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Identification of novel methylation markers in hepatocellular carcinoma using a methylation array.

Shin SH, Kim BH, Jang JJ, Suh KS, Kang GH - J. Korean Med. Sci. (2010)

Bottom Line: Twenty-four new genes have been identified as hypermethylated at their promoter CpG island loci in a cancer-specific manner.Of these, TNFRSF10C, HOXA9, NPY, and IRF5 were frequently hypermethylated in hepatocellular carcinoma tissue samples and their methylation was found to be closely associated with inactivation of gene expression.Further study will be required to elucidate the clinicopathological implications of these newly found DNA methylation markers in hepatocellular carcinoma.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Epigenetics, Cancer Research Institute and Brain Korea 2nd Stage, Seoul National University, Seoul, Korea.

ABSTRACT
Promoter CpG island hypermethylation has become recognized as an important mechanism for inactivating tumor suppressor genes or tumor-related genes in human cancers of various tissues. Gene inactivation in association with promoter CpG island hypermethylation has been reported to be four times more frequent than genetic changes in human colorectal cancers. Hepatocellular carcinoma is also one of the human cancer types in which aberrant promoter CpG island hypermethylation is frequently found. However, the number of genes identified to date as hypermethylated for hepatocellular carcinoma (HCC) is fewer than that for colorectal cancer or gastric cancer, which can be attributed to fewer attempts to perform genome-wide methylation profiling for HCC. In the present study, we used bead-array technology and coupled methylation-specific PCR to identify new genes showing cancer-specific methylation in HCC. Twenty-four new genes have been identified as hypermethylated at their promoter CpG island loci in a cancer-specific manner. Of these, TNFRSF10C, HOXA9, NPY, and IRF5 were frequently hypermethylated in hepatocellular carcinoma tissue samples and their methylation was found to be closely associated with inactivation of gene expression. Further study will be required to elucidate the clinicopathological implications of these newly found DNA methylation markers in hepatocellular carcinoma.

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Analysis of mRNA expression of candidate genes in hepatocellular carcinoma cell lines by quantitative RT-PCR. Cells were either mock-treated (PBS) or treated with 5-aza-dC (1AZA [1 µM] or 5AZA [5 µM] for 96 hr) as indicated. The expression patterns and re-expression patterns for each gene after 5-aza-dC treatment are shown for the cell lines that were methylated. Numbers along the horizontal axis indicate the cell lines. Gene expression levels were determined by quantitative RT-PCR and normalized to GAPDH levels.
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Figure 6: Analysis of mRNA expression of candidate genes in hepatocellular carcinoma cell lines by quantitative RT-PCR. Cells were either mock-treated (PBS) or treated with 5-aza-dC (1AZA [1 µM] or 5AZA [5 µM] for 96 hr) as indicated. The expression patterns and re-expression patterns for each gene after 5-aza-dC treatment are shown for the cell lines that were methylated. Numbers along the horizontal axis indicate the cell lines. Gene expression levels were determined by quantitative RT-PCR and normalized to GAPDH levels.

Mentions: For hypermethylated and transcriptionally silenced genes, the DNA demethylating agent 5-aza-dC is known to induce gene re-expression (14, 15). To identify whether promoter CpG island hypermethylation was closely associated with gene expression in the 24 newly identified cancer-specific methylated genes, we treated the 8 HCC cell lines with 5-aza-dC (1 and 5 µM for 96 hr) and performed quantitative RT-PCR of the mock-treated and 5-aza-dC treated cell lines. TNFRSF10C, NPY, TIAM1, and HOXA9 were methylated in all cell lines and showed up-regulation of mRNA expression in all cell lines with 5-aza-dC, although TIAM1 was not re-expressed in two cell lines, SNU-878 and Huh7. Most of the methylated genes exhibited re-expression after 5-aza-dC treatment; in contrast, SNU-886 in PDGFRB; SNU-878 in IRF5; SNU-398, HepG2, and Huh7 in HIC2; HepG2 and Huh7 in HS3ST2; SNU-878 in ADCYAP1; and SNU-886 in NOTCH3 were down-regulated by 5-aza-dC. In addition, HepG2 and SNU-761 in WNT2, HepG2 in ADAMTS12, and SNU-878 in FLT3 were not re-expressed (Fig. 6). In such cases, histone modification may be related to transcription suppression (16, 17).


