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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

Comparison of ALU and LINE-1 repeats between methylated and unmethylated genes. For ALU counting, the promoter sequence of a specific gene was divided into 20 bins of 1-kb sequence each (10 bins upstream and 10 bins downstream of each gene transcription start site), and the presence of ALU was annotated for each bin. We counted bins containing ALU within a 1-kb sequence. For LINE-1 counting, the promoter sequence of a specific gene was divided into 7 bins of 1-kb sequence each (2 bins upstream and 5 bins downstream of each gene transcription site), and the presence of LINE-1 was annotated for each bin. Bins containing LINE-1 within a 1-kb sequence were counted. Student's t-test was performed to determine the statistical significance of the difference of means between 2 groups.
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Figure 5: Comparison of ALU and LINE-1 repeats between methylated and unmethylated genes. For ALU counting, the promoter sequence of a specific gene was divided into 20 bins of 1-kb sequence each (10 bins upstream and 10 bins downstream of each gene transcription start site), and the presence of ALU was annotated for each bin. We counted bins containing ALU within a 1-kb sequence. For LINE-1 counting, the promoter sequence of a specific gene was divided into 7 bins of 1-kb sequence each (2 bins upstream and 5 bins downstream of each gene transcription site), and the presence of LINE-1 was annotated for each bin. Bins containing LINE-1 within a 1-kb sequence were counted. Student's t-test was performed to determine the statistical significance of the difference of means between 2 groups.

Mentions: To identify whether the candidate genes (n = 443) are methylated in their promoter CpG island loci, we tried to design MSP primers by using the MSPprimer or MethPrimer software programs and successfully designed optimal primers for 380 genes. We analyzed the methylation status of these 380 genes in 8 HCC cell lines (SNU398, SNU475, SNU739, SNU761, SNU878, SNU886, HepG2, and Huh7) using MSP and found that 239 of 380 genes were methylated in one or more cell lines (Fig. 3), and that 167 genes were methylated in at least four cell lines. To identify the factors that might influence in their predisposition to DNA methylation, we compared the occupancy rate of Polycomb proteins (EED2 and SUZ12) and the frequency of the H3K27me3 modification between methylated genes and unmethylated genes in HCC cell lines by using the occupancy maps published for embryonic stem cells (27). Genes methylated in HCC cell lines had a higher frequency of SUZ12, EED, and H3K27me3 targets compared to genes not methylated in HCC cell lines (Fig. 4). Next, we counted the frequency of LINE-1 and ALU in upstream and downstream sequences around the transcription start site of the 380 genes and then compared the number of LINE-1 or ALU repeats between methylated and unmethylated genes in HCC cell lines. The number of ALU repeats was significantly higher in unmethylated genes than in methylated genes, whereas the number of LINE-1 was similar (Fig. 5).


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)

Comparison of ALU and LINE-1 repeats between methylated and unmethylated genes. For ALU counting, the promoter sequence of a specific gene was divided into 20 bins of 1-kb sequence each (10 bins upstream and 10 bins downstream of each gene transcription start site), and the presence of ALU was annotated for each bin. We counted bins containing ALU within a 1-kb sequence. For LINE-1 counting, the promoter sequence of a specific gene was divided into 7 bins of 1-kb sequence each (2 bins upstream and 5 bins downstream of each gene transcription site), and the presence of LINE-1 was annotated for each bin. Bins containing LINE-1 within a 1-kb sequence were counted. Student's t-test was performed to determine the statistical significance of the difference of means between 2 groups.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Comparison of ALU and LINE-1 repeats between methylated and unmethylated genes. For ALU counting, the promoter sequence of a specific gene was divided into 20 bins of 1-kb sequence each (10 bins upstream and 10 bins downstream of each gene transcription start site), and the presence of ALU was annotated for each bin. We counted bins containing ALU within a 1-kb sequence. For LINE-1 counting, the promoter sequence of a specific gene was divided into 7 bins of 1-kb sequence each (2 bins upstream and 5 bins downstream of each gene transcription site), and the presence of LINE-1 was annotated for each bin. Bins containing LINE-1 within a 1-kb sequence were counted. Student's t-test was performed to determine the statistical significance of the difference of means between 2 groups.
Mentions: To identify whether the candidate genes (n = 443) are methylated in their promoter CpG island loci, we tried to design MSP primers by using the MSPprimer or MethPrimer software programs and successfully designed optimal primers for 380 genes. We analyzed the methylation status of these 380 genes in 8 HCC cell lines (SNU398, SNU475, SNU739, SNU761, SNU878, SNU886, HepG2, and Huh7) using MSP and found that 239 of 380 genes were methylated in one or more cell lines (Fig. 3), and that 167 genes were methylated in at least four cell lines. To identify the factors that might influence in their predisposition to DNA methylation, we compared the occupancy rate of Polycomb proteins (EED2 and SUZ12) and the frequency of the H3K27me3 modification between methylated genes and unmethylated genes in HCC cell lines by using the occupancy maps published for embryonic stem cells (27). Genes methylated in HCC cell lines had a higher frequency of SUZ12, EED, and H3K27me3 targets compared to genes not methylated in HCC cell lines (Fig. 4). Next, we counted the frequency of LINE-1 and ALU in upstream and downstream sequences around the transcription start site of the 380 genes and then compared the number of LINE-1 or ALU repeats between methylated and unmethylated genes in HCC cell lines. The number of ALU repeats was significantly higher in unmethylated genes than in methylated genes, whereas the number of LINE-1 was similar (Fig. 5).

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