Genome-wide DNA methylation profiles by high-throughput techniques in alcohol-related hepatocellular carcinoma: identification of epigenetic signatures in liver tissue and peripheral blood cells DNA as potentially useful biomarkers of disease

DNA methylation is the major epigenetic feature of eukaryotic cell DNA and consists in the covalent binding of a methyl group to the 5’ carbon of a cytosine in a CpG dinucleotide sequence and acts regulating gene expression. Methyl-transfer reactions occur within one-carbon metabolism pathway that takes place principally in the liver: hepatic tissue that is, therefore, to be considered among the most interesting target tissues for DNA methylation analysis. Moreover alcohol, a major risk factor for hepatic cancer, is known to disturb one-carbon metabolism but the mechanisms underlying the alcohol-related liver carcinogenesis are still incompletely understood. We, therefore, designed this study to investigate DNA methylation profiles in alcohol-related hepatocellular carcinoma (HCC) by high-throughput techniques for genome-wide analysis. Main scope of the present project was to define a possible role for DNA methylation in the development of non-viral alcohol-related HCC in DNA obtained either from liver tissue and peripheral blood mononuclear cells (PBMCs) with the final goal of identifying potentially useful epigenetic biomarkers for HCC from an easily accessible DNA source, namely PBMCs. The methylation status and the transcriptional levels of all the annotated genes were assessed on liver HCC tissue compared to homologous non-neoplastic tissue using a genome-wide, array-based approach in eight patients undergoing curative surgery. The merging of DNA methylation and gene expression data allowed identifying hypermethylated and transcriptional repressed genes among which six genes belonging to retinol metabolism (ADH1A, ADH1B, ADH6, CYP3A43, CYP4A22 and RDH16). Among other hypermethylated-repressed genes, was detected a key gene of one-carbon metabolism, SHMT1, ESR1, a transcription factor with an hormone-binding domain involved in cell cycle regulation, and hepcidin, a liver peptide hormone involved in iron homeostasis were also identified as epigenetically regulated through DNA methylation inducing transcriptional repression. Interestingly, the gene expression analysis on RNA extracted from buffy coat of HCC patients, alcoholic patients without liver cancer and healthy subjects revealed that transcriptional repression of RDH16 was significantly associated with hepatic cancer. Moreover, the down-regulation of RDH16, SHMT1 and ESR1 was associated to chronic alcohol intake compared to controls. These findings suggest that expression profiles of specific genes obtained from PBMCs may be useful biomarkers for HCC.