Abstract: Objective:To study the differentially expressed genes and regulatory signaling pathways related to hypoxia and immune mechanisms in ischemic stroke(IS), so as to identify potential biomarkers for the diagnosis of IS.Methods:The IS expression profile dataset GSE58294 was downloaded from the GEO database.Singlesample GSEA(ssGSEA) and t-distributed stochastic neighbor embedding (t-SNE) algorithms were employed to estimate the immune and hypoxia statuses of subjects.The"limma"software package was used to screen differentially expressed genes (DEGs).DAVID online database was used for gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis, least absolute shrinkage and selection operator(LASSO)was utilized to screen IS key(hub)genes, and then real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to verify the differential expressions of key genes.Results:60 hypoxia-immune-IS-related DEGs were screened.KEGG results revealed that the DEGs were enriched in PI3K-Akt signaling pathway.6 key genes-CHPF2, MBOAT2, IL18RAP, TIMM8A, ALDOAP2, and LPAR4 were obtained using the LASSO regression.CHPF2, MBOAT2, and IL18RAP were up-regulated (all P< 0.001), while TIMM8A, ALDOAP2 and LPAR4 were down-regulated (all P< 0.001).The ROC curve showed that the AUC of these 6 key genes ranged from 0.8941 to 0.9943.RT-qPCR results showed that the expressions of MBOAT2 and IL18RAP in transient middle cerebral artery occlusion (tMCAO) mice were significantly higher than those in sham operation mice(all P< 0.001).Conclusion:IL18RAP and MBOAT2 may be hypoxia-immune-IS-related DEGs and may serve as potential biomarkers for the diagnosis of IS.