Discovery of DNA Methylation-Driven Genes in Hepatocellular Carcinoma via Multi-Omics Integration and Functional Role of PHYHD1

Objective: Hepatocellular carcinoma (HCC) is characterized by profound epigenetic dysregulation, particularly in DNA methylation. This study leverages integrated multi-omics to systematically identify key methylation-driven genes in HCC, with a subsequent focus on characterizing the expression patterns and biological functions of a previously understudied candidate, PHYHD1 . Methods: We collected five pairs of HCC tissues and matched adjacent non-tumor counterparts for integrated multi-omics profiling, including whole-genome bisulfite sequencing (WGBS), RNA sequencing (RNA-seq), and TMT-based quantitative proteomics. Differentially methylated regions (DMRs), differentially expressed genes (DEGs), and differentially expressed proteins (DEPs) were identified. Bioinformatic analyses, including functional enrichment and protein-protein interaction (PPI) network construction, were used to prioritize candidate genes, which were further validated using public datasets (TCGA, GEO). Methylation status and expression of PHYHD1 were verified using RT-qPCR, Western blot, and immunohistochemistry (IHC) in cell lines, patient tissues, and a DEN/CCl₄-induced murine HCC model. Functional impact of PHYHD1 on HCC cell proliferation, apoptosis, and tumorigenicity was assessed via in vitro assays (CCK-8, colony formation, flow cytometry) and an in vivo Phyhd1 knockout mouse model. Results: WGBS revealed global hypo-methylation in HCC, identifying 97,523 DMRs. Integration of methylome, transcriptome, and proteome data yielded 19 genes with consistent differential status across all three levels. Functional annotation showed enrichment in metabolic pathways, including retinol, tyrosine, and pyruvate metabolism. Although PHYHD1 was hyper-methylated and down-regulated at both mRNA and protein levels in HCC, its gain- or loss-of-function did not significantly affect cell proliferation, colony formation, apoptosis in vitro , or liver tumorigenesis in vivo . Conclusion: Our integrated multi-omics approach identified a panel of potential DNA methylation-driven genes in HCC. PHYHD1 was confirmed as an epigenetically silenced gene in HCC, but its manipulation did not alter classic malignant phenotypes, suggesting it may not act as a canonical driver gene. Its silencing may represent a passenger event or influence tumor progression through non-cell-autonomous mechanisms. The biological role of PHYHD1 warrants further investigation.