Analysis of Temporal Expression Variations of Hub Genes in Myocardial Ischemia/Reperfusion Injury: Integrating bioinformatics analysis and experimental validation

Abstract BACKGROUND Morbidity from myocardial ischemia/reperfusion injury (MIRI) has increased. Blood flow restoration can damage ischemic myocardium. No effective treatment exists for MIRI, and its mechanisms remain poorly understood. Our aim was to identify the key genes and pathways implicated in the development of MIRI, as these may represent promising biomarkers and therapeutic targets for this condition. METHODS Using GSE160516 dataset comparing sham and myocardial ischemia-reperfusion injury at 6, 24, and 72 hours post-reperfusion, we identified differentially expressed genes via GEO2R. We analyzed functional annotation and pathways using DAVID, examined protein interactions through STRING and Cytoscape, and evaluated miRNA predictions, transcription factors, drug-gene interactions, ROC curves, and immune cell infiltration. Gene validation used RT-qPCR and external datasets. RESULTS 341 differentially expressed genes were identified at 6 hours, 399 at 24 hours, and 431 at 72 hours post-reperfusion, with 61 common across groups. GO and KEGG analyses showed DEGs enriched in 'inflammatory response', 'cytokine-cytokine receptor interaction', and 'chemokine signaling pathway'. PPI network analysis identified Itgam, IL-6, and Ccl2 as hub genes. mmu-let-7c-1-3p, mmu-miR-466l-3p, Nfkb1, and hydrocortisone butyrate interacted with most hub genes. ROC analysis showed diagnostic value of hub genes for MIRI. Immune cell analysis showed increased resting dendritic cells and decreased neutrophil infiltration. External validation and RT-qPCR confirmed upregulation of SERPINE1, CCL2, IL-6, and CXCL10, and downregulation of PTGS2 and CD68 post-reperfusion. Conclusion Under reperfusion conditions, the expression variations of hub genes in HL-1 cells identified six genes—Serpine1, Ccl2, Il6, Cxcl10, Ptgs2, and Cd68—with significant differences, suggesting their potential relevance in the treatment of myocardial ischemia-reperfusion injury (MIRI).