| Objective:In this study,we investigated the gene expression microarray data set of membranous nephropathy in the GEO database,applied bioinformatics methods to analyze and identify key genes associated with M2 macrophages,and verified their diagnostic value.Subsequently,the biological function of each key gene was analyzed to provide new ideas for MN pathogenesis research and diagnosis and treatment.Methods:The microarray datasets GSE104948 and GSE108109 were obtained by downloading from the GEO database,where GSE108109 served as validation dataset.Differential expression analysis was performed between MN and normal controls using the limma package in R software(version 4.1.1),and GO functional analysis and KEGG signaling pathway analysis were performed on differentially expressed genes(DEGs)obtained from the screening using the online database Metascape.Immuno-infiltration analysis of MN and normal controls using the CIBERSORT algorithm was also performed and M2 macrophages were found to be significantly different between MN and normal controls.Therefore,weighted gene co-expression network analysis(WGCNA)was performed using M2 macrophages as a phenotype to screen M2macrophage-associated hub genes.Meanwhile,we used Metascape to perform enrichment analysis of Hub genes again,and the method was consistent with the enrichment analysis of differentially expressed genes described above.Then,the intersection of differentially expressed genes and Hub genes was taken to identify potential key genes.Second,key genes were identified by validating the expression levels of potential key genes in dataset GSE108109.Afterwards,its diagnostic value was assessed by ROC curve analysis.Finally,gene set enrichment analysis(GSEA)was used to explore the biological functions involved in the above key genes.Results:567 DEGs were selected by limma package,including 297 up-regulated genes and270 down-regulated genes.Pathway and functional enrichment analysis showed that up-regulated differentially expressed genes were mainly associated with the body ’s response to external stimuli as well as the immune system,while down-regulated differentially expressed genes were mainly related to molecular metabolism.Immune cell infiltration analysis showed that six immune cell infiltration levels were significantly differentially expressed compared to normal controls,with activated natural killer cells and monocytes infiltrating at higher levels in the MN group,while M2 macrophages,activated dendritic cells,na?ve B cells,and neutrophils infiltrating at lower levels in MN samples.Using M2 macrophages as a phenotype for WGCNA,a total of 38 Hub genes were selected.GO functional analysis showed that Hub genes were mainly enriched in "small molecule catabolic processes,oxidoreductase activity,cellular response to oxidative stress,and cellular response to chemical stress".KEGG enrichment analysis showed that Hub genes were mainly enriched in the "chemical carcinogen-reactive oxygen species" signaling pathway.Then,nine potentially critical genes were obtained by taking the intersection of DEGs and Hub genes.The expression levels of potential key genes were verified in dataset GSE108109,which finally resulted in six key genes: HLF,PPARGC1 A,ESRRG,HGD,KCNJ16,and SULT1C2.The diagnostic value was assessed by ROC curve analysis,and the AUC values of the six genes were greater than0.85,which had a high diagnostic value.GSEA analysis showed that these key genetic changes can cause arginine and proline metabolism,glycine,serine and threonine metabolism,fatty acid metabolism,complement and coagulation cascade,peroxisomes,PPAR signaling pathway and other pathways up-regulated.At the same time,these key genes also downregulate pathways such as antigen processing and presentation,FCγR-mediated phagocytosis,and natural killer cell-mediated cytotoxicity.Conclusion:This study shows that PPARGC1 A,ESRRG,KCNJ16,HLF,HGD and SULT1C2 are key genes related to M2 macrophages in MN.This discovery may provide new insights for the influence of M2 macrophages on MN. |
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