Frontiers in genetics · 2026
Epigenetic silencing of PHYHD1 in hepatocellular carcinoma revealed by integrated multi-omics analysis.
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Guo Tianfu, Li Yafei, He Tiansheng, Luo Hui, Liu Yuwen, Xie Binhui, Liu Zhiping
Аннотация
INTRODUCTION: Hepatocellular carcinoma (HCC) is characterized by extensive epigenetic alterations, particularly DNA methylation. Through integrated multi-omics analysis, we identified methylation-driven genes in HCC, including PHYHD1-a previously understudied candidate whose role in hepatocarcinogenesis remains unexplored. This study aimed to validate and functionally characterize PHYHD1 in HCC.
METHODS: Five pairs of HCC and adjacent non-tumor tissues were analyzed using whole-genome bisulfite sequencing (WGBS), RNA-seq, and TMT-based proteomics. We identified 97,523 differentially methylated regions (DMRs), 875 differentially expressed genes (DEGs), and 225 differentially expressed proteins (DEPs). PHYHD1 hypermethylation was confirmed by public methylation datasets, and its mRNA/protein expression was validated by RT-qPCR, Western blot, and immunohistochemistry in cell lines, patient tissues (n = 76 pairs), and a DEN/CCl4-induced murine HCC model. Functional effects were assessed via in vitro assays (CCK-8, colony formation, flow cytometry) and an in vivo Phyhd1 knockout model.
RESULTS: Integration of methylome, transcriptome, and proteome data revealed 19 consistently altered genes enriched in metabolic pathways. PHYHD1 was hypermethylated and downregulated at both mRNA and protein levels in HCC. However, gain- or loss-of-function of PHYHD1 had no detectable effect on proliferation, apoptosis, or tumorigenicity in vitro or in vivo (all P > 0.05).
DISCUSSION: Integrated multi-omics analysis identified PHYHD1 as an epigenetically silenced gene in HCC, but functional studies indicate that its silencing does not act as a canonical driver of malignant phenotypes. Instead, PHYHD1 hypermethylation may represent a passenger event or a context-dependent modulator that only exerts effects under specific stress conditions (e.g., hypoxia or metabolic inflammation). These findings suggest that not all methylation-driven genes in HCC are functional drivers, and they highlight the need for further investigation into passenger epigenetic alterations in tumor biology.
METHODS: Five pairs of HCC and adjacent non-tumor tissues were analyzed using whole-genome bisulfite sequencing (WGBS), RNA-seq, and TMT-based proteomics. We identified 97,523 differentially methylated regions (DMRs), 875 differentially expressed genes (DEGs), and 225 differentially expressed proteins (DEPs). PHYHD1 hypermethylation was confirmed by public methylation datasets, and its mRNA/protein expression was validated by RT-qPCR, Western blot, and immunohistochemistry in cell lines, patient tissues (n = 76 pairs), and a DEN/CCl4-induced murine HCC model. Functional effects were assessed via in vitro assays (CCK-8, colony formation, flow cytometry) and an in vivo Phyhd1 knockout model.
RESULTS: Integration of methylome, transcriptome, and proteome data revealed 19 consistently altered genes enriched in metabolic pathways. PHYHD1 was hypermethylated and downregulated at both mRNA and protein levels in HCC. However, gain- or loss-of-function of PHYHD1 had no detectable effect on proliferation, apoptosis, or tumorigenicity in vitro or in vivo (all P > 0.05).
DISCUSSION: Integrated multi-omics analysis identified PHYHD1 as an epigenetically silenced gene in HCC, but functional studies indicate that its silencing does not act as a canonical driver of malignant phenotypes. Instead, PHYHD1 hypermethylation may represent a passenger event or a context-dependent modulator that only exerts effects under specific stress conditions (e.g., hypoxia or metabolic inflammation). These findings suggest that not all methylation-driven genes in HCC are functional drivers, and they highlight the need for further investigation into passenger epigenetic alterations in tumor biology.