The DNA methylation profile of liver tumors in C3H mice and identification of differentially methylated regions involved in the regulation of tumorigenic genes

Abstract

Background: C3H 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.

Methods: We 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.

Results: We 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.

Conclusions: Our 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.

Keywords: 5-aza-2'-deoxycytidine; C3H mice; DNA methylation; Liver tumors; Reduced representation bisulfite sequencing (RRBS).

Fig. 1

Outline of DNA methylation status of normal and tumor liver tissues in C3H mice. a Hierarchical clustering of methylation profiles of normal and tumor tissues using 1-Peason’s correlation distance. b Horizontal bar plots showing the percentage of hyper- and hypo-DMCs in all DMCc (left) and in all CpG (right) per chromosome. c Pie charts showing percentage of DMCs in the region between − 5000 bp and TSS, exon, intron and intergenic. d Pie charts showing percentage of DMCs in CpG islands, CpG island shores (defined as 2 kb flanks of CpG islands) and other regions. e The scatter plot of DNA methylation differences of DMRs and gene expression ratios between the normal and tumor tissues. The data of 118 expressing genes with the promoter DMRs were used. The relationship between DNA methylation differences and gene expression ratios of normal and liver tumors was determined by Spearman correlation analysis. 28 DMRs whose associated genes are up- or downregulated by hypo- or hyper-DMRs more than 2-fold in tumor tissues and appear in a PubMed search for the term “cancer” were shown in square, and listed in Table 1. Triangle plots indicate other DMRs

Fig. 2

Mst1r associated with hypo-DMR is up-regulated in the liver tumor tissues of C3H mice. a Schematic view of the Mst1r locus. Methylation levels of CpG sites were visualized using Integrative Genomics Viewer (IGV) software. The position of TSS, DMR and the positions assessed by bisulfite sequencing (BS-1 and BS-2) were indicated. The magnified view indicates 8 CpG positions in the DMR and predicted transcription factor binding sites. b The average methylation values of the CpG sites in normal and tumor tissues. The CpG sites with ≥10 reads on both strands (≥20 reads in total) are indicated. Statistical significance between normal and tumor tissue was analyzed by Student’s t-test. **, *** Significantly different at p < 0.01 and 0.001, respectively. c Validation of RRBS data for normal and tumor liver tissues of C3H mice by bisulfite sequencing. Methylated and unmethylated cytosine are shown as ● and ○ respectively. The numbers above the circles indicate the position of CpG shown in Fig. 1a. d Validation of gene expression levels of Mst1r by real-time PCR in the normal (n = 6) and tumor tissues (n = 11) of C3H mice. The expression of Mst1r is normalized to the expression of rRNA. Statistical significance between the two groups was analyzed by Student’s t-test. *** Significantly different at p < 0.001

Fig. 3

Slpi associated with hypo-DMR is up-regulated in the liver tumor tissues of C3H mice. a Schematic view of the Slpi locus. The position of TSS, DMR and the position assessed by bisulfite sequencing (BS) were indicated. The magnified view indicates 7 CpG positions in the DMR and predicted transcription factor binding sites. b The average methylation values of the CpG sites in normal and tumor tissues. The CpG sites with ≥10 reads on both strands are indicated. Statistical significance between normal and tumor tissue was analyzed by Student’s t-test. * Significantly different at p < 0.05. cValidation of RRBS data for normal and tumor liver tissues of C3H mice by bisulfite sequencing. Methylated and unmethylated cytosine are shown as ● and ○ respectively. d Validation of gene expression levels of Slpi by real-time PCR in the normal (n = 6) and tumor tissues (n = 11) of C3H mice. The expression of Slpi is normalized to the expression of rRNA. Statistical significance between the two groups was analyzed by the Student’s t-test. *** Significantly different at p < 0.001

Fig. 4

Extl1 associated with hyper-DMR is down-regulated in the liver tumor tissues of C3H mice. a Schematic representation of the Extl1 locus. The position of TSS, DMR and the position assessed by bisulfite sequencing (BS) were indicated. The magnified view indicates 27 CpG positions in the DMR and predicted transcription factor binding sites. b The average methylation values of the CpG sites in normal and tumor tissues. The CpG sites with ≥10 reads on both strands are indicated. Statistical significance between normal and tumor tissue was analyzed by Student’s t-test. *, **, *** Significantly different at p < 0.05, 0.01 and 0.001, respectively. c Validation of RRBS data for normal and tumor liver tissues of C3H mice by bisulfite sequencing. Methylated and unmethylated cytosine are shown as ● and ○ respectively. d Validation of gene expression levels of Extl1 by real-time PCR in the normal (n = 6) and tumor tissues (n = 11) of C3H mice. The expression of Extl1 is normalized to the expression of rRNA. Statistical significance between the two groups was analyzed by the Student’s t-test. * Significantly different at P < 0.05

Fig. 5

Reduced DNA methylation of DMRs of Mst1r, Slpi, and Extl1 after 5-aza-dC treatment are associated with up-regulation of these genes in hepa1c1c7 cells and Hepa1-6 cells. Hepa1-6 cells (b, d, e) and Hepa1c1c7 cells (a, c) were cultured with 0, 0.1 or 1 μM of 5-aza-dC, and 0, 50 or 100 μM of 5-aza-dC for 72 h, respectively. Left figures: the results of bisulfite sequencing of CpGs detected in the DMRs of liver tumors in C3H mice. ●: methylated cytosine, ○: unmethylated cytosine. Right figures: the expressions of Mst1r, Slpi, and Extl were measured by real-time PCR and normalized to the expression of β-actin or rRNA (n = 3). Statistical significance was analyzed by one-way ANOVA followed by Turkey-Kramer test as a post hoc comparison. *^, **^, *** Significantly different at p < 0.05, 0.01, and 0.001, respectively

Fig. 6

Overexpression of Mst1r induces IL33 in Hepa1c1c7 cells. Overexpression of Mst1r (a) and upregulation of IL33 by Mst1r overexpression (b) in Hepa1c1c7 cells was confirmed by real-time PCR. *** Significantly different at p < 0.001 (n = 3). (c) Confirmation by real-time PCR of upregulation of IL33 in the tumor tissues (n = 11) compared to the normal tissues (n = 6) of C3H mice. Statistical significance between the two groups was analyzed by the Student’s t-test. *** Significantly different at p < 0.001

Fig. 7

Human database searches showed hypomethylation of downstream regions of MST1R and SLPI with upregulation of their expressions. RNA-seq dataset and DNA methylation dataset of 41 paired normal and tumor tissues of human livers were downloaded from TCGA. a, b Average gene expression ratio (tumor tissues/normal tissues) was calculated using 41 paired data from TCGA. *^, ** Significantly different at P < 0.05, and 0.01, respectively. c The difference in DNA methylation β-value (tumor – normal) for each CpG around TSS was calculated using 41 paired data from TCGA. d The correlation between expressions of MST1R and IL33 in the HCC tissues highly expressing MST1R