Aberrant DNA methylation of imprinted loci in hepatocellular carcinoma and after in vitro exposure to common risk factors - PubMed (original) (raw)
Aberrant DNA methylation of imprinted loci in hepatocellular carcinoma and after in vitro exposure to common risk factors
Marie-Pierre Lambert et al. Clin Epigenetics. 2015.
Abstract
Background: Hepatocellular carcinoma (HCC) is among the most frequent human malignancies and a major cause of cancer-related death worldwide. It is characterized by late detection and fast progression, and it is believed that epigenetic disruption may be one of the molecular mechanisms leading to hepatocarcinogenesis. Previous studies from our group revealed that HCC tumors exhibit specific DNA methylation signatures associated with major risk factors and tumor progression. Imprinted genes are mono-allelically expressed in a parent-of-origin-dependent manner and have been suggested to be more susceptible to deregulation in cancer. To test this notion, we performed a targeted analysis of DNA methylation in known imprinted genes, using HCC samples and in vitro models of carcinogenic exposure.
Results: Analysis of HCC DNA methylation in two independent datasets showed that differentially methylated loci are significantly enriched in imprinted genes. Most of the promoters of imprinted genes were found hypomethylated in HCC tumors compared to surrounding tissues, contrasting with the frequent promoter hypermethylation observed in tumors. We next investigated the status of methylation of the imprinting control region (ICR) of different imprinted clusters and found that the 15q11-13 ICR was significantly hypomethylated in tumors relative to their surrounding tissues. In addition, expression of imprinted genes within this cluster was frequently deregulated in a gene-specific manner, suggesting distinct mechanisms of regulation in this region. Finally, primary human hepatocytes and hepatocyte-like HepaRG cells displayed higher methylation variability in certain imprinted loci after natural hepatitis B virus (HBV) infection and after lipid accumulation, respectively.
Conclusion: The methylation status of a large panel of imprinted genes was found deregulated in HCC, suggesting a major role of this mechanism during hepatocarcinogenesis. In vitro models support the hypothesis of imprinted gene methylation as a potential marker of environmental exposures.
Keywords: Imprinting; Liver cancer; Methylome; Promoter methylation.
Figures
Figure 1
Imprinted genes are differentially methylated in HCC. (A) Volcano plot for the comparison of HCC tumors vs. adjacent tissues (surrounding/tumor). The horizontal line defines the P value threshold of 0.001. Two representative hypermethylated (APC and RASSF1) and hypomethylated (GABRA5 and MEST) genes are shown respectively in red and blue fonts. (B) Distribution of differentially methylated CpG sites (n = 244) according to chromosomal location (blue bars). Reference proportion for all probes is shown as red bars. (C) Heatmap of differentially methylated imprinted CpG sites (n = 43), with high methylation represented in red and low methylation in blue. Annotations in the lower bars correspond to tumor vs. surrounding tissues, associated risk factors, and tumor stage. No data is shown for surrounding tissues (NA). (D) Estimated proportion of imprinted genes in the total dataset (upper pie chart) and the differentially methylated CpG sites (lower pie chart). Preferential maternal and paternal expressions are shown separately. (E) Validation of selected imprinted genes was assessed by pyrosequencing using 5 tumor/surrounding pairs. *P value <0.05.
Figure 2
Deregulation of the 15q11-13 cluster in HCC. (A) Pyrosequencing assays to assess Tumor vs. Surrounding methylation at imprinting control regions (ICR) for MEST and KCNQ1 (n = 5 pairs). The ICR for the 15q11-13 cluster, composed of the Prader-Willi Syndrome region (PWS-SRO) and the Angelman Syndrome region (AS-SRO), was also assessed. (B) Diagram of the 15q11-13 cluster showing the different regions of study, including the ICR and two control non-imprinted (ni) genes (APBA2 and TJP1). (C) qRT-PCR for selected genes in the 15q11-13 cluster comparing tumors and adjacent tissues (n = 5 tumor/surrounding pairs). *P value <0.05.
Figure 3
TCGA replication analysis. HM450 genome-wide methylation data was downloaded from TCGA (LIHC) for 47 available HCC tumor/surrounding pairs (see Methods). (A) Multi-dimensional scaling (MDS) plot showing differential clustering of control vs. tumor tissues. (B) Differential methylation analysis using paired (tumor/surrounding) linear regression identified 1,328 differentially methylated positions (DMPs); 30 sites (out of 1,328) corresponding to known imprinted loci were used to build an unsupervised cluster. The corresponding heatmap shows the normalized methylation data in a blue-red scale (from lower to higher methylation). Tumor grade is shown in the annotation panel as G1, G2, or G3. (C) Example plots of methylation data for 6 of the top differentially methylated imprinted sites in the TCGA HCC tumor vs. surrounding comparison (n = 47 tumor/surrounding pairs). (D) Enrichment analysis comparing the proportion of differentially methylated positions (DMPs) found in imprinted loci for dataset 1 (our original GoldenGate analysis) and dataset 2 (TCGA HM450 analysis). The barplot shows a significant enrichment in imprinted loci in both datasets. The data used to calculate enrichment and the corresponding P values (Fisher’s exact test) are shown in the lower panel. *P value <0.05.
Figure 4
In vitro models of risk factor exposure. Primary human hepatocytes were naturally infected with HBV. Efficiency was monitored by qRT-PCR of HBx transcript (upper panel). DNA extracted at different time points was used for pyrosequencing analysis of selected imprinted regions comparing mock to HBV-infected hepatocytes (lower panels).
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