Genome-wide DNA methylation analysis during non-alcoholic steatohepatitis-related multistage hepatocarcinogenesis: comparison with hepatitis virus-related carcinogenesis

Abstract

The aim of this study was to clarify the significance of DNA methylation alterations during non-alcoholic steatohepatitis (NASH)-related hepatocarcinogenesis. Single-CpG-resolution genome-wide DNA methylation analysis was performed on 264 liver tissue samples using the Illumina Infinium HumanMethylation450 BeadChip. After Bonferroni correction, 3331 probes showed significant DNA methylation alterations in 113 samples of non-cancerous liver tissue showing NASH (NASH-N) as compared with 55 samples of normal liver tissue (NLT). Principal component analysis using the 3331 probes revealed distinct DNA methylation profiles of NASH-N samples that were different from those of NLT samples and 37 samples of non-cancerous liver tissue showing chronic hepatitis or cirrhosis associated with hepatitis B virus (HBV) or hepatitis C virus (HCV) infection (viral-N). Receiver operating characteristic curve analysis identified 194 probes that were able to discriminate NASH-N samples from viral-N samples with area under the curve values of more than 0.95. Jonckheere-Terptsra trend test revealed that DNA methylation alterations in NASH-N samples from patients without hepatocellular carcinoma (HCC) were inherited by or strengthened in NASH-N samples from patients with HCC, and then inherited by or further strengthened in 22 samples of NASH-related HCC (NASH-T) themselves. NASH- and NASH-related HCC-specific DNA methylation alterations, which were not evident in viral-N samples and 37 samples of HCC associated with HBV or HCV infection, were observed in tumor-related genes, such as WHSC1, and were frequently associated with mRNA expression abnormalities. These data suggested that NASH-specific DNA methylation alterations may participate in NASH-related multistage hepatocarcinogenesis.

Figure 1.

Histopathological appearance of a liver biopsy specimen from a representative patient with non-alcoholic steatohepatitis. (A) Steatosis involves about 35% of hepatocytes in this case: Representative large lipid droplets (macrovesicular steatosis) and small lipid droplets (microvesicular steatosis) are indicated by arrows and arrowheads, respectively. A focus of lobular inflammation (circle) is also observed. Scale bar, 100 μm (hematoxylin and eosin staining). (B) Bridging fibrosis (arrow) and perivenular/pericellular fibrosis (square) are observed. High-power view of the area marked with the square (inset) indicates that hepatocytes (*) are surrounded and downsized by fibrosis. Scale bar, 100 μm (azan staining).

Figure 2.

DNA methylation profiles of NLT, non-cancerous liver tissue showing non-alcoholic steatohepatitis (NASH-N), and non-cancerous liver tissue showing chronic hepatitis or cirrhosis associated with HBV or HCV infection (viral-N). PCA was performed using the 3331 probes showing significant DNA methylation alterations in NASH-N samples relative to NLT samples, in NLT samples (n = 55, black), NASH-N samples (n = 113, red) and viral-N samples (n = 37, blue).

Figure 3.

Scattergrams of DNA methylation levels for representative probes showing area under the curve values of more than 0.95 in ROC curve analysis for discrimination between non-cancerous liver tissue showing non-alcoholic steatohepatitis (NASH-N) and non-cancerous liver tissue showing chronic hepatitis or cirrhosis associated with HBV or HCV infection (viral-N). Using each probe and its cutoff value (CV) described in Supplementary Table 5, available at Carcinogenesis Online, NASH-N samples (n = 113, red) were discriminated from viral-N samples (n = 37, blue) with sufficient sensitivity and specificity (97.2-100%).

Figure 4.

Ordered differences in DNA methylation levels for representative probes listed in Supplementary Table 4, available at Carcinogenesis Online, which were aberrantly methylated in non-cancerous liver tissue showing non-alcoholic steatohepatitis (NASH-N) relative to NLT and showed significant differences in DNA methylation levels between NASH-N and non-cancerous liver tissues showing chronic hepatitis or cirrhosis associated with HBV or HCV infection (viral-N). NASH-specific alterations of DNA methylation relative to NLT samples (n = 55) were observed in NASH-N samples from biopsy specimens from patients with morbid obesity but without hepatocellular carcinoma (HCC) (NASH-N-B, n = 91), and inherited by or strengthened in NASH-N samples from partial hepatectomy specimens from patients with HCC (NASH-N-H, n = 22), and then inherited by or further strengthened in NASH-related HCC (NASH-T, n = 22) itself. (A) Representative probes showing DNA hypermethylation by Jonckheere-Terpstra (JT) trend test. (B) Representative probes showing DNA hypomethylation by JT trend test.

Figure 5.

DNA methylation levels and mRNA expression levels for representative probes that were included in the 80 NASH-specific probes (Supplementary Table 8, available at Carcinogenesis Online) for which DNA methylation alterations were inherited by NASH-related hepatocellular carcinoma (HCC) (NASH-T) and showed significant differences in DNA methylation levels between NASH-T samples and HCC associated with HBV or HCV infection (viral-T). (A) DNA methylation levels in NASH-T samples (n = 22) and viral-T samples (n = 37). Each gene symbol is shown under the Infinium probe ID. (B) Levels of mRNA expression for the WHSC1 gene in NASH-T samples (n = 21) and viral-T samples (n = 31).