Global analysis of DNA methylation in early-stage liver fibrosis - PubMed (original) (raw)

Global analysis of DNA methylation in early-stage liver fibrosis

Yoko Komatsu et al. BMC Med Genomics. 2012.

Abstract

Background: Liver fibrosis is caused by chemicals or viral infection. The progression of liver fibrosis results in hepatocellular carcinogenesis in later stages. Recent studies have revealed the importance of DNA hypermethylation in the progression of liver fibrosis to hepatocellular carcinoma (HCC). However, the importance of DNA methylation in the early-stage liver fibrosis remains unclear.

Methods: To address this issue, we used a pathological mouse model of early-stage liver fibrosis that was induced by treatment with carbon tetrachloride (CCl4) for 2 weeks and performed a genome-wide analysis of DNA methylation status. This global analysis of DNA methylation was performed using a combination of methyl-binding protein (MBP)-based high throughput sequencing (MBP-seq) and bioinformatic tools, IPA and Oncomine. To confirm functional aspect of MBP-seq data, we complementary used biochemical methods, such as bisulfite modification and in-vitro-methylation assays.

Results: The genome-wide analysis revealed that DNA methylation status was reduced throughout the genome because of CCl4 treatment in the early-stage liver fibrosis. Bioinformatic and biochemical analyses revealed that a gene associated with fibrosis, secreted phosphoprotein 1 (Spp1), which induces inflammation, was hypomethylated and its expression was up-regulated. These results suggest that DNA hypomethylation of the genes responsible for fibrosis may precede the onset of liver fibrosis. Moreover, Spp1 is also known to enhance tumor development. Using the web-based database, we revealed that Spp1 expression is increased in HCC.

Conclusions: Our study suggests that hypomethylation is crucial for the onset of and in the progression of liver fibrosis to HCC. The elucidation of this change in methylation status from the onset of fibrosis and subsequent progression to HCC may lead to a new clinical diagnosis.

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Figures

Figure 1

Figure 1

Validation of a mouse model of CCl 4 -induced early-stage liver fibrosis. (A) The effect of CCl4 treatment on enzymatic activities of serum Alt and Ast. The vertical axis represents each enzymatic activity (U/I) plus the standard deviation (**_p_-value < 0.01, n = 3-4). (B) The effect of CCl4 treatment on mRNA levels of collagen-related genes. mRNA levels of ECM-related genes, such as αSma, Col1a2, and Timp1, were determined by qPCR. Cyclophilin was used as the normalization control. The vertical axes represent the relative mRNA quantity plus the standard deviation (**_p_-value < 0.01, n = 3-4). (C) Histological changes induced by CCl4 in mouse livers. Paraffin-fixed liver section stained with H&E, MT, and Sirius-red (original magnification ×400).

Figure 2

Figure 2

DNA methylation profile of the liver genome. (A) Pie chart representing the proportions of genomic features with CCl4- and control-specific peaks. Gene body was defined as transcribed region, involving exon and intron. Promoter was as 20 kbp regions upstream from transcriptional start sites (TSS). 3'-flanking region was as 20 kbp regions downstream from transcription termination sites (TTS). These definitions were determined by using the UCSC Genome browser database. Intergenic region represented other region these defined regions. (B) Genome-wide distribution of methylated sites. Red and blue bars represent methylated sites of the CCl4- and control-specific peaks, respectively. (C) Number of methylated sites in each chromosome. Red and blue bars represent the CCl4- and control-specific peaks, respectively. (D) Relative number of methylated sites in all chromosomes. CCl4-specific peaks in all chromosomes (red) were relatively compared with control ones (blue).

Figure 3

Figure 3

In silico functional analysis of methylated sites. IPA analysis of all methylation peaks (A), and annotated peaks among the promoters (B). The horizontal axis represents the significance scores for each function as -log (_p_-value). Red and blue bars represent CCl4 and control samples, respectively.

Figure 4

Figure 4

Validation of the methylation site located upstream of the Spp1 TSS. (A) Epigenetic features of the methylation site located upstream of the Spp1 TSS assessed using the UCSC Genome browser. The Spp1 gene body is shown in dark blue. The methylation site upstream of the Spp1 TSS is shown as a red rectangle on the chromosome diagram and on the custom track. ChIP-seq signals for H3K4 me1 and H3K4 me4 in the liver (orange), for RNAP II in the liver and p300 in MEL leukemia (light blue), and for the DNase I hotspot in the liver (green) are represented as density plots. (B) MBP-IP assay of the methylation status of the site located upstream of the Spp1 TSS. The CCl4 sample was normalized to the control sample. The vertical axis represents fold changes plus the standard deviation (**_p_-value < 0.01, n = 3-4). (C) Bisulfite modification assay of the methylation status of the site located upstream of the Spp1 TSS. The CCl4 sample was normalized to the control sample. The vertical axis represents percentage of methylated sites in the site located upstream of the Spp1 TSS (n = 10).

Figure 5

Figure 5

Effect of the methylation site located upstream of the Spp1 TSS on Spp1 expression. (A) _In-vitro_-methylation assay of the site located upstream of the Spp1 TSS. The fragment DNA of the site located upstream of the Spp1 TSS (chr5:104846058-104846328) was enzymatically methylated in vitro by Sss1 before ligation into pGL3-Basic. The vertical axis represents the luciferase activity plus the standard deviation. (*_p_-value < 0.05, n = 3). (B) qPCR assay of Spp1 mRNA expression. Cyclophilin was used as the normalization control. The vertical axis represents the relative mRNA quantity plus the standard deviation (*_p_-value < 0.05, n = 3-4). (C) The effect of 5-dAza-C treatment on expression of Spp1 mRNA. mRNA levels of Spp1 were determined by qPCR. Gapdh was used as the normalization control. The vertical axis represents the relative mRNA quantity plus the standard deviation (*_p_-value < 0.05, n = 3).

Figure 6

Figure 6

Oncomine analysis of Spp1 mRNA expression in human HCC. Box-plot diagrams were analyzed to compare the Spp1 mRNA levels in normal liver tissue with that in HCC using the Oncomine dataset from studies reported by Mas et al. (**_p_-value < 0.01), Chen et al. (**_p_-value < 0.01), and Wurmbach et al. (**_p_-value < 0.01). The vertical axis represents the log2 median value. The upper (75%) and lower (25%) quartiles are represented by the upper and lower borders of the boxes, respectively.

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