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

<b>Background:</b> 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. <b>Methods:</b> 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. <b>Results:</b> 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 <i>CNTN1</i>, <i>NCAM1</i>, <i>PLP1</i>, <i>GPM6B</i>, <i>SLC1A3</i>, and <i>PMP2</i>. 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 <i>CNTN1</i> promotes HC cell invasion in vitro and metastasis in vivo. Mechanistically, <i>CNTN1</i> exerts its pro-invasive effects by activating the PI3K-AKT signaling pathway and inducing epithelial-mesenchymal transition (EMT). <b>Conclusions:</b> Our integrated analysis identifies <i>CNTN1</i> as a critical oncogenic driver in HC, promoting metastasis through PI3K-AKT-mediated EMT. These findings nominate <i>CNTN1</i> as a potential prognostic biomarker and therapeutic target in HC.