Integrated Transcriptomic Analysis and Functional Validation Identify CNTN1 as a Novel Metastatic Driver in Hilar...

Background: Hilar cholangiocarcinoma (HC) is a highly aggressive malignancy with a poor prognosis, highlighting the urgent need to elucidate its molecular drivers. This study aimed to systematically identify and functionally validate key genes and pathways driving HC pathogenesis. Methods: RNA sequencing (RNA-seq) was performed on paired primary HC tumors and matched adjacent non-tumorous tissues to identify differentially expressed genes (DEGs). Subsequent bioinformatic analyses, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction (PPI) network construction, were conducted to characterize the functional landscape and identify hub genes. Transwell assays and orthotopic metastatic models were used to investigate the functions of Contactin-1 (CNTN1) in HC invasion in vitro and metastasis in vivo. Results: RNA-seq analysis identified 35 DEGs in HC, mainly involved in cell adhesion, cytoskeletal regulation, and axon development. PPI network analysis identified six hub genes, including CNTN1, NCAM1, PLP1, GPM6B, SLC1A3, and PMP2. Furthermore, we demonstrated that CNTN1, a neuronal membrane glycoprotein, was markedly up-regulated in HC at both mRNA and protein levels, and its elevated expression correlated with poor prognosis. Gain- and loss-of-function studies demonstrated that CNTN1 promotes HC cell invasion in vitro and metastasis in vivo. Mechanistically, CNTN1 exerts its pro-invasive effects by activating the PI3K-AKT signaling pathway and inducing epithelial-mesenchymal transition (EMT). Conclusions: Our integrated analysis identifies CNTN1 as a critical oncogenic driver in HC, promoting metastasis through PI3K-AKT-mediated EMT. These findings nominate CNTN1 as a potential prognostic biomarker and therapeutic target in HC.