Abstract
Background: Hepatocellular carcinoma (HCC) remains one of the deadliest malignancies globally, posing substantial health threats and economic burdens. Despite advances in therapeutic modalities-including surgical resection, liver transplantation, and targeted therapies-HCC prognosis remains unsatisfactory. This highlights an urgent need for novel biomarkers to improve prognostic stratification. Senescence-related genes (SRGs) have emerged as promising candidates for HCC prognostic exploration, making their association with clinical outcomes the focus of this study.. Methods: Comprehensive machine learning was applied to screen SRGs associated with HCC survival, identifying key prognostic genes. A clinical nomogram based on SRG scores was constructed and validated. NAP1L4 expression in HCC was verified using GEPIA2.0, UALCAN, HPA databases, and clinical samples from 40 patients at the First Affiliated Hospital of Anhui Medical University. In vitro, NAP1L4 was knocked down in HCCLM3 cells to assess proliferation and migration changes. RNA-seq analyzed differentially expressed genes post-knockdown, followed by GSEA/KEGG enrichment to identify relevant pathways. Drug sensitivity and Mendelian randomization analyses explored therapeutic implications and gut microbiota-related HCC risk modulation.. Results: Machine learning identified NAP1L4 as a pivotal SRG, with the SRG score-based nomogram strongly associated with poor overall survival. High NAP1L4 expression in HCC was confirmed by multi-omics and clinical samples. NAP1L4 knockdown in HCCLM3 cells significantly inhibited proliferation and migration. RNA-seq revealed 129 upregulated and 101 downregulated genes post-knockdown, with GSEA/KEGG highlighting the NOD-like receptor pathway as critical for NAP1L4-mediated HCC effects. Elevated NAP1L4 correlated with adverse prognosis and tumor microenvironment alterations (increased M0 macrophages and regulatory T cells). Reduced NAP1L4 expression enhanced sensitivity to cisplatin and axitinib. Mendelian randomization showed ANGPT1 modulates HCC risk by increasing gut Faecalitalea cylindroides abundance.. Conclusion: SRGs, particularly NAP1L4, play critical roles in HCC progression. High NAP1L4 expression associates with poor prognosis, tumor microenvironment remodeling, and altered drug sensitivity. These findings support SRGs as potential prognostic biomarkers and therapeutic targets to enhance HCC treatment efficacy.