Functional and Clinical Validation of tsRNA-Defined Molecular Subtypes Guides Precision Therapy in Gastric Cancer

Ben Liu^1*Ye Tian¹Xin Hu²Yuan Liu¹Wenqi Wu¹Yanxin Yao¹Huahuan Liu¹Wei Wang¹Hongji Dai¹Yubei Huang¹Changyu Sun¹Yan Cui¹Zun Li¹Xiannan Zhang¹Liqing Jia¹Fubing Wang¹Fengju Song¹Kexin Chen¹Yuan Pan¹

• ¹Tianjin Medical University Cancer Institute and Hospital, Tianjin, China
• ²Zhongnan Hospital of Wuhan University, Wuhan, China

Gastric cancer (GC) is characterized by marked molecular and clinical heterogeneity, complicating prognosis and treatment. Here, we profiled transfer RNA-derived small RNAs (tsRNAs) in GC and identified three distinct molecular subtypes, each defined by unique tsRNA expression signatures and tumor microenvironment features. The Stromal_L subtype exhibited the most favorable prognosis, while the Stromal_H subtype was associated with a higher frequency of DNA repair gene mutations and poorer survival. Multi-omics characterization further revealed subtype-specific pathway dysregulation, including hyperactivated G2M checkpoint and fatty acid metabolism in Stromal_L.To enable clinical translation, we leveraged 25 tsRNA-associated hub genes and ten machine learning algorithms to construct a prognostic model. The random survival forest (RSF)-optimized GCtsRNAscore effectively stratified patients into high-risk and low-risk groups (https://github.com/huxintmu/GCtsRNAscore). High-risk patients demonstrated increased sensitivity to targeted agents (axitinib, bexarotene, dasatinib), whereas low-risk patients showed enhanced response to immunotherapy. Among six subtype-discriminatory tsRNAs, tsRNA-Asp-3-0024, a previously unannotated fragment, was significantly upregulated in GC tissues and cell lines via Pandora-seq and qRT-PCR validation. Clinically, elevated tsRNA-Asp-3-0024 expression independently predicted adverse prognosis (multivariate Cox P<0.001). Functional studies confirmed its tumor-promotive role: knockdown suppressed proliferation, induced apoptosis in GC cells, and recapitulated the phenotypes in patient-derived organoids. Our work establishes tsRNAs as key regulators of GC heterogeneity, providing a clinically applicable subtyping framework and nominating tsRNA-Asp-3-0024 as a novel therapeutic target.