Knockdown of cullin 3 inhibits progressive phenotypes and increases chemosensitivity in cholangiocarcinoma cells.

Abstract

Cholangiocarcinoma (CCA) is an extremely aggressive malignancy arising from the epithelial cells lining the bile ducts. It presents a substantial global health issue, with the highest incidence rates, ranging from 40‑100 cases/100,000 individuals, found in Southeast Asia, where liver fluke infection is endemic. In Europe and America, incidence rates range from 0.4‑2 cases/100,000 individuals. Globally, mortality rates range from 0.2‑2 deaths/100,000 person‑years and are increasing in most countries. Chemotherapy is the primary treatment for advanced CCA due to limited options from late‑stage diagnosis, but its efficacy is hindered by drug‑resistant phenotypes. In a previous study, proteomics analysis of drug‑resistant CCA cell lines (KKU‑213A‑FR and KKU‑213A‑GR) and the parental KKU‑213A line identified cullin 3 (Cul3) as markedly overexpressed in drug‑resistant cells. Cul3, a scaffold protein within CUL3‑RING ubiquitin ligase complexes, is crucial for ubiquitination and proteasome degradation, yet its role in drug‑resistant CCA remains to be elucidated. The present study aimed to elucidate the role of Cul3 in drug‑resistant CCA cell lines. Reverse transcription‑quantitative PCR and western blot analyses confirmed significantly elevated Cul3 mRNA and protein levels in drug‑resistant cell lines compared with the parental control. Short interfering RNA‑mediated Cul3 knockdown sensitized cells to 5‑fluorouracil and gemcitabine and inhibited cell proliferation, colony formation, migration and invasion. In addition, Cul3 knockdown induced G₀/G₁ cell cycle arrest and suppressed key cell cycle regulatory proteins, cyclin D, cyclin‑dependent kinase (CDK)4 and CDK6. Bioinformatics analysis of CCA patient samples using The Cancer Genome Atlas data revealed Cul3 upregulation in CCA tissues compared with normal bile duct tissues. STRING analysis of upregulated proteins in drug‑resistant CCA cell lines identified a highly interactive Cul3 network, including COMM Domain Containing 3, Ariadne RBR E3 ubiquitin protein ligase 1, Egl nine homolog 1, Proteasome 26S Subunit Non‑ATPase 13, DExH‑box helicase 9 and small nuclear ribonucleoprotein polypeptide G, which showed a positive correlation with Cul3 in CCA tissues. Knocking down Cul3 significantly suppressed the mRNA expression of these genes, suggesting that Cul3 may act as an upstream regulator of them. Gene Ontology analysis revealed that the majority of these genes were categorized under binding function, metabolic process, cellular anatomical entity, protein‑containing complex and protein‑modifying enzyme. Taken together, these findings highlighted the biological and clinical significance of Cul3 in drug resistance and progression of CCA.