Identification of Key Genes and Pathways in Lenvatinib-resistant Hepatocellular Carcinoma using Bioinformatic Analysis and Experimental Validation

Background: Resistance to lenvatinib poses a serious threat to the therapy of patients with Hepatocellular Carcinoma (HCC). The mechanism by which HCC develops resistance to lenvatinib is currently unknown. Objective: The aim of this study was to identify key genes and pathways involved in lenvatinib resistance in HCC using bioinformatic analysis and experimental validation. Methods: Differentially expressed genes (DEGs) were identified from the GSE186191 gene expression profile, comparing HCC cell lines with lenvatinib-resistant HCC cell lines. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were then carried out using DAVID. A protein-protein interaction network was constructed to visualize DEGs and identify hub genes. The expression and prognostic significance of these hub genes were further examined. Additionally, genomic enrichment analysis (GSEA) was utilized to investigate the potential functions of key genes. Following this, the presence of AHSG was validated in both the original Huh7 cells and the lenvatinib-resistant Huh7 (Huh7LR) cells resistant to lenvatinib through the utilization of quantitative real-time PCR (qRT-PCR). Results: A total of 232 DEGs were identified between HCC cell lines and those that are resistant to lenvatinib. These DEGs were significantly associated with arrhythmogenic right ventricular cardiomyopathy, hypertrophic cardiomyopathy, dilated cardiomyopathy, and mucin-type O-glycan biosynthesis. Three hub genes, including AHSG, C6, and ORM1, were identified. The low expression of AHSG showed a poorer prognosis in HCC. GSEA demonstrated a significant correlation between low AHSG expression and pathways involving fatty acid metabolism, ribosome function, glycine, serine, and threonine metabolism, peroxisome activity, and bile acid biosynthesis. The expression of AHSG was notably reduced in Huh7LR cells (p = 0.006) compared to Huh7 cells. Conclusion: Diminished AHSG expression is strongly associated with lenvatinib resistance in HCC, suggesting that it may have implications for developing effective strategies to overcome this resistance.