Analysis of diagnostic biomarkers for ischemic stroke and experimental validation of targeted cuproptosis related genes

Chen Ying; Guo Xiaojing; Mo Xueni; Ma Wei; Wu Shangzhi; Li Xiangling; Xie Tingting

doi:10.12307/2025.957

Abstract：BACKGROUND:Studies have shown that immune cells are involved in all processes of ischemic stroke,in which cuproptosis also plays a key role. OBJECTIVE:To screen diagnostic biomarkers related to the progression of ischemic stroke through bioinformatics,and analyze and validate cuproptosis-related genes closely related to the occurrence and development of ischemic stroke. METHODS:The GSE16561 microarray was obtained from the GEO database,containing data from 39 cases of ischemic stroke(ischemic stroke group)and 24 controls(control group).Differentially expressed genes from the ischemic stroke microarray data were analyzed.Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed.By using LASSO and Random Forest methods,key genes affecting the occurrence and development of ischemic stroke were screened,and a diagnostic model was established and validated.Differential gene analysis was performed through immune cell infiltration and weighted gene co expression network.The differentially expressed immune-related genes were intersected with cuproptosis genes to obtain the hub genes related to cuproptosis immunity.In vitro cell experiments were conducted to divide rat hippocampal neurons into a normal group and an ischemic stroke group,and qPCR experiments were performed to verify the results. RESULTS AND CONCLUSION:(1)573 differentially expressed genes were obtained by differential analysis.Differentially expressed genes were mainly enriched in biological processes,such as positive regulation of immune response,and signaling pathways such as lipid and atherosclerosis.(2)Machine learning methods were used to screen diagnostic genes such as MFN2,PKM2,CREG1,and FOXO3A,which may have some diagnostic value for ischemic stroke.(3)Immune infiltration analysis revealed resting plasma cells,NK cells,macrophages,etc.,indicating that immune cells play a certain role in the pathogenesis of ischemic stroke.(4)Weighted gene co-expression network analysis combined with immune infiltration analysis obtained 118 key module genes,which were intersected with cuproptosis genes to obtain 2 cuproptosis and immune characteristic genes.The correlation analysis between four diagnostic genes and Hub genes showed that the expression of FOXO3A and MFN2,PKM2 and BCL2L1,MTF1 and MFN2,ATP7B and BCL2L1 were correlated.(5)The qPCR results showed significant differences in the genes MTF1 and ATP7B between the ischemic stroke group and the blank group.To conclude,ATP7B and MTF1 can serve as characteristic genes for cuproptosis in ischemic stroke.It is possible to improve ischemic stroke by intervening in ATP7B and MTF1 to regulate cuproptosis.