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

## TL;DR

This study identifies specific microRNAs involved in gallbladder cancer progression and resistance to treatment, offering potential new therapeutic targets.

## Contribution

The study reveals stage-specific miRNA profiles linked to EMT and EGFR-TKI resistance in gallbladder cancer.

## Key findings

- miR-200 family is overexpressed in early-stage gallbladder cancer but downregulated in advanced stages.
- miR-361-3p and miR-423-5p are associated with tumor grade and lymph node involvement.
- The EGFR tyrosine kinase inhibitor resistance pathway is enriched in both early and advanced stages.

## Abstract

Gallbladder cancer is one of the most aggressive cancers, often detected late, when treatment is less effective. To better understand how it progresses from early to advanced stages, we studied small molecules called microRNAs (miRNAs) that control how genes work. Using tissue samples from patients with gallstones, early-stage gallbladder cancer, and advanced-stage cancer, we compared miRNA levels. We found that some miRNAs that help stop cancer spread (like the miR-200 family) were active in early stages but decreased in advanced cancer. Other miRNAs, such as miR-361-3p and miR-423-5p, were linked to tumor grade and lymph node involvement. Our analysis also revealed that a key cell growth pathway, related to EGFR resistance, was active in both stages. These findings help explain how gallbladder cancer develops and could guide future research to design new treatments that target specific miRNAs involved in cancer growth and spread.

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.