Bile‐Derived Exosomal <scp>miR</scp> ‐196a/‐196b as Diagnostic Biomarkers Associated With Tumor Progression in Biliary Tract Cancer

Bile-based liquid biopsy presents a promising approach for identifying novel biomarkers for biliary tract cancer (BTC) given its proximity to tumor sites. The aim of this study was to identify bile-derived exosomal microRNAs (miRNAs) with diagnostic potential for BTC. The candidate miRNAs were initially screened by RNA sequencing of exosomal RNA from the bile samples of 10 BTC patients and 10 controls, resulting in the identification of miR-196a, miR-196b, and miR-424. Subsequent validation using TaqMan PCR in an independent cohort (50 BTC, 53 controls) confirmed that miR-196a and miR-196b were specifically elevated in the bile of BTC patients, with miR-196a also elevated in early-stage BTC. MiR-196a achieved an area under the receiver operating characteristic curve (AUROC) of 0.79 for the diagnosis of BTC. The combination of miR-196a and CA19-9 increased the AUROC to 0.86, and further improvement to 0.88 was observed upon the addition of CEA. Furthermore, serum levels of these miRNAs were analyzed in 64 BTC patients and 79 controls; serum miR-196a levels were significantly elevated in patients with metastatic BTC. Analysis of public datasets confirmed that both miR-196a and miR-196b were significantly upregulated in invasive BTC tissues compared to normal biliary epithelium and biliary intraepithelial neoplasia. Functional assays demonstrated that miR-196a and miR-196b promoted the proliferation and invasion of cholangiocarcinoma cells (p < 0.05). In conclusion, bile-derived exosomal miR-196a and miR-196b are novel BTC-specific biomarkers. MiR-196a may contribute to early BTC detection, enhance diagnostic accuracy in combination with serum markers, and assist in detecting metastasis.