Integrated bulk and single-cell transcriptomics with virtual knockout analysis identifies FOS as an...

Chronic atrophic gastritis (CAG) is a precancerous lesion characterized by epithelial injury, glandular atrophy, and impaired gastric function. Molecular targets linking epithelial alterations with potential traditional Chinese medicine-derived compounds remain unclear. GEO bulk transcriptomic datasets were analyzed to identify differentially expressed genes (DEGs), which were intersected with CAG-related genes for enrichment analysis. Protein-protein interaction (PPI) network analysis and reverse network pharmacology were used to identify key targets and construct a drug-component-target network. Single-cell RNA sequencing, virtual knockout, molecular docking, CCK-8 assay, and RT-qPCR in 1‑Methyl‑3‑nitro‑1‑nitrosoguanidine (MNNG)-treated GES-1 cells were then integrated to prioritize and evaluate FOS. A total of 502 DEGs and 54 intersected CAG-related genes were identified, with enrichment in wound healing, epithelial junction organization, tight junctions, cAMP signaling, gastric cancer, and digestive or secretory pathways. Five key genes-APOA1, CFTR, FOS, HMOX1, and VIM-were identified, all with AUCs above 0.80. Among 21,679 single cells, FOS was the only key gene predominantly enriched in gastric epithelial cells. Virtual knockout predicted associations between FOS-network perturbation and five genes involved in hormone processing and gastric secretory pathways. Docking generated candidate fisetin-FOS and kaempferol-FOS poses for preliminary compound prioritization. MNNG reduced GES-1 cell viability and significantly increased FOS mRNA expression. Integrated computational analyses highlighted FOS as an epithelial-associated candidate gene in CAG and suggested exploratory associations with gastric secretory pathways. These findings support further functional and compound-intervention studies of the FOS/AP-1 axis.