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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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Flow chart for selection of candidate genes. Screening of candidate tumor suppressor genes (TSGs) was performed in 5 hepatocellular carcinoma (HCC) cell lines treated with 5 µM 5-aza-2'-deoxycytidine (AZA) or 300 nM trichostatin A (TSA) by using a 24,526-oligonucleotide mRNA microarray. We obtained 793 candidates whose gene expression did not increase with TSA treatment (< 1.4-fold) but increased more than 2-fold after AZA treatment. We excluded genes that do not harbor CpG islands in their promoters or whose methylation status in HCC tumors had already been reported in the literature. We further excluded genes for which adequate oligonucleotide primers could not be designed by using the MSPprimer or MethPrimer software programs. As a result, we selected 380 genes to be examined for their methylation status in HCC cell lines by using methylation-specific PCR (MSP).
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Figure 1: Flow chart for selection of candidate genes. Screening of candidate tumor suppressor genes (TSGs) was performed in 5 hepatocellular carcinoma (HCC) cell lines treated with 5 µM 5-aza-2'-deoxycytidine (AZA) or 300 nM trichostatin A (TSA) by using a 24,526-oligonucleotide mRNA microarray. We obtained 793 candidates whose gene expression did not increase with TSA treatment (< 1.4-fold) but increased more than 2-fold after AZA treatment. We excluded genes that do not harbor CpG islands in their promoters or whose methylation status in HCC tumors had already been reported in the literature. We further excluded genes for which adequate oligonucleotide primers could not be designed by using the MSPprimer or MethPrimer software programs. As a result, we selected 380 genes to be examined for their methylation status in HCC cell lines by using methylation-specific PCR (MSP).

Mentions: The expression profiles of 5 HCC cell lines (SNU398, SNU761, SNU878, HepG2, and Huh7) were obtained before and after treatment with either AZA or TSA by using a microarray platform (Fig. 1). To identify genes undergoing hypermethylation-dependent expression changes, we determined the expression fold changes of individual genes between mock-treated and TSA- or AZA-treated cells, and plotted TSA- and AZA-related fold changes on the x- and y-axis, respectively (Fig. 2). The DNA demethylating agent (AZA) induces reexpression of densely hypermethylated and transcriptionally inactive genes, whereas the class I and II histone deacetylase inhibitor (TSA) does not induce reexpression (25, 26). Of the genes that did not show an increase in expression with TSA treatment (< 1.4-fold), subsets of genes displayed a peak of AZA-induced gene expression (> 2-fold). We considered the genes showing both < 1.4-fold expression with TSA treatment and > 2-fold expression with AZA treatment as candidate genes that might be inactivated by hypermethylation. In at least one of 5 cell lines, 793 genes were found to meet the selection criteria. Of these, genes that have no CpG islands in their promoters and proximal transcriptional start sites (TSS) as well as those genes whose methylation status in HCC had already been reported, were excluded from subsequent analysis (Fig. 1).


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)

Flow chart for selection of candidate genes. Screening of candidate tumor suppressor genes (TSGs) was performed in 5 hepatocellular carcinoma (HCC) cell lines treated with 5 µM 5-aza-2'-deoxycytidine (AZA) or 300 nM trichostatin A (TSA) by using a 24,526-oligonucleotide mRNA microarray. We obtained 793 candidates whose gene expression did not increase with TSA treatment (< 1.4-fold) but increased more than 2-fold after AZA treatment. We excluded genes that do not harbor CpG islands in their promoters or whose methylation status in HCC tumors had already been reported in the literature. We further excluded genes for which adequate oligonucleotide primers could not be designed by using the MSPprimer or MethPrimer software programs. As a result, we selected 380 genes to be examined for their methylation status in HCC cell lines by using methylation-specific PCR (MSP).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Flow chart for selection of candidate genes. Screening of candidate tumor suppressor genes (TSGs) was performed in 5 hepatocellular carcinoma (HCC) cell lines treated with 5 µM 5-aza-2'-deoxycytidine (AZA) or 300 nM trichostatin A (TSA) by using a 24,526-oligonucleotide mRNA microarray. We obtained 793 candidates whose gene expression did not increase with TSA treatment (< 1.4-fold) but increased more than 2-fold after AZA treatment. We excluded genes that do not harbor CpG islands in their promoters or whose methylation status in HCC tumors had already been reported in the literature. We further excluded genes for which adequate oligonucleotide primers could not be designed by using the MSPprimer or MethPrimer software programs. As a result, we selected 380 genes to be examined for their methylation status in HCC cell lines by using methylation-specific PCR (MSP).
Mentions: The expression profiles of 5 HCC cell lines (SNU398, SNU761, SNU878, HepG2, and Huh7) were obtained before and after treatment with either AZA or TSA by using a microarray platform (Fig. 1). To identify genes undergoing hypermethylation-dependent expression changes, we determined the expression fold changes of individual genes between mock-treated and TSA- or AZA-treated cells, and plotted TSA- and AZA-related fold changes on the x- and y-axis, respectively (Fig. 2). The DNA demethylating agent (AZA) induces reexpression of densely hypermethylated and transcriptionally inactive genes, whereas the class I and II histone deacetylase inhibitor (TSA) does not induce reexpression (25, 26). Of the genes that did not show an increase in expression with TSA treatment (< 1.4-fold), subsets of genes displayed a peak of AZA-induced gene expression (> 2-fold). We considered the genes showing both < 1.4-fold expression with TSA treatment and > 2-fold expression with AZA treatment as candidate genes that might be inactivated by hypermethylation. In at least one of 5 cell lines, 793 genes were found to meet the selection criteria. Of these, genes that have no CpG islands in their promoters and proximal transcriptional start sites (TSS) as well as those genes whose methylation status in HCC had already been reported, were excluded from subsequent analysis (Fig. 1).

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