Genome-wide DNA methylation profiling reveals novel epigenetically regulated genes and non-coding RNAs in human testicular cancer

Abstract

BACKGROUND: Testicular germ cell tumour (TGCT) is the most common malignant tumour in young males. Although aberrant DNA methylation is implicated in the pathophysiology of many cancers, only a limited number of genes are known to be epigenetically changed in TGCT. This report documents the genome-wide analysis of differential methylation in an in vitro model culture system. Interesting genes were validated in TGCT patient samples. METHODS: In this study, we used methylated DNA immunoprecipitation (MeDIP) and whole-genome tiling arrays to identify differentially methylated regions (DMRs). RESULTS: We identified 35 208 DMRs. However, only a small number of DMRs mapped to promoters. A genome-wide analysis of gene expression revealed a group of differentially expressed genes that were regulated by DNA methylation. We identified several candidate genes, including APOLD1, PCDH10 and RGAG1, which were dysregulated in TGCT patient samples. Surprisingly, APOLD1 had previously been mapped to the TGCT susceptibility locus at 12p13.1, suggesting that it may be important in TGCT pathogenesis. We also observed aberrant methylation in the loci of some non-coding RNAs (ncRNAs). One of the ncRNAs, hsa-mir-199a, was downregulated in TGCT patient samples, and also in our in vitro model culture system. CONCLUSION: This report is the first application of MeDIP-chip for identifying epigenetically regulated genes and ncRNAs in TGCT. We also demonstrated the function of intergenic and intronic DMRs in the regulation of ncRNAs.

Regulation of Axl receptor tyrosine kinase expression by miR-34a and miR-199a/b in solid cancer

“…Epigenetic regulation by CpG methylation of these miRs could be cell line specific or other epigenetic mechanisms and specific transcriptional factors might interfere with the expression. Bisulfite conversion and methylation-specific PCR supported the existing data and also revealed that miR-34a, miR-199a1, miR-199a2 and miR-199b promoters were hyper-methylated and inversely correlated with their expression in a panel of cancer cells (Lodygin et al, 2008;Garzia et al, 2009;Cheung et al, 2010). Our data shows in particular that miR-199a1 and miR-199b promoters were hypermethylated in NSCLC, BRC and CRC cell lines.…”

“…In general, it implies different stages of controlling mechanisms of a gene, especially of an oncogene. Moreover, miR-34a and miR-199a2 are reported that they are epigenetically controlled by CpG methylation (Lodygin et al, 2008;Cheung et al, 2010). 5-Aza treatment significantly induced the expression of either one or all of the miRs (miR-34a, miR-199a and b) in the screened cell lines.…”

“…Our data shows in particular that miR-199a1 and miR-199b promoters were hypermethylated in NSCLC, BRC and CRC cell lines. CpG methylation might account for the loss of these three miRs in a substantial portion of carcinomas (Bommer et miR-34a and 199a/b regulates Axl expression G Mudduluru et al Lodygin et al, 2008;Cheung et al, 2010). Our data show that Axl-targeting miRs are epigenetically silenced in cancer cell lines/tumors, and that miR-34a and miR199a/b are regulating Axl expression at mRNA levels by degrading it and also inhibiting translation, which is corroborating the Axl overexpression in different types of cancers (Hafizi and Dahlback, 2006).…”

Assessment of methylation level prediction accuracy in methyl-DNA immunoprecipitation and sodium bisulfite based microarray platforms

“…MeDIP-CGI-arrays, however, are relatively reliable in identifying high DNA methylation differences within the same region between different samples, as was expected. 38 These arrays perform less reliably in distinguishing intermediate methylation differences between samples (r = 0.56 for IQR, Fig. 1B) and in classifying different CGIs in the same sample according to their methylation levels.…”

Make way for the ‘next generation’: application and prospects for genome-wide, epigenome-specific technologies in endocrine research

“…In addition to the specific genes subject to DNA methylationmediated silencing reviewed by these authors, other studies on endocrine tumours have adopted genomewide approaches to identify potential novel epigenetically silenced genes. These types of studies on testicular (Cheung et al 2010) and ovarian cancer (Michaelson--Cohen et al 2011) have utilised MeDIP enrichment and on pancreatic tumours and ovarian cancer have used pharmacologic unmasking approaches (Kang et al 2010, Shimizu et al 2011. Still other studies on pituitary and thyroid tumours have described drug-induced unmasking techniques of histone tail modification that impact upon gene expression in these tumour types (reviewed in Farrell (2006), Ezzat (2008) and Yacqub-Usman et al (2012)).…”