CDT1-Mediated Regulation of DNA Damage Repair and Immune-Related Target Genes Underlies Hepatocellular Carcinoma Development

Background Hepatocellular carcinoma (HCC) progression is tightly driven by genomic instability and dysregulated DNA damage repair (DDR) pathways. The replication licensing factor Chromatin Licensing and DNA Replication Factor 1 (CDT1) is frequently overexpressed in HCC tissues; however, the molecular mechanisms by which CDT1 coordinates DDR programs and modulates tumor immune microenvironment remain poorly elucidated. This study aimed to systematically characterize CDT1-centered regulatory networks in HCC, delineate their dual impacts on DDR machinery and immune-related signaling pathways, and validate the clinical translational value of CDT1 as a potential biomarker or therapeutic target. Methods A stable CDT1 knockdown (CDT1-KD) cell model was established in Huh7 HCC cells using lentiviral short hairpin RNA (shRNA), with silencing efficiency verified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot analysis. DNA double-strand breaks (DSBs) were quantified via γ-H2AX immunofluorescence staining. RNA sequencing (RNA-seq) was performed to profile CDT1-dependent transcriptional alterations, followed by differential expression analysis using DESeq2, Gene Ontology (GO) functional annotation, and Gene Set Enrichment Analysis (GSEA). Chromatin immunoprecipitation sequencing (ChIP-seq) was employed to map genome-wide CDT1 chromatin occupancy, which was further integrated with the transcriptome data to prioritize direct CDT1 target genes. The involvement of CDT1 in immune regulation was investigated by intersecting CDT1-bound and CDT1-regulated genes with manually curated immune-related gene sets. Finally, the clinical relevance of CDT1 expression in HCC was analyzed using public databases, including The Cancer Genome Atlas (TCGA), UALCAN, and the Human Protein Atlas (HPA). Results CDT1 silencing significantly increased the number of γ-H2AX foci in Huh7 cells, indicating exacerbated accumulation of DNA DSBs. RNA-seq analysis identified 4,581 CDT1-dependent differentially expressed genes (DEGs), including 2,739 upregulated and 1,842 downregulated transcripts. Functional enrichment analysis of downregulated DEGs revealed significant enrichment in biological processes related to DNA replication initiation, homologous recombination (HR), and broader DDR cascades—consistent with GSEA results showing suppressed activity of DDR-related gene sets in CDT1-KD cells. ChIP-seq data demonstrated that CDT1 primarily binds to intergenic and intronic regions of the genome; integration with RNA-seq data identified 328 CDT1-bound DEGs, which were enriched in signal transduction and cell proliferation pathways (e.g., HNRNPD, FBXL4, DROSHA). Intersection with immune gene catalogs yielded 307 immune-related CDT1 target genes; notably, CDT1 knockdown led to reduced expression of key immune regulators such as SEMA3D, CHUK, and PIK3R3, implicating CDT1 in the modulation of HCC immune signaling. Clinically, CDT1 expression was significantly upregulated in HCC tissues compared to adjacent non-tumor tissues, and high CDT1 expression was independently associated with poorer overall survival in HCC patients. Conclusions CDT1 functions as a pivotal oncogenic regulator in HCC by coupling maintenance of DDR proficiency with control of immune-related gene expression. Its dual roles in sustaining DNA replication/repair programs and shaping the tumor immune microenvironment highlight CDT1 as a promising prognostic biomarker and a potential therapeutic target for HCC. Targeting CDT1 may not only disrupt tumor genomic stability but also enhance responsiveness to immunotherapy, thereby addressing the challenge of tumor heterogeneity in HCC treatment.