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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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Methylation frequencies of 33 DNA methylation markers in hepatocellular carcinoma (HCC) tissue samples (n = 95). DNA methylation markers were distributed along the x-axis according to the decreasing order of methylation frequency.
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Figure 9: Methylation frequencies of 33 DNA methylation markers in hepatocellular carcinoma (HCC) tissue samples (n = 95). DNA methylation markers were distributed along the x-axis according to the decreasing order of methylation frequency.

Mentions: Genes with methylation frequencies higher in HCC than in nonneoplastic liver tissues are more likely to play an important role in hepatocarcinogenesis. To identify a possible correlation of gene hypermethylation with clinicopathological features of HCC patients, we selected 33 genes showing a methylation difference higher than 19% between HCC and nonneoplastic liver samples (Fig. 8) and analyzed their methylation statuses in another set of HCC tissue samples (n = 95) by MSP. Hypermethylation was detected in 1 or more genes in all HCC samples. RNASE4 showed the highest methylation frequency (90%), followed by DUOXA1 (82%), CRABP2 (66%), and NETO2 (61%) (Fig. 9). The methylation status of the DNA methylation markers was correlated with clinicopathological features such as age, gender, tumor size, AFP and GGT levels, microscopic vascular invasion, and clinical outcome. The results of these association studies are summarized in Table 2. The number of methylated genes was significantly higher in older (≥ 55 yr) than in younger patients (11.7 vs 8.8, P = 0.030 by Student's t-test). NETO2, DHDH, MAP6D1, C1orf59, CYB5R2, ULBP1, SPINT1, and ZNF342 were more frequently hypermethylated in tumors from patients older than 55 yr than in tumors from patients younger than 55 yr (P < 0.05 for all genes). HSPA12B was more frequently methylated in tumors from female patients than in tumors from male patients (P = 0.008). RNASE4 was more frequently methylated in tumors smaller than 6 cm than in HCC tumors bigger than 6 cm (P = 0.018). The methylation frequencies of NETO2, DHDH, and SPINT1 were significantly higher in HCC cases with AFP production smaller than 100 ng/mL than in HCC cases with AFP production higher than 100 ng/mL, whereas the methylation frequency of RASGRP2 and HSPA12B was significantly lower in HCCs with low AFP production (< 100 ng/mL). INA, PPP1R14A, and ECEL1 were more frequently methylated in HCC patients with low serum GGT levels (< 56 ng/mL) than in HCC patients with high serum GGT levels (≥ 56 ng/mL) (P < 0.05 for all genes). The methylation frequencies of C1orf59, OXTR, and CYB5R2 were significantly lower in HCC tumors with microscopic vein invasion than in HCCs without microscopic vein invasion (P < 0.05 for all genes).


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 frequencies of 33 DNA methylation markers in hepatocellular carcinoma (HCC) tissue samples (n = 95). DNA methylation markers were distributed along the x-axis according to the decreasing order of methylation frequency.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Methylation frequencies of 33 DNA methylation markers in hepatocellular carcinoma (HCC) tissue samples (n = 95). DNA methylation markers were distributed along the x-axis according to the decreasing order of methylation frequency.
Mentions: Genes with methylation frequencies higher in HCC than in nonneoplastic liver tissues are more likely to play an important role in hepatocarcinogenesis. To identify a possible correlation of gene hypermethylation with clinicopathological features of HCC patients, we selected 33 genes showing a methylation difference higher than 19% between HCC and nonneoplastic liver samples (Fig. 8) and analyzed their methylation statuses in another set of HCC tissue samples (n = 95) by MSP. Hypermethylation was detected in 1 or more genes in all HCC samples. RNASE4 showed the highest methylation frequency (90%), followed by DUOXA1 (82%), CRABP2 (66%), and NETO2 (61%) (Fig. 9). The methylation status of the DNA methylation markers was correlated with clinicopathological features such as age, gender, tumor size, AFP and GGT levels, microscopic vascular invasion, and clinical outcome. The results of these association studies are summarized in Table 2. The number of methylated genes was significantly higher in older (≥ 55 yr) than in younger patients (11.7 vs 8.8, P = 0.030 by Student's t-test). NETO2, DHDH, MAP6D1, C1orf59, CYB5R2, ULBP1, SPINT1, and ZNF342 were more frequently hypermethylated in tumors from patients older than 55 yr than in tumors from patients younger than 55 yr (P < 0.05 for all genes). HSPA12B was more frequently methylated in tumors from female patients than in tumors from male patients (P = 0.008). RNASE4 was more frequently methylated in tumors smaller than 6 cm than in HCC tumors bigger than 6 cm (P = 0.018). The methylation frequencies of NETO2, DHDH, and SPINT1 were significantly higher in HCC cases with AFP production smaller than 100 ng/mL than in HCC cases with AFP production higher than 100 ng/mL, whereas the methylation frequency of RASGRP2 and HSPA12B was significantly lower in HCCs with low AFP production (< 100 ng/mL). INA, PPP1R14A, and ECEL1 were more frequently methylated in HCC patients with low serum GGT levels (< 56 ng/mL) than in HCC patients with high serum GGT levels (≥ 56 ng/mL) (P < 0.05 for all genes). The methylation frequencies of C1orf59, OXTR, and CYB5R2 were significantly lower in HCC tumors with microscopic vein invasion than in HCCs without microscopic vein invasion (P < 0.05 for all genes).

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