Integrative transcriptomic analysis and experimental validation identify GPR97 and PROK2 as novel genes upregulated...

Acute myocardial infarction (AMI) is a major global health burden. Current diagnostic reliance on high-sensitivity cardiac troponin (hs-cTn) is limited by its lack of disease specificity and inability to reveal upstream molecular and immune mechanisms. This study aimed to identify robust core genes and their functional networks in AMI via integrated multi-dataset analysis and experimental validation. Three peripheral blood transcriptomic datasets (GSE60993, GSE61144, GSE97320) were obtained from GEO. Differentially expressed genes (DEGs) were identified using limma, with common DEGs extracted. Diagnostic performance was evaluated via ROC curves. Functional annotation used GO, KEGG and GSEA. Immune infiltration was analyzed with CIBERSORT. Key genes were validated in H9C2 cells under oxygen-glucose deprivation (OGD) using RT-qPCR and Western blot. Eight core genes were consistently upregulated in AMI peripheral blood, all showing high diagnostic accuracy (AUC > 0.80). They were enriched in immune-inflammatory pathways. GSEA identified three activated pathways: adipocytokine signaling, leukocyte transendothelial migration and insulin signaling, with ACSL1, MMP9 and PYGL as key drivers. Immune analysis revealed increased neutrophils and decreased γδ T cells, with all eight genes positively correlating with neutrophil infiltration. The OGD model in H9C2 cells confirmed specific upregulation of GPR97, PROK2, ALPL and PADI4. This integrated computational and experimental study identifies a set of high-value diagnostic core genes in AMI. It first validates the specific upregulation of novel genes (GPR97, PROK2) in myocardial ischemia, placing them within a pathological network involving metabolic, immune and protective pathways. These findings provide new molecular insights and a foundation for novel diagnostics and therapeutic targets.