Identification of novel methylation markers in hepatocellular carcinoma using a methylation array.

Shin SH, Kim BH, Jang JJ, Suh KS, Kang GH - J. Korean Med. Sci. (2010)

Analysis of mRNA expression of candidate genes in hepatocellular carcinoma cell lines by quantitative RT-PCR. Cells were either mock-treated (PBS) or treated with 5-aza-dC (1AZA [1 µM] or 5AZA [5 µM] for 96 hr) as indicated. The expression patterns and re-expression patterns for each gene after 5-aza-dC treatment are shown for the cell lines that were methylated. Numbers along the horizontal axis indicate the cell lines. Gene expression levels were determined by quantitative RT-PCR and normalized to GAPDH levels.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2908783&req=5

Figure 6: Analysis of mRNA expression of candidate genes in hepatocellular carcinoma cell lines by quantitative RT-PCR. Cells were either mock-treated (PBS) or treated with 5-aza-dC (1AZA [1 µM] or 5AZA [5 µM] for 96 hr) as indicated. The expression patterns and re-expression patterns for each gene after 5-aza-dC treatment are shown for the cell lines that were methylated. Numbers along the horizontal axis indicate the cell lines. Gene expression levels were determined by quantitative RT-PCR and normalized to GAPDH levels.
Mentions: For hypermethylated and transcriptionally silenced genes, the DNA demethylating agent 5-aza-dC is known to induce gene re-expression (14, 15). To identify whether promoter CpG island hypermethylation was closely associated with gene expression in the 24 newly identified cancer-specific methylated genes, we treated the 8 HCC cell lines with 5-aza-dC (1 and 5 µM for 96 hr) and performed quantitative RT-PCR of the mock-treated and 5-aza-dC treated cell lines. TNFRSF10C, NPY, TIAM1, and HOXA9 were methylated in all cell lines and showed up-regulation of mRNA expression in all cell lines with 5-aza-dC, although TIAM1 was not re-expressed in two cell lines, SNU-878 and Huh7. Most of the methylated genes exhibited re-expression after 5-aza-dC treatment; in contrast, SNU-886 in PDGFRB; SNU-878 in IRF5; SNU-398, HepG2, and Huh7 in HIC2; HepG2 and Huh7 in HS3ST2; SNU-878 in ADCYAP1; and SNU-886 in NOTCH3 were down-regulated by 5-aza-dC. In addition, HepG2 and SNU-761 in WNT2, HepG2 in ADAMTS12, and SNU-878 in FLT3 were not re-expressed (Fig. 6). In such cases, histone modification may be related to transcription suppression (16, 17).

Bottom Line: Twenty-four new genes have been identified as hypermethylated at their promoter CpG island loci in a cancer-specific manner.Of these, TNFRSF10C, HOXA9, NPY, and IRF5 were frequently hypermethylated in hepatocellular carcinoma tissue samples and their methylation was found to be closely associated with inactivation of gene expression.Further study will be required to elucidate the clinicopathological implications of these newly found DNA methylation markers in hepatocellular carcinoma.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Epigenetics, Cancer Research Institute and Brain Korea 2nd Stage, Seoul National University, Seoul, Korea.

ABSTRACT
Promoter CpG island hypermethylation has become recognized as an important mechanism for inactivating tumor suppressor genes or tumor-related genes in human cancers of various tissues. Gene inactivation in association with promoter CpG island hypermethylation has been reported to be four times more frequent than genetic changes in human colorectal cancers. Hepatocellular carcinoma is also one of the human cancer types in which aberrant promoter CpG island hypermethylation is frequently found. However, the number of genes identified to date as hypermethylated for hepatocellular carcinoma (HCC) is fewer than that for colorectal cancer or gastric cancer, which can be attributed to fewer attempts to perform genome-wide methylation profiling for HCC. In the present study, we used bead-array technology and coupled methylation-specific PCR to identify new genes showing cancer-specific methylation in HCC. Twenty-four new genes have been identified as hypermethylated at their promoter CpG island loci in a cancer-specific manner. Of these, TNFRSF10C, HOXA9, NPY, and IRF5 were frequently hypermethylated in hepatocellular carcinoma tissue samples and their methylation was found to be closely associated with inactivation of gene expression. Further study will be required to elucidate the clinicopathological implications of these newly found DNA methylation markers in hepatocellular carcinoma.

Show MeSH
Related in: MedlinePlus