Urinary TAGMe methylation enables preoperative risk stratification and reveals enhancer‑mediated regulation in urothelial carcinoma

Accurate risk stratification in urothelial carcinoma (UC) remains challenging due to marked molecular heterogeneity, underscoring the potential value of non‑invasive biomarkers for improving risk stratification. TAGMe is an intergenic urinary DNA methylation marker that we previously developed for UC detection. Here, we investigated its potential value for preoperative prognostic stratification and explored its underlying biological function in UC. Preoperative urinary TAGMe methylation was measured by methylation-sensitive restriction enzyme quantitative PCR in 375 patients with UC and analyzed in relation to clinicopathological features and survival outcomes using Kaplan–Meier and Cox regression analyses. TAGMe was integrated with conventional clinicopathological variables to develop prognostic models and a nomogram, which were evaluated using the concordance index and time-dependent ROC analysis. Multi-omics analyses integrating ATAC-seq, Hi-C, ChIP-seq, CUT&RUN, and TCGA data were used to characterize the regulatory properties of the TAGMe locus. Dual-luciferase, DNA demethylation, and gain-of-function experiments were subsequently performed to validate TAGMe enhancer activity and investigate the functional role of BHLHE40-AS1. Preoperative TAGMe levels were associated with adverse pathological features and poorer recurrence-free, progression-free, and overall survival. Incorporating TAGMe into a prognostic nomogram improved risk discrimination beyond conventional clinicopathological variables and refined stratification within established pathological subgroups. Integrative epigenomic analyses revealed that TAGMe resides within an active enhancer‑like region marked by H3K27ac and H3K4me1 and physically interacts with the lncRNA BHLHE40‑AS1 locus. TAGMe activated BHLHE40‑AS1 transcription in a methylation‑dependent manner, and TAGMe hypermethylation correlated with reduced BHLHE40‑AS1 expression. Functional assays demonstrated that BHLHE40‑AS1 suppresses proliferation, migration, and epithelial–mesenchymal transition in bladder cancer cells. Urinary TAGMe methylation may complement conventional clinicopathological factors for preoperative risk stratification in UC, while the methylation-sensitive TAGMe–BHLHE40-AS1 enhancer axis provides a mechanistic link between intergenic DNA methylation and tumor progression.