An R-loop-related gene signature predicts prognosis and shapes the immune microenvironment in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) features a poor prognosis and unclear pathogenesis, and the role of R-loops, key regulators of genome stability, has not been systematically characterized in this disease. Transcriptomic and single-cell RNA sequencing data of ESCC were integrated with published R-loop-related genes (RLRGs) to construct an RLRG scoring system. The associations between the RLRG score and prognosis, tumor immune microenvironment, and tumor stemness were analyzed via multiple bioinformatics algorithms. The expression of core genes was verified using reverse transcription quantitative polymerase chain reaction (RT-qPCR), Western Blot, immunohistochemistry, and immunofluorescence. Seven key RLRGs related to the prognosis of the ESCC patients were identified, among which three core genes—BRIX1, CHEK1, and NUP155—were significantly upregulated in ESCC tissues compared with controls, as confirmed by both bioinformatic analysis and experimental validation. Notably, BRIX1, CHEK1, and NUP155 were co-enriched in DNA replication, pyrimidine metabolism, RNA degradation, and MYC target pathways, which are closely linked to R-loop biology and suggest potential crosstalk between these genes and R-loop regulatory networks. Meanwhile, the low RLRGs score group exhibited significantly worse overall survival but displayed a more active yet dysfunctional immune microenvironment. Single-cell analysis identified SquamousEpithelium cells as the key cell type with the highest RLRG scores across five independent algorithms. Malignant SquamousEpithelium cells with high RLRG scores showed enhanced stemness and activation of pathways including JAK-STAT, hypoxia, and DNA repair. This study provided the first systematic characterization of the R-loop landscape in ESCC, suggests that SquamousEpithelium cells may play a key role in R-loop-associated progression and are identified as potential prognostic biomarkers with possible therapeutic relevance that requires functional validation.