Clinical utility of cell-free urine miR-93-5p, miR-191-5p, miR-31-5p for invasive urothelial carcinoma detection and immune signature-based subtyping

This study aimed to explore the potential value of urinary cell-free microRNA (miR)-93-5p, miR-191-5p, and miR-31-5p levels in the non-invasive prediction of basal-like and luminal-like invasive urothelial carcinoma immune signature phenotypes according to the molecular classification of bladder cancer, by comparing their expression in patients and healthy controls. The study included morning urine samples from 49 bladder cancer patients and 43 controls. A quantitative image-based immunohistochemical analysis classified tumor cases into basal-like and luminal-like subtypes. Quantitative real-time PCR (qRT-PCR) was used to measure cell-free miR-93-5p, miR-191-5p, and miR-31-5p expression levels in urine. MiR-191-5p was significantly downregulated in bladder cancer patients compared to healthy controls (p < 0.001), with a 24-fold decrease. Notably, miR-191-5p levels were markedly lower in the luminal-like subtype relative to the control group (p < 0.001). In contrast, miR-93-5p was significantly upregulated in the basal-like subtype, showing a 4.15-fold increase compared to controls (p < 0.001). Elevated levels of miR-93-5p were also observed in high-grade tumors (3.4-fold, p = 0.004), in tumors exhibiting necrosis (3.4-fold, p < 0.001), and in the presence of carcinoma in situ (CIS) (2.6-fold, p = 0.02). Furthermore, miR-93-5p levels showed a positive correlation with tumor size (r = 0.41, p < 0.001). In this exploratory cohort, miR-93-5p also demonstrated strong discriminatory performance in identifying CK5/6-positive tumors, with a receiver operating characteristic (ROC) curve cut-off value of 1.57 for 2^ΔΔCT value. In this single-center, exploratory study, urinary cell-free miR-93-5p and miR-191-5p showed potential utility as non-invasive biomarkers for rapid molecular subtype identification in bladder cancer. miR-93-5p was associated with basal-like (aggressive) tumor features, while miR-191-5p was inversely associated with invasive urothelial carcinoma. miR-31-5p appears to serve as a complementary marker, especially in cases of CIS.