Gene Expression and DNA Methylation Alterations in the Glycine N-Methyltransferase Gene in Diet-Induced Nonalcoholic Fatty Liver Disease-Associated Carcinogenesis

Abstract

Nonalcoholic fatty liver disease (NAFLD) is becoming a major etiological risk factor for hepatocellular carcinoma (HCC) in the United States and other Western countries. In this study, we investigated the role of gene-specific promoter cytosine DNA methylation and gene expression alterations in the development of NAFLD-associated HCC in mice using (1) a diet-induced animal model of NAFLD, (2) a Stelic Animal Model of nonalcoholic steatohepatitis-derived HCC, and (3) a choline- and folate-deficient (CFD) diet (CFD model). We found that the development of NAFLD and its progression to HCC was characterized by down-regulation of glycine N-methyltransferase (Gnmt) and this was mediated by progressive Gnmt promoter cytosine DNA hypermethylation. Using a panel of genetically diverse inbred mice, we observed that Gnmt down-regulation was an early event in the pathogenesis of NAFLD and correlated with the extent of the NAFLD-like liver injury. Reduced GNMT expression was also found in human HCC tissue and liver cancer cell lines. In in vitro experiments, we demonstrated that one of the consequences of GNMT inhibition was an increase in genome methylation facilitated by an elevated level of S-adenosyl-L-methionine. Overall, our findings suggest that reduced Gnmt expression caused by promoter hypermethylation is one of the key molecular events in the development of NAFLD-derived HCC and that assessing Gnmt methylation level may be useful for disease stratification.

Glycine Metabolism and Its Alterations in Obesity and Metabolic Diseases

“…By contrast, decreased GNMT expression is observed in NAFLDs, both in animal models receiving a high-fat diet [48] and in patients with hepatic steatosis [49]. Furthermore, DNA hypermethylation represses GNMT gene expression in the pathogenesis of NAFLD [146] and hepatocellular carcinoma [145,147]. The loss of GNMT in hepatocellular carcinoma may favor the appearance of aberrant DNA methylation patterns on some promoters [145].…”

Aberrant DNA methylation results in altered gene expression in non-alcoholic steatohepatitis-related hepatocellular carcinomas

“…In the light of these facts, it would be informative to understand the significance of DNA methylation alterations in NASH-related hepatocarcinogenesis. Due to the difficulty in obtaining appropriate human samples, many previous studies of NASH-related HCC have been conducted using animal models (Borowa-Mazgaj et al 2019 ). Even in studies using human clinical samples, DNA methylation status has been examined only for individual tumor-related genes (Tian et al 2015 ), and studies employing genome-wide DNA methylation analysis have been very limited.…”

The use of cell free DNA in the diagnosis of HCC

“…To date, several studies have showed that altered DNA methylation at several genes are associated with the initiation and progression of HCC, including p15 and p16 [64] , APC [65] , SPINT2 [66] , SFRP1 [67] , TFP12 [68] , GSTP1 [69] and RASSF1A [70] . NAFLD-related HCC is associated with hypermethylation of the glycine N-methyltransferase ( GNMT ) promoter, resulting in reduced gene expression [71] . Differential DNA hypomethylation has also been seen in HCC.…”