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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

Effect of 5-aza-2'-deoxycytidine (AZA) and trichostatin A (TSA) on gene expression. RNA was isolated from hepatocellular carcinoma (HCC) cell lines left untreated or treated with AZA, TSA, or a combination of AZA and TSA. mRNA was reverse-transcribed, and gene expression was quantitated by real-time PCR and normalized to GAPDH expression.
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Figure 7: Effect of 5-aza-2'-deoxycytidine (AZA) and trichostatin A (TSA) on gene expression. RNA was isolated from hepatocellular carcinoma (HCC) cell lines left untreated or treated with AZA, TSA, or a combination of AZA and TSA. mRNA was reverse-transcribed, and gene expression was quantitated by real-time PCR and normalized to GAPDH expression.

Mentions: To ensure that the microarray data represent real changes in expression, we selected 7 genes (CRABP2, EFEMP2, HIST1H2AE, INA, NETO2, RASGRP2, and TP53I13) and analyzed their expression changes in HCC cell lines treated with AZA, TSA, or a combination of AZA and TSA using semi-quantitative real-time PCR (Fig. 7). Treatment with AZA alone resulted in induction of mRNA expression of these 7 genes. In some cases, the effect of combined AZA and TSA treatment was stronger than that of AZA or TSA alone. These data implicate that histone deacetylation may also be involved in the inhibition of gene transcription by DNA methylation.


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)

Effect of 5-aza-2'-deoxycytidine (AZA) and trichostatin A (TSA) on gene expression. RNA was isolated from hepatocellular carcinoma (HCC) cell lines left untreated or treated with AZA, TSA, or a combination of AZA and TSA. mRNA was reverse-transcribed, and gene expression was quantitated by real-time PCR and normalized to GAPDH expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Effect of 5-aza-2'-deoxycytidine (AZA) and trichostatin A (TSA) on gene expression. RNA was isolated from hepatocellular carcinoma (HCC) cell lines left untreated or treated with AZA, TSA, or a combination of AZA and TSA. mRNA was reverse-transcribed, and gene expression was quantitated by real-time PCR and normalized to GAPDH expression.
Mentions: To ensure that the microarray data represent real changes in expression, we selected 7 genes (CRABP2, EFEMP2, HIST1H2AE, INA, NETO2, RASGRP2, and TP53I13) and analyzed their expression changes in HCC cell lines treated with AZA, TSA, or a combination of AZA and TSA using semi-quantitative real-time PCR (Fig. 7). Treatment with AZA alone resulted in induction of mRNA expression of these 7 genes. In some cases, the effect of combined AZA and TSA treatment was stronger than that of AZA or TSA alone. These data implicate that histone deacetylation may also be involved in the inhibition of gene transcription by DNA methylation.

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