Abstract
Chronic liver inflammation precedes the majority of hepatocellular carcinomas (HCC). Here, we explore the connection between chronic inflammation and DNA methylation in the liver at the late precancerous stages of HCC development in Mdr2(-/-) (Mdr2/Abcb4-knockout) mice, a model of inflammation-mediated HCC. Using methylated DNA immunoprecipitation followed by hybridization with "CpG islands" (CGIs) microarrays, we found specific CGIs in 76 genes which were hypermethylated in the Mdr2(-/-) liver compared to age-matched healthy controls. The observed hypermethylation resulted mainly from an age-dependent decrease of methylation of the specific CGIs in control livers with no decrease in mutant mice. Chronic inflammation did not change global levels of DNA methylation in Mdr2(-/-) liver, but caused a 2-fold decrease of the global 5-hydroxymethylcytosine level in mutants compared to controls. Liver cell fractionation revealed, that the relative hypermethylation of specific CGIs in Mdr2(-/-) livers affected either hepatocyte, or non-hepatocyte, or both fractions without a correlation between changes of gene methylation and expression. Our findings demonstrate that chronic liver inflammation causes hypermethylation of specific CGIs, which may affect both hepatocytes and non-hepatocyte liver cells. These changes may serve as useful markers of an increased regenerative activity and of a late precancerous stage in the chronically inflamed liver.
Highlights
Hepatocellular carcinoma (HCC) typically develops on a background of chronic inflammation induced by viruses or other risk factors that damage the liver and cause compensatory proliferation resulting in hepatocarcinogenesis, a multistep process with accumulation of genetic and epigenetic alterations [1]
Using methylated DNA immunoprecipitation followed by hybridization with “CpG islands” (CGIs) microarrays, we found specific CGIs in 76 genes which were hypermethylated in the mouse gene encoding the multidrug resistance-2 protein (Mdr2)-/- liver compared to age-matched healthy controls
The Tet1 expression was significantly increased in the liver of Mdr2-KO compared to Mdr2-/+ mice (Figure 1C), while the Tet2 expression was similar in both groups (Figure 1D); the expression of the Tet3 gene in the tested livers was too low for a reliable quantification
Summary
Hepatocellular carcinoma (HCC) typically develops on a background of chronic inflammation induced by viruses or other risk factors that damage the liver and cause compensatory proliferation resulting in hepatocarcinogenesis, a multistep process with accumulation of genetic and epigenetic alterations [1]. In order to explore gene methylation and expression patterns in cell fractions of the chronically inflamed liver, we used the Mdr2-knockout (Mdr2-KO) mice, a well-characterized model of chronic inflammation-mediated HCC [18] These mutants lack the Mdr2/Abcb P-glycoprotein (the murine ortholog of human MDR3) which is responsible for phosphatidylcholine transport across the hepatocyte’s canalicular membrane. This causes a dramatic decrease of phospholipids in bile resulting in bile regurgitation into portal tracts [19] and the development of chronic cholestatic hepatitis at an early age (starting from 2 months) and HCC with a high incidence in the adult age (between 12 and 18 months) [18]. This is the first study exploring the genome-scale liver DNA methylation at the late precancerous stage in a murine model of chronic inflammation-mediated hepatocarcinogenesis
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