Abstract
BackgroundEpigenome-wide studies in hepatocellular carcinoma (HCC) have identified numerous genes with aberrant DNA methylation. However, methods for triaging functional candidate genes as useful biomarkers for epidemiological study have not yet been developed.MethodsWe conducted targeted next-generation bisulfite sequencing (bis-seq) to investigate associations of DNA methylation and mRNA expression in HCC. Integrative analyses of epigenetic profiles with DNA copy number analysis were used to pinpoint functional genes regulated mainly by altered DNA methylation.ResultsSignificant differences between HCC tumor and adjacent non-tumor tissue were observed for 28 bis-seq amplicons, with methylation differences varying from 12% to 43%. Available mRNA expression data in Oncomine were evaluated. Two candidate genes (GRASP and TSPYL5) were significantly under-expressed in HCC tumors in comparison with precursor and normal liver tissues. The expression levels in tumor tissues were, respectively, 1.828 and − 0.148, significantly lower than those in both precursor and normal liver tissue. Validations in an additional 42 paired tissues showed consistent under-expression in tumor tissue for GRASP (−7.49) and TSPYL5 (−9.71). A highly consistent DNA hypermethylation and mRNA repression pattern was obtained for both GRASP (69%) and TSPYL5 (73%), suggesting that their biological function is regulated by DNA methylation. Another two genes (RGS17 and NR2E1) at Chr6q showed significantly decreased DNA methylation in tumors with loss of DNA copy number compared to those without, suggesting alternative roles of DNA copy number losses and hypermethylation in the regulation of RGS17 and NR2E1.ConclusionsThese results suggest that integrative analyses of epigenomic and genomic data provide an efficient way to filter functional biomarkers for future epidemiological studies in human cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-015-0105-1) contains supplementary material, which is available to authorized users.
Highlights
Epigenome-wide studies in hepatocellular carcinoma (HCC) have identified numerous genes with aberrant DNA methylation
Comparison of DNA methylation results from targeted bis-seq and Illumina arrays A total of 29 CpG amplicons covering 20 genes (15 hyper- and 5 hypo-methylated genes) were sequenced by targeted bis-seq
Among the six hypermethylated and under-expressed genes (CLCN1, General receptor for phosphoinositides 1-associated scaffold protein (GRASP), MAST1, SPAG6, TRIL and Testis-specific protein Y-encoded-like 5 (TSPYL5)) without losses of copy number, we further examined whether miRNAs that target those genes are over-expressed in HCC tumor tissue
Summary
Epigenome-wide studies in hepatocellular carcinoma (HCC) have identified numerous genes with aberrant DNA methylation. Epigenome-wide association studies [1,2,3,4,5,6,7], including ours [8, 9] have identified large panels of genes with aberrant DNA methylation in hepatocellular carcinoma (HCC). Comprehensive analyses of available genetic and epigenetic data together may help us to better understand the functions of genes identified as being mainly regulated by aberrant DNA methylation, and narrow down the number of crucial methylation markers involved in hepatocarcinogenesis for future large epidemiological studies, including those which might involve using blood as an indirect surrogate, or cell-free tumor-derived DNA as a directly relevant analyte
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