Integrative analysis of Helicobacter pylori-driven stomach adenocarcinoma reveals epigenetic deregulation, immune evasion, and therapeutic resistance.

BACKGROUND: Helicobacter pylori (H. pylori) infection is a major etiological factor for stomach adenocarcinoma (STAD), yet the key molecular drivers linking infection to tumor progression remain insufficiently defined. This study aimed to identify H. pylori-related hub genes in STAD and validate their functional relevance using integrated bioinformatics and experimental approaches. METHODOLOGY: Differentially expressed genes (DEGs) were identified from two microarray datasets (GSE13911 and GSE54129) comparing H. pylori-positive STAD samples with controls. Common DEGs were used to construct a protein-protein interaction (PPI) network via STRING and Cytoscape, and hub genes were ranked using CytoHubba. Transcriptomic validation was conducted using TCGA-STAD data, followed by analyses of enrichment pathways, promoter methylation, somatic mutations, CNVs, immune subtype associations, and drug sensitivity using GSCA, UALCAN, cBioPortal, and CTRP datasets. miRNA-mRNA regulatory interactions were predicted using miRNet and validated in vitro. Experimental validation included RT-qPCR, Western blotting, CCK-8 proliferation assays, colony formation, and wound-healing assays in MKN45 and AGS cells following siRNA-mediated knockdown of key hub genes. Additionally, AGS cells were infected with live H. pylori to directly assess infection-induced changes in gene expression and malignant phenotypes. RESULTS: Four hub genes (THBS2, CTNNB1, COL4A1, and E2F3) were identified as commonly upregulated in H. pylori-positive STAD samples and were further validated as highly expressed in STAD tissues and cell lines. Promoter hypomethylation and CNV gains contributed to their overexpression. Pathway analyses linked the hub genes to EMT, cell cycle progression, immune suppression, and oncogenic signaling. miRNA profiling identified hsa-miR-9-3p and hsa-miR-9-5p as common regulators with diagnostic potential. Importantly, H. pylori infection of AGS cells induced strong upregulation of COL4A1 and CTNNB1 and significantly increased proliferation, clonogenicity, and migration, demonstrating a direct infection-driven oncogenic response. Conversely, siRNA-mediated silencing of COL4A1 or CTNNB1 markedly reduced proliferation, colony formation, and wound closure, confirming their functional roles in STAD progression. Immune correlation and drug sensitivity analyses further linked high hub-gene expression to immunosuppressive microenvironments and resistance to multiple therapeutic agents. CONCLUSION: This study identifies THBS2, CTNNB1, COL4A1, and E2F3 as key H. pylori-associated oncogenic drivers in STAD. Functional assays demonstrate that H. pylori enhance malignant phenotypes through COL4A1 and CTNNB1, while gene silencing reverses these effects. These findings highlight the hub genes and their regulatory miRNAs as promising diagnostic biomarkers and potential therapeutic targets in H. pylori-related gastric cancer.