Integrated analyses of DNA methylation and hydroxymethylation reveal tumor suppressive roles of ECM1, ATF5, and EOMES in human hepatocellular carcinoma.

Fei Gao,Weilong Liu,Ying Ou,Junwen Wang,Yudong Xia,Xing Liu,Zhilong Lin,Xiuqing Zhang,Huijuan Luo,Jian Huang,Baojin Zhou,Boping Zhou,Bo Wen

doi:10.1186/s13059-014-0533-9

Abstract

BackgroundDifferences in 5-hydroxymethylcytosine, 5hmC, distributions may complicate previous observations of abnormal cytosine methylation statuses that are used for the identification of new tumor suppressor gene candidates that are relevant to human hepatocarcinogenesis. The simultaneous detection of 5-methylcytosine and 5-hydroxymethylcytosine is likely to stimulate the discovery of aberrantly methylated genes with increased accuracy in human hepatocellular carcinoma.ResultsHere, we performed ultra-performance liquid chromatography/tandem mass spectrometry and single-base high-throughput sequencing, Hydroxymethylation and Methylation Sensitive Tag sequencing, HMST-seq, to synchronously measure these two modifications in human hepatocellular carcinoma samples. After identification of differentially methylated and hydroxymethylated genes in human hepatocellular carcinoma, we integrate DNA copy-number alterations, as determined using array-based comparative genomic hybridization data, with gene expression to identify genes that are potentially silenced by promoter hypermethylation.ConclusionsWe report a high enrichment of genes with epigenetic aberrations in cancer signaling pathways. Six genes were selected as tumor suppressor gene candidates, among which, ECM1, ATF5 and EOMES are confirmed via siRNA experiments to have potential anti-cancer functions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0533-9) contains supplementary material, which is available to authorized users.

Highlights

Differences in 5-hydroxymethylcytosine, 5hmC, distributions may complicate previous observations of abnormal cytosine methylation statuses that are used for the identification of new tumor suppressor gene candidates that are relevant to human hepatocarcinogenesis
We identified three new genes (ECM1, ATF5, and EOMES) with potential anti-cancer functions that may promote the understanding of the molecular mechanisms of Hepatocellular carcinoma (HCC) development and progression and potentiate the future clinical applications of 5hmC detection
Increased 5mC but decreased 5hmC levels at genomic CCGG loci in HCC We first performed UPLC-MS/MS to investigate global 5mC and 5hmC levels in 16 pairs of HCC and Non-cancerous liver tissues (non-HCC) samples and two HCC cell lines (97 L and LM6 cells). We found that both 5mC and 5hmC were frequently decreased in 71% and 100% of the HCC specimens, respectively, compared with non-HCC specimens

Summary

Introduction

Differences in 5-hydroxymethylcytosine, 5hmC, distributions may complicate previous observations of abnormal cytosine methylation statuses that are used for the identification of new tumor suppressor gene candidates that are relevant to human hepatocarcinogenesis. The simultaneous detection of 5-methylcytosine and 5-hydroxymethylcytosine is likely to stimulate the discovery of aberrantly methylated genes with increased accuracy in human hepatocellular carcinoma. Surgical and chemotherapeutic treatment of HCC is evolving, surgical resection remains the treatment of choice for many patients. A number of studies have reported that the hypermethylation of tumor suppressor genes (TSGs) contributes to HCC pathogenesis [9,10,11].

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