Stage-Specific miRNA Profiling Reveals Key Regulators of EMT and EGFR-TKI Resistance in Gallbladder Cancer

Background: Gallbladder cancer (GBC) is a highly aggressive malignancy characterized by a poor prognosis, particularly in its advanced stages. While microRNAs (miRNAs) regulate cancer progression, their specific role in the transition from early to advanced GBC is poorly understood. Methods: We performed miRNA expression profiling on 41 formalin-fixed paraffin-embedded (FFPE) tissues, including 10 gallstone disease (GSD) controls, 14 early-stage GBC (stage I and II), and 17 advanced-stage GBC cases (stage III and IV), using the NanoString nCounter platform. Differentially expressed miRNAs (DEMs) were identified followed by miRNA target identification using miRTarBase. Results: We identified 43 significantly dysregulated miRNAs in early-stage and 46 in advanced-stage GBC compared to controls. Based on the literature search, we found EMT-inhibiting miRNAs (miR-200 family) to be overexpressed in early stage and downregulated in advanced stages (miR-574-3p, miR-195-5p) in our study. Pathway analysis revealed significant enrichment of the 'EGFR tyrosine kinase inhibitor resistance' pathway in both the stages. The correlation of DEMs with clinicopathological features revealed that the expression of miR-361-3p and miR-423-5p was significantly associated with tumor grade (r = -0.605, p = 0.0003) and lymph node status (r = -0.621, p = 0.0001), respectively. Conclusions: This study identifies distinct miRNA signatures associated with GBC initiation and progression, offering insights into the molecular pathogenesis of the disease. Furthermore, functional studies of the miRNAs implicated in EMT and EGFR-TKI resistance may be conducted using GBC cell lines to dissect the precise roles of key miRNAs and explore their potential as novel therapeutic targets in GBC.