Abstract
Background: We aimed to identify key molecular drivers of gastric cancer progression and poor prognosis by integrating multi-omics analyses with experimental validation. Methods: Single-cell RNA-seq data were clustered to delineate major cell types. InferCNV identified tumor epithelial cells, and reclustering revealed a malignant subset with poor prognosis. The overlap between subset markers and The Cancer Genome Atlas Stomach Adenocarcinoma (TCGA-STAD) upregulated differentially expressed genes (DEGs) was modeled with univariate, LASSO-, and multivariate Cox to derive a prognostic signature. Patients were stratified according to signature scores, and group differences in survival and immunologic features were compared. Spatial transcriptomics defined the localization patterns of key signature genes. In vitro functional assays (CCK-8, colony formation, EdU incorporation, flow cytometry, Transwell migration and invasion, and wound healing) confirmed the pivotal role of SRI. Results: Reclustering of tumor epithelial cells yielded seven subsets (C0-C6), with C5 displaying marked malignant features and correlating with poor prognosis in multiple cohorts. Intersecting 208 genes yielded a five-gene signature (ASCL2, REPIN1, CXCL3, TMEM176A, SRI). The signature stratified patients into high- and low-risk groups. The high-risk cohort exhibited significantly poorer survival, distinct immune-infiltration patterns, elevated immune-evasion scores, and a reduced predicted response to immunotherapy. Single-cell and spatial transcriptomics localized TMEM176A to fibroblasts and SRI to the tumor epithelium. Finally, in vitro knockdown of SRI inhibited tumor cell proliferation, migration and invasion. Conclusions: Our multi-omics approach identified a malignant epithelial subset, C5, and a five-gene signature that stratifies gastric cancer prognosis and immune response. Functional assays showed that SRI knockdown impairs tumor cell growth, migration and invasion.