Abstract
BACKGROUND: R-loops, RNA-DNA hybrid structures, play essential roles in maintaining genomic stability and regulating transcription. This study aims to identify key R-loop regulatory genes as prognostic markers for LUAD and explore their associations with immunotherapy response and drug sensitivity, supporting personalized treatment strategies. METHODS: We integrated 1771 R-loops genes with differentially expressed genes in LUAD. Through univariate Cox, LASSO, and multivariate Cox analyses, we constructed an R-loops prognostic risk score (R-loops Score) and validated it in three independent GEO cohorts. Correlations with clinical variables, immune features, and drug response were examined. Key genes were further evaluated by qPCR and Western blot in LUAD cell lines and tumor tissues. RESULTS: Patients with high R-loops Scores had significantly poorer overall survival compared with low-score patients. A nomogram combining the R-loops Score and clinical factors achieved AUCs of 0.732, 0.713, and 0.719 for predicting 1-, 2-, and 5-year OS, respectively. Pathway enrichment indicated that high-score tumors were enriched in cell cycle regulation, phase separation, and epithelial-mesenchymal transition. High R-loops Scores were associated with male sex, advanced stages, immune evasion and immunotherapy resistance, but increased chemotherapy and targeted therapy sensitivity. EIF3B was further validated as a key gene through analysis of a local patient cohort. CONCLUSION: The R-loops Score represents a promising prognostic tool for LUAD, offering valuable insights into survival outcomes, immune characteristics and drug responsiveness. Notably, qPCR and Western blot validation consistently confirmed EIF3B as a key gene, further supporting their potential as biomarkers. These results support future research and serve as a reference for the personalized precision treatment of LUAD.