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Pharmacological unmasking microarray approach-based discovery of novel DNA methylation markers for hepatocellular carcinoma.

Jung N, Won JK, Kim BH, Suh KS, Jang JJ, Kang GH - J. Korean Med. Sci. (2012)

Bottom Line: Of the 239 promoter CpG island loci hypermethylated in HCC cell lines (as revealed by methylation-specific PCR), 221 loci were found to be hypermethylated in HCC or nonneoplastic liver tissues.In conclusion, we identified 221 novel DNA methylation markers for HCC.One promising prognostic marker, HIST1H2AE, should be further validated in the prognostication of HCC patients.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Epigenetics, Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea.

ABSTRACT
DNA methylation is one of the main epigenetic mechanisms and hypermethylation of CpG islands at tumor suppressor genes switches off these genes. To find novel DNA methylation markers in hepatocellular carcinoma (HCC), we performed pharmacological unmasking (treatment with 5-aza-2'-deoxycytidine or trichostatin A) followed by microarray analysis in HCC cell lines. Of the 239 promoter CpG island loci hypermethylated in HCC cell lines (as revealed by methylation-specific PCR), 221 loci were found to be hypermethylated in HCC or nonneoplastic liver tissues. Thirty-three loci showed a 20% higher methylation frequency in tumors than in adjacent nonneoplastic tissues. Correlation of individual cancer-related methylation markers with clinicopathological features of HCC patients (n = 95) revealed that the number of hypermethylated genes in HCC tumors was higher in older than in younger patients. Univariate and multivariate survival analysis revealed that the HIST1H2AE methylation status is closely correlated with the patient's overall survival (P = 0.022 and P = 0.010, respectively). In conclusion, we identified 221 novel DNA methylation markers for HCC. One promising prognostic marker, HIST1H2AE, should be further validated in the prognostication of HCC patients.

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Related in: MedlinePlus

Methylation-specific PCR (MSP) analysis of 239 genes in 10 pairs of tumor and surrounding nontumor tissue. The methylated and unmethylated status is indicated by a gray and a white box, respectively. Genes were divided into subgroups based on the methylation pattern in hepatocellular carcinoma (HCC) tumors and surrounding nonneoplastic liver tissues (see main text).
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Figure 6: Methylation-specific PCR (MSP) analysis of 239 genes in 10 pairs of tumor and surrounding nontumor tissue. The methylated and unmethylated status is indicated by a gray and a white box, respectively. Genes were divided into subgroups based on the methylation pattern in hepatocellular carcinoma (HCC) tumors and surrounding nonneoplastic liver tissues (see main text).

Mentions: In order to confirm whether CpG island loci methylated in cell lines are also methylated in primary tumor tissues, we analyzed 10 paired HCC and adjacent nonneoplastic liver tissues for their methylation status in 239 genes using MSP (Fig. 6). We subdivided all the genes (n = 360) into 6 groups (named 0-5) based on the comparison of methylation frequencies of individual genes between HCC and nonneoplastic liver tissues: 1) higher methylation frequency in HCC than in nonneoplastic liver (group 1, n = 56), 2) fully methylated in both HCC and non-neoplastic liver (group 2, n = 96), 3) not fully methylated but identical methylation frequencies in HCC and non-neoplastic liver (group 3, n = 17), 4) genes not methylated in HCC or non-neoplastic liver (group 4, n = 18), 5) genes less frequently methylated in HCC than in non-neoplastic livers (group 5, n = 52), and 6) unmethylated genes in HCC cell lines (group 0, n = 141) (Fig. 6). To identify key biological functions associated with a list of genes within each group, we performed functional annotation analysis using the DAVID tool (http://david.abcc.ncifcrf.gov/) (Table 1). Group 1 genes showed a significant association with the neuropeptide signaling pathway, positive regulation of amine transport, and acid secretion, whereas group 5 genes exhibited a significant association with nucleosome assembly, chromatin assembly, protein-DNA complex assembly, chromatin assembly or disassembly, and tissue development.


