Disturbance of DNA methylation patterns in the early phase of hepatocarcinogenesis induced by a choline-deficient L-amino acid-defined diet in rats

Abstract

The authors investigated the DNA methylation patterns of the E-cadherin, Connexin 26 (Cx26), Rassf1a and c-fos genes in the early phase of rat hepatocarcinogenesis induced by a choline-deficient L-amino acid-defined (CDAA) diet. Six-week-old F344 male rats were continuously fed with the CDAA diet, and three animals were then killed at each of 4 and 8 days and 3 weeks. Genomic DNA was extracted from livers for assessment of methylation status in the 5' upstream regions of E-cadherin, Cx26, Rassf1a and c-fos genes by bisulfite sequencing, compared with normal livers. The livers of rats fed the CDAA diet for 4 and 8 days and 3 weeks were methylated in E-cadherin, Cx26 and Rassf1a genes, while normal livers were all unmethylated. In contrast, normal livers were highly methylated in c-fos gene. Although the livers at 4 days were weakly methylated, those at 8 days and 3 weeks were markedly unmethylated. Methylation patterns of CpG sites in E-cadherin, Cx26 and Rassf1a were sparse and the methylation was not associated with gene repression. These results indicate that gene-specific DNA methylation patterns were found in livers of rats after short-term feeding of the CDAA diet, suggesting gene-specific hypermethylation might be involved in the early phase of rat hepatocarcinogenesis induced by the CDAA diet.

Figure 1

Methylation analysis of the 5′ upstream region of the E‐cadherin gene in rat by bisulfite sequencing. The primer pair used for bisulfite sequencing is shown. The transcription start site was defined as +1. Methylated CpG sites are represented by closed circles and unmethylated CpG sites are represented by open circles. NRL, normal liver tissue.

Figure 2

Methylation analysis of the 5′ upstream region of the Cx26 gene in rat by bisulfite sequencing. The primer pair used for bisulfite sequencing is shown. The transcription start site was defined as +1. Methylated CpG sites are represented by closed circles and unmethylated CpG sites are represented by open circles. NRL, normal liver tissue.

Figure 3

Methylation analysis of the 5′ upstream region of the Rassf1a gene in rat by bisulfite sequencing. The primer pair used for bisulfite sequencing is shown. The transcription start site was defined as +1. Methylated CpG sites are represented by closed circles and unmethylated CpG sites are represented by open circles. NRL, normal liver tissue.

Figure 4

Methylation analysis of the 5′ upstream region of the c‐fos gene in rat by bisulfite sequencing. The primer pair used for bisulfite sequencing is shown. The transcription start site was defined as +1. Methylated CpG sites are represented by closed circles and unmethylated CpG sites are represented by open circles. NRL, normal liver tissue.

Figure 5

Expression levels of E‐cadherin, Cx26, Rassf1a, c‐fos and Dnmt1 mRNAs relative to Gapdh mRNA using semiquantitative reverse transcription–polymerase chain reaction analysis. N, normal liver tissue.