Identification of Key Genes Mediated by N6-Methyladenosine Methyltransferase METTL3 in Ischemic Stroke via Bioinformatics Analysis and Experiments.

Abstract

The N6-methyladenosine (m6A) methyltransferase METTL3 has been demonstrated to function in mediating m6A modification, but its role in ischemic stroke (IS) has not been fully elucidated. This study aimed to explore the downstream mechanism of METTL3-mediated m6A modification in IS. GSE16561 and GSE22255 were downloaded from the Gene Expression Omnibus database for analysis of differentially expressed genes (DEGs), and it was found that METTL3 mRNA was downregulated in IS. Then quantitative real-time polymerase chain reaction was used to verify the downregulation of METTL3 mRNA in the peripheral blood of IS patients and the cortexes of transient middle cerebral artery occlusion mice. By combining DEGs with the m6A-downregulated genes in GSE142386 which performed methylated RNA immunoprecipitation sequencing (MeRIP-seq) on METTL3-deficient and control endothelial cells, a total of 131 genes were identified as the METTL3-mediated m6A-modified genes in IS. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the genes were mainly involved in cytokine-cytokine receptor interaction, MAPK signaling pathway and NF-kappa B signaling pathway. CTSS and SBK1 were further screened as the key METTL3-mediated m6A-modified genes by random forest model and PCR validation. The ROC curve analysis showed that the combination with CTSS and SBK1 was of good diagnostic value for IS, with the AUC of 0.810, sensitivity of 0.780, and specificity of 0.773. Overall, we found that METTL3-mediated m6A modification may influence the occurrence and development of IS by participating in inflammation-related biological processes, and two key m6A-modified genes mediated by METTL3 (CTSS and SBK1) can be used as diagnostic biomarkers for IS.