23Functional and mechanistic role of lncRNA-A1 in cholangiocarcinoma
A Tinahones-Ruano, J Blázquez-Vicens, A Aparicio-Rey, E M Esquinas-Román, I Maseda, C Riobello, A Capelo-Diz, J Cañas-Martín, E Larraona, S Perdikari, C García, M García-Fernandez de Barrena, B Porteiro, J Lozano, M Coll, P Sancho-Bru, E Gonzalez-Sanchez, J M Bañales, P Milkiewicz, M Milkiewicz, L Bujanda-Fernández, M A Avila, A Aransay, R Nogueiras, B Pelacho, J Amengual, I Fabregat, J Vaquero, R Entrialgo-Cadiero, S Vicent, M E Guicciardi, G J Gores, A Arana, L E Sanchez Piñon, M Fildalgo, A Woodhoo, M Varela-ReyAbstract
Background and Aims
Cholangiocarcinoma (CCA) represents a highly heterogeneous group of malignant biliary tumors that can arise throughout the biliary tree. Their silent onset, aggressive biology, and resistance to chemotherapy contribute to the alarming mortality associated with CCA, accounting for 2% of all annual cancer-related deaths worldwide. Long non-coding RNAs (lncRNAs) interact with DNA, RNA, and proteins to regulate global gene expression, acting as versatile modulators of numerous biological processes and playing central roles in the pathogenesis of diverse disorders. Nevertheless, their specific contribution to cholangiocarcinoma development remains largely uncharacterized. In this study, our main objective is to investigate the biological function and molecular mechanism of lncRNA-A1 in CCA progression.
Methods
We analyzed lncRNA-A1 expression in liver samples from patients with primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC), as well as in mouse models of cholestasis, cholangiocarcinoma, and several CCA cell lines. Functional studies were subsequently performed through in vitro and in vivo targeting of lncRNA-A1 in selected experimental systems.
Results
lncRNA-A1 was markedly upregulated in liver samples from PBC and PSC patients, and consistently elevated across three distinct cholestasis mouse models at different time points: bile duct ligation (BDL), mice fed with a cholic acid diet, and MDR2-KO mice. At the cellular level, we observed specific upregulation of lncRNA-A1 in the biliary tree of MDR2-KO mice. In vitro, EGF stimulation increased lncRNA-A1 expression, while its inhibition impaired EGF-driven growth of primary cholangiocyte organoids. Remarkably, lncRNA-A1 was also broadly overexpressed in CCA cells, and its levels were consistently elevated in tumoral liver tissue from three independent CCA animal models (SB1-Syngeneic, AKT-NICD, and AKT-YAP), compared with non-tumoral counterparts. Silencing of lncRNA-A1 induced cell death and significantly reduced viability and migration in several CCA cell lines. Importantly, in vivo inhibition of lncRNA-A1 led to a substantial reduction in tumor size, further supporting its role in tumor progression. Finally, RNA-seq analysis following lncRNA-A1 silencing revealed regulation of oncogenic pathways, particularly those involving mTOR and c-Myc signaling, underscoring its potential contribution to CCA pathogenesis. Additionally, a synergistic effect was observed between chemotherapeutic CCA agents, such as gemcitabine and cisplatin, and gene silencing. In resistant cell lines, this combined approach led to significant cell death, indicating that targeting this gene may enhance therapeutic efficacy and overcome drug resistance.
Conclusion
Taken together, these findings indicate that lncRNA-A1 promotes CCA progression by activating c-Myc and mTOR signaling pathways, highlighting its potential as a therapeutic target. Also, gene silencing significantly reduced tumor growth in mouse models, highlighting its potential as a therapeutic target for patients with gemcitabine and cisplatin resistant tumors.