Pharmacological unmasking microarray approach-based discovery of novel DNA methylation markers for hepatocellular carcinoma.

Jung N, Won JK, Kim BH, Suh KS, Jang JJ, Kang GH - J. Korean Med. Sci. (2012)

Methylation-specific PCR (MSP) analysis of 239 genes in 10 pairs of tumor and surrounding nontumor tissue. The methylated and unmethylated status is indicated by a gray and a white box, respectively. Genes were divided into subgroups based on the methylation pattern in hepatocellular carcinoma (HCC) tumors and surrounding nonneoplastic liver tissues (see main text).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369444&req=5

Figure 6: Methylation-specific PCR (MSP) analysis of 239 genes in 10 pairs of tumor and surrounding nontumor tissue. The methylated and unmethylated status is indicated by a gray and a white box, respectively. Genes were divided into subgroups based on the methylation pattern in hepatocellular carcinoma (HCC) tumors and surrounding nonneoplastic liver tissues (see main text).
Mentions: In order to confirm whether CpG island loci methylated in cell lines are also methylated in primary tumor tissues, we analyzed 10 paired HCC and adjacent nonneoplastic liver tissues for their methylation status in 239 genes using MSP (Fig. 6). We subdivided all the genes (n = 360) into 6 groups (named 0-5) based on the comparison of methylation frequencies of individual genes between HCC and nonneoplastic liver tissues: 1) higher methylation frequency in HCC than in nonneoplastic liver (group 1, n = 56), 2) fully methylated in both HCC and non-neoplastic liver (group 2, n = 96), 3) not fully methylated but identical methylation frequencies in HCC and non-neoplastic liver (group 3, n = 17), 4) genes not methylated in HCC or non-neoplastic liver (group 4, n = 18), 5) genes less frequently methylated in HCC than in non-neoplastic livers (group 5, n = 52), and 6) unmethylated genes in HCC cell lines (group 0, n = 141) (Fig. 6). To identify key biological functions associated with a list of genes within each group, we performed functional annotation analysis using the DAVID tool (http://david.abcc.ncifcrf.gov/) (Table 1). Group 1 genes showed a significant association with the neuropeptide signaling pathway, positive regulation of amine transport, and acid secretion, whereas group 5 genes exhibited a significant association with nucleosome assembly, chromatin assembly, protein-DNA complex assembly, chromatin assembly or disassembly, and tissue development.

Bottom Line: Of the 239 promoter CpG island loci hypermethylated in HCC cell lines (as revealed by methylation-specific PCR), 221 loci were found to be hypermethylated in HCC or nonneoplastic liver tissues.In conclusion, we identified 221 novel DNA methylation markers for HCC.One promising prognostic marker, HIST1H2AE, should be further validated in the prognostication of HCC patients.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Epigenetics, Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea.

ABSTRACT
DNA methylation is one of the main epigenetic mechanisms and hypermethylation of CpG islands at tumor suppressor genes switches off these genes. To find novel DNA methylation markers in hepatocellular carcinoma (HCC), we performed pharmacological unmasking (treatment with 5-aza-2'-deoxycytidine or trichostatin A) followed by microarray analysis in HCC cell lines. Of the 239 promoter CpG island loci hypermethylated in HCC cell lines (as revealed by methylation-specific PCR), 221 loci were found to be hypermethylated in HCC or nonneoplastic liver tissues. Thirty-three loci showed a 20% higher methylation frequency in tumors than in adjacent nonneoplastic tissues. Correlation of individual cancer-related methylation markers with clinicopathological features of HCC patients (n = 95) revealed that the number of hypermethylated genes in HCC tumors was higher in older than in younger patients. Univariate and multivariate survival analysis revealed that the HIST1H2AE methylation status is closely correlated with the patient's overall survival (P = 0.022 and P = 0.010, respectively). In conclusion, we identified 221 novel DNA methylation markers for HCC. One promising prognostic marker, HIST1H2AE, should be further validated in the prognostication of HCC patients.

Show MeSH
Related in: MedlinePlus