Experimental verification and comprehensive analysis of m7G methylation regulators in the subcluster classification of ischemic stroke

Abstract

Background: Ischemic stroke (IS) is a fatal cerebrovascular disease involving several pathological mechanisms. Modification of 7-methylguanosine (m7G) has multiple regulatory functions. However, the expression pattern and mechanism of m7G in IS remain unknown. Herein, we aimed to explore the effect of m7G modification on IS.Methods: We screened significantly different m7G-regulated genes in Gene Expression Omnibus datasets, GSE58294 and GSE22255. The random forest (RF) algorithm was selected to identify key m7G-regulated genes that were subsequently validated using the middle cerebral artery occlusion (MCAO) model and quantitative polymerase chain reaction (qPCR). A risk model was subsequently generated using key m7G-regulated genes. Then, “ConsensusClusterPlus” package was used to distinguish different m7G clusters of patients with IS. Simultaneously, between two m7G clusters, differentially expressed genes (DEGs) and immune infiltration differences were also explored. Finally, we investigated functional enrichment and the mRNA–miRNA–transcription factor network of DEGs.Results: RF and qPCR confirmed that EIF3D, CYFIP2, NCBP2, DCPS, and NUDT1 were key m7G-related genes in IS that could accurately predict clinical risk (area under the curve = 0.967). NCBP2 was the most significantly associated gene with immune infiltration. Based on the expression profiles of these key m7G-related genes, the IS group could be divided into two clusters. According to the single-sample gene set enrichment analysis algorithm, four types of immune cells (immature dendritic cells, macrophages, natural killer T cells, and TH1 cells) were significantly different in the two m7G clusters. The functional enrichment of 282 DEGs between the two clusters was mainly concentrated in the “regulation of apoptotic signaling pathway,” “cellular response to DNA damage stimulus,” “adaptive immune system,” and “pyroptosis.” The miR-214–LTF–FOXJ1 axis may be a key regulatory pathway for IS.Conclusion: Our findings suggest that EIF3D, CYFIP2, NCBP2, DCPS, and NUDT1 may serve as potential diagnostic biomarkers for IS and that the m7G clusters developed by these genes provide more evidence for the regulation of m7G in IS.