Screening and mechanistic investigation of key radioresistance genes in oral squamous cell carcinoma

To identify key candidate genes associated with radioresistance in oral squamous cell carcinoma (OSCC), elucidate their regulatory mechanisms and molecular pathways. This study integrated bulk transcriptomic data from TCGA-OSCC and GEO databases with single-cell RNA sequencing (scRNA-seq) data. Key prognostic genes were identified through differential expression analysis, protein-protein interaction (PPI) network analysis, and Cox and Lasso regression modeling. Single-cell analysis was performed to validate gene expression patterns and cellular mechanisms. The expression levels of the selected genes were subsequently verified by RT-qPCR and Western blot (WB) analyses. From the intersection of 7695 OSCC-related and 66 radiotherapy-related differentially expressed genes, we identified 39 radioresistance-associated candidate genes. Subsequent Cox and Lasso regression analyses revealed two key prognostic genes: TNFRSF19 (protective factor, HR < 1) and CCNA1 (risk factor, HR > 1). The risk prediction model demonstrated moderate but significant predictive performance, with AUCs of 0.62, 0.64, and 0.64 for 1-year, 3-year, and 5-year survival, respectively. Single-cell analysis revealed that macrophages represent a pivotal cell type mediating radioresistance. Pseudotime trajectory analysis showed that TNFRSF19 expression increased during macrophage maturation, promoting anti-tumor polarization, while CCNA1 expression decreased, facilitating functional differentiation. RT-qPCR revealed a dose-dependent increase in TNFRSF19 mRNA and a concomitant decrease in CCNA1 mRNA with escalating radiation, which was corroborated at the protein level by Western blotting. TNFRSF19 and CCNA1 serve as critical regulators of radioresistance in OSCC, exerting their effects through modulation of macrophage differentiation and function. These findings provide novel biomarkers for predicting radiotherapy response and potential therapeutic targets for synergistic radiosensitization strategies.