Abstract
BackgroundC3H mice have been frequently used in cancer studies as animal models of spontaneous liver tumors and chemically induced hepatocellular carcinoma (HCC). Epigenetic modifications, including DNA methylation, are among pivotal control mechanisms of gene expression leading to carcinogenesis. Although information on somatic mutations in liver tumors of C3H mice is available, epigenetic aspects are yet to be clarified.MethodsWe performed next generation sequencing-based analysis of DNA methylation and microarray analysis of gene expression to explore genes regulated by DNA methylation in spontaneous liver tumors of C3H mice. Overlaying these data, we selected cancer-related genes whose expressions are inversely correlated with DNA methylation levels in the associated differentially methylated regions (DMRs) located around transcription start sites (TSSs) (promoter DMRs). We further assessed mutuality of the selected genes for expression and DNA methylation in human HCC using the Cancer Genome Atlas (TCGA) database.ResultsWe obtained data on genome-wide DNA methylation profiles in the normal and tumor livers of C3H mice. We identified promoter DMRs of genes which are reported to be related to cancer and whose expressions are inversely correlated with the DNA methylation, including Mst1r, Slpi and Extl1. The association between DNA methylation and gene expression was confirmed using a DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-aza-dC) in Hepa1c1c7 cells and Hepa1-6 cells. Overexpression of Mst1r in Hepa1c1c7 cells illuminated a novel downstream pathway via IL-33 upregulation. Database search indicated that gene expressions of Mst1r and Slpi are upregulated and the TSS upstream regions are hypomethylated also in human HCC. These results suggest that DMRs, including those of Mst1r and Slpi, are involved in liver tumorigenesis in C3H mice, and also possibly in human HCC.ConclusionsOur study clarified genome wide DNA methylation landscape of C3H mice. The data provide useful information for further epigenetic studies of mice models of HCC. The present study particularly proposed novel DNA methylation-regulated pathways for Mst1r and Slpi, which may be applied not only to mouse HCC but also to human HCC.
Highlights
C3H mice have been frequently used in cancer studies as animal models of spontaneous liver tumors and chemically induced hepatocellular carcinoma (HCC)
According to the recent reports showing that DNA methylation levels within ± 1000 bp regions [13] or ± 2000 bp regions [11, 16, 17] of Transcription start site (TSS) showed a strong correlation with gene repression, we focused on Differentially methylated region (DMR) within ± 2000 bp of TSS
22–55 million sequence read pairs were obtained for the representation bisulfite sequencing (RRBS) libraries and 63– 71% of the reads were aligned to unique genomic locations
Summary
C3H mice have been frequently used in cancer studies as animal models of spontaneous liver tumors and chemically induced hepatocellular carcinoma (HCC). C3H mice have been employed for numerous studies of carcinogenesis The mice, their males, are predisposed to spontaneously develop liver tumors in adulthood and have been used as an animal model of spontaneous hepatocellular carcinoma (HCC) [1, 2]. Somatic point mutations of ras genes (Ha-ras, K-ras, and N-ras) were detected in a variety of human and animal tumors7 These somatic mutations activate Ras proteins and lead to activation of tumor augmenting pathways, including the RAF/MEK/ERK kinase cascade [7]. Another somatic mutation was reported in B-raf, a member of the RAF kinase family in about 20% of liver tumors in C3H mice administered DEN at 2 weeks of age [8]. B-raf mutation augments the kinase activity leading to activation of the MEK/ERK cascade [5, 8]
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