Bioinformatics Analysis and Mechanistic Validation of ERCC1, SYK and GNAS in Acute Ischemic Stroke
ZHENG Jianbiao1, ZHANG Jinbiao2*, FENG Nana3
The core pathophysiological feature of AIS (acute ischemic stroke) is local cerebral ischemia/hypoxia and neuronal damage resulting from acute interruption of cerebral blood flow. ERCC1 (excision repair cross-complementing group 1), SYK (spleen tyrosine kinase), and GNAS [guanine nucleotide-binding protein G(s) subunit alpha] are involved in DNA damage repair, immune-inflammatory response, and G protein signaling, respectively, and are all closely associated with the pathological process of AIS; however, their co-expression characteristics and integrated regulatory mechanisms in AIS remain unclear. This study integrated gene expression profile data from the GEO databases (GSE37587, GSE140275) and the GeneCards database of AIS patients, employed bioinformatics approaches to screen for DEGs (differentially expressed genes), and performed GO/KEGG pathway enrichment analysis, immune cell infiltration analysis, and multi-layered regulatory network construction. Meanwhile, the correlations between target genes and thrombosis-associated molecular markers were investigated using the GTEx database. Furthermore, a rat model of middle cerebral artery occlusion (MCAO) was established, and in vivo experiments were performed to validate the above bioinformatics analysis results. A total of 14 key AIS-related DEGs were identified. GO and KEGG analyses revealed their involvement in DNA damage repair, immune regulation, and G protein-coupled signaling pathways. Literature review confirmed the close association of ERCC1, SYK, and GNAS with AIS pathophysiology. TIMER analysis demonstrated significant correlations between these genes and immune infiltration levels (P<0.05). GTEx further suggested their regulatory relationships with multiple thrombosis-related markers. Multi-layered network analysis identified five transcription factors (MXD3, KLF9, DPF2, MAZ, ATF1) co-targeting ERCC1 and GNAS, and three transcription factors (KLF4, SUZ12, EZH2) co-targeting GNAS and SYK, as well as six miRNAs (hsa-miR-7-5p, hsa-miR-15a-5p, hsa-miR-18a-5p, hsa-miR-218-5p, hsa-let-7a-5p, hsa-let-7b-5p) commonly targeting all three genes. Protein-protein interaction analysis revealed that UBC was the sole shared interactor among the three proteins, while CALM1 was the shared interactor between SYK and GNAS. Animal experiments showed that, compared to controls, MCAO rats exhibited significantly higher neurological deficit scores at 24 h post-surgery, increased infarct volumes (P<0.001), and typical ischemic pathology (HE staining: neuronal vacuolar degeneration and inflammatory infiltration). Western blot revealed markedly elevated ERCC1, SYK, and GNAS protein levels in MCAO rat brains (P<0.001). These results indicate that ERCC1, SYK, and GNAS are coordinately upregulated in AIS and participate in its pathophysiological process through regulating immune inflammation, DNA repair, and vascular homeostasis.
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