Abstract
PURPOSE: The aim of this study was to investigate DNA methylation alterations in non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinomas (HCCs). METHODS: Genome-wide DNA methylation analysis was performed using the Infinium Human Methylation 450 K BeadChip, and levels of mRNA expression were analyzed by quantitative reverse transcription-PCR. RESULTS: Compared to 36 samples of normal control liver tissue (C), DNA methylation alterations were observed on 19,281 probes in 22 samples of cancerous tissue (T) obtained from patients showing histological features compatible with NASH in their non-cancerous liver tissue (N). Among those probes, 1396 were located within CpG islands or their shores and shelves, designed around the transcription start sites of 726 genes. In representative genes, such as DCAF4L2, CKLF, TRIM4, PRC1, UBE2C and TUBA1B, both DNA hypomethylation and mRNA overexpression were observed in T samples relative to C samples, and the levels of DNA methylation and mRNA expression were inversely correlated with each other. DNA hypomethylation occurred even in N samples at the precancerous NASH stage, and this was inherited by or further strengthened in T samples. DNA hypomethylation of DCAF4L2, CKLF and UBE2C was observed in both NASH-related and viral hepatitis-related HCCs, whereas that of TRIM4, PRC1 and TUBA1B occurred in a NASH-related HCC-specific manner. DNA hypomethylation and/or mRNA overexpression of these genes was frequently associated with the necroinflammatory grade of NASH and was correlated with poorer tumor differentiation. CONCLUSION: DNA methylation alterations may occur under the necroinflammatory conditions characteristic of NASH and participate in NASH-related hepatocarcinogenesis through aberrant expression of tumor-related genes.