activated natural killer cells,monocytes,and resting dendritic cells decreased,while the expression abundance of memory B cells and activated memory T cells increased(P<0.05).Compared with the early stage,the proportion of initial B cells,initial CD4 T cells,and monocytes decreased in the advanced stage,and the proportion of memory B cells and M2 macrophages increased(P<0.05).Conclusion: Compared with normal tissues,the formation of atherosclerosis is mainly related to genes such as MMP9,ITGAX,and CD163,and is mainly involved in biological processes such as the classical pathway of complement activation and circulating immunoglobulin-mediated humoral immune response.Compared with the early stage of atherosclerosis,the differential genes in the advanced stage are mainly TYROBP,FCGR2 B,CSF1R,ITGB2,involved in leukocyte migration,regulation of inflammatory response,regulation of immune effect process,positive regulation of cytokine production and receptor-mediated internal swallow.Compared with the normal group,the expression ratio of memory B cells and activated CD4 memory T cells in the atherosclerosis group increased;during the progression of atherosclerosis,the proportion of memory B cells and M2 macrophages increased.