Identification Of Biomarkers Of Ischemic Stroke And Their Potential Molecular Mechanisms Based On Weighted Gene Co-Expression Network Analysis

Objective:Ischemic stroke is one of the most common cerebrovascular diseases,which seriously threatens people’s health.In this study,Weighted gene co-expression network analysis(WGCNA)and other bioinformatics methods were used to explore the potential molecular mechanism of ischemic stroke.Methods :(1)The Gene Expression profile data that met the inclusion and exclusion criteria were downloaded from the Gene Expression Omnibus(GEO)database,and the data were normalized and corrected.(2)Differential analysis of gene expression profile data was performed to screen differentially expressed genes.(3)WGCNA was used to analyze the gene expression profile data and screen the gene modules and core genes most associated with ischemic stroke.(4)Gene module enrichment was analyzed using Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).Gene Set Enrichment Analysis(GSEA)was used to enrich gene expression profile data.(5)The Receiver Operating Characteristic(ROC)curves were analyzed for core genes.(6)To predict miRNA of core genes and construct miRNA-mRNA interaction network.(7)The immune cells in CIBERSORT website(https://cibersort.stanford.edu)data for the reference,using R language software calculation gene expression profile data of each sample in the immune cell infiltration.(8)q PCR was used to detect the expression levels of core genes in patients with ischemic stroke and normal people.Results:(1)A total of 191 differentially expressed genes were screened out,including81 up-regulated genes and 110 down-regulated genes.(2)WGCNA analysis obtained 8 gene modules,among which the blue module had the highest correlation coefficient with ischemic stroke,and four core genes CD6,ICAM2,CUTA and GIMAP5 were screened out.(3)GO enrichment analysis results focused on immune-related biological processes such as leukocyte mediated immunity,cell activation involved in immune response,leukocyte activation involved in immune response,regulation of T cell activation,immune response-regulating signaling pathway,positive regulation of T cell activation,immune response-regulating cell surface receptor signaling pathway,and positive regulation of leukocyte activation.KEGG enrichment showed that Th1 and Th2 cell differentiation,Th17 cell differentiation,antigen processing and presentation,T cell receptor signaling pathway,cell apoptosis,NF-kappa B signaling pathway,necroptosis and p53 signaling pathway were related to immunity and cell death.GSEA in the ischemic stroke group was enriched in inflammatory response,hypoxia response,coagulation response,apoptosis and other signaling pathways.(4)We predicted the miRNAs of four core genes,and obtained 18 mRNAs,including miR-1229-3p,miR-3162-3p,miR-3960,miR-4437,miR-1268 a,miR-1268 b and miR-4508.(5)The results of immune infiltration showed that the levels of monocytes and neutrophils in the ischemic stroke group were significantly higher than those in the normal control group,while CD8+T cells were the opposite.The results of correlation analysis between immune cells and four core genes were as follows: The expression of CD6 was positively correlated with CD8+T cells(r = 0.41,P <0.01)and activated NK cells(r = 0.37,P = 0.02),and negatively correlated with neutrophils(r =-0.43,P < 0.01).CUTA expression was positively correlated with CD8+T cells(r = 0.35,P=0.03)and negatively correlated with neutrophils(r =-0.59,P < 0.001).The expression of GIMAP5 was positively correlated with CD8+T cells(r = 0.34,P=0.03)and negatively correlated with neutrophils(r =-0.41,P < 0.01).ICAM2 was positively correlated with CD8+T cells(r= 0.39,P=0.01)and negatively correlated with neutrophils(r=-0.50,P < 0.01).(6)q PCR results showed that the expression levels of CD6,CUTA,GIMAP5 and ICAM2 in the ischemic stroke group were significantly lower than those in the control group(P < 0.05).Conclusion:(1)This study found that CD6,CUTA,GIMAP5 and ICAM2 in peripheral blood could be used as biomarkers of ischemic stroke.(2)This study found that peripheral blood neutrophils,CD8+T cells and monocytes play an important role in the occurrence and development of ischemic stroke and may become immunotherapeutic targets for ischemic stroke.(3)This study found that the expression levels of CD6,CUTA,GIMAP5 and ICAM2 in peripheral blood of ischemic stroke may be correlated with the levels of neutrophils and CD8+T cell infiltration.(4)This study found that 18 miRNAs,including miR-1229-3p,miR-3162-3p,miR-3960,miR-4437,miR-1268 a,miR-1268 b and miR-4508,may regulate CD6,CUTA,GIMAP5 and ICAM2.And then involved in the pathological process of ischemic stroke.(5)This study further elucidated the underlying molecular mechanism of ischemic stroke and provided a potential theoretical basis for the treatment of ischemic stroke.