Integration of Multi-Omics Data Identifies the Role of the Selenium-Related Gene PNPO and Pre-exhausted CD127 ⁻ CD8 ⁺ T Cells in Laryngeal Carcinoma

Objective: To elucidate mechanistic links among selenium measures, selenium-related genes, and immune traits in laryngeal carcinoma (LC) by integrating Mendelian randomization, bulk transcriptomic, and single-cell analyses. Methods: Two-sample Mendelian randomization (MR) was used to evaluate the causal effects of selenium measures and genetically predicted expression of selenium-related genes instrumented by eQTLs on LC risk, and immune traits were screened as candidate mediators via a directionality-based filter. We then examined PNPO expression in relation to pathway activity and immune infiltration in the TCGA and GEO cohorts. Single-cell RNA sequencing was further analyzed to characterize PNPO-associated programs in malignant epithelial and immune cells. The scPagwas and deconvolution algorithms were applied to prioritize disease-relevant T-cell subsets, and hdWGCNA was used to identify phenotype-associated gene modules. Intercellular communication, signaling activity, developmental trajectories, and transcription factor programs were evaluated in complementary analyses. The reporting of this study conforms to the STROBE-MR guideline for Mendelian randomization studies. Results: Genetically predicted selenium measures showed no evidence of a causal association with LC risk. In contrast, PNPO showed evidence consistent with involvement in LC susceptibility, potentially involving CD127 − CD8 + T cells. In bulk transcriptomic cohorts, tumors with low PNPO expression were enriched for oncogenic pathways and were associated with poorer survival. PNPO expression correlated positively with inferred CD8 + T-cell infiltration. At single-cell resolution, PNPO − malignant epithelial cells displayed transcriptional features consistent with stemness, drug resistance, and immune-evasion programs. We further identified a pre-exhausted CD127 − CD8 + T-cell subset with distinct molecular and functional characteristics. Conclusion: These analyses implicate PNPO-linked vitamin B6 metabolism and a pre-exhausted CD127 − CD8 + T-cell state in LC, highlighting candidates for mechanistic validation and potential therapeutic exploration.