J Korean Med Sci.  2010 Aug;25(8):1152-1159. 10.3346/jkms.2010.25.8.1152.

Identification of Novel Methylation Markers in Hepatocellular Carcinoma using a Methylation Array

Affiliations
  • 1Laboratory of Epigenetics, Cancer Research Institute and Brain Korea 2nd Stage, Seoul National University, Seoul, Korea. ghkang@snu.ac.kr
  • 2Department of Pathology, Korea University Medical School, Seoul, Korea.
  • 3Department of Pathology, Seoul National University College of Medicine, Seoul, Korea.
  • 4Department of Surgery, Seoul National University College of Medicine, 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.

Keyword

Bead Array; CpG Islands; DNA Methylation; Carcinoma, Hepatocellular

MeSH Terms

Antimetabolites, Antineoplastic/therapeutic use
Azacitidine/analogs & derivatives/therapeutic use
Carcinoma, Hepatocellular/drug therapy/*genetics
Cell Line, Tumor
CpG Islands
*DNA Methylation
GPI-Linked Proteins/genetics
Gene Expression Profiling
Homeodomain Proteins/genetics
Humans
Interferon Regulatory Factors/genetics
Liver Neoplasms/drug therapy/*genetics
Neuropeptide Y/genetics
Oligonucleotide Array Sequence Analysis
Promoter Regions, Genetic
Tumor Necrosis Factor Decoy Receptors/genetics

Figure

  • Fig. 1 Flow chart for selection of candidate methylation markers. We used 5 paired hepatocellular carcinoma/normal tissues to screen for candidate methylation markers using a methylation array. We obtained 72 candidates that showed significant hypermethylation in hepatocellular carcinoma tissues. We removed genes with no CpG island loci in their promoters, imprinted genes, and genes for which methylation status was already known for hepatocellular carcinoma. Thus, we selected 36 genes to further examine for methylation analysis using methylation-specific PCR.

  • Fig. 2 Representative examples of MSP analysis of DST, FLT3, PTCH2 and TIAM1 in 8 HCC cell lines. DNA extracted from 8 HCC cell lines were amplified with primers specific to the methylated (M) or unmethylated (UM) CpG islands of each gene after modification with sodium bisulfite. +, positive control; DW, distilled water. Positive controls for methylated MSP and unmethylated DNA are M.SssI-treated placental DNA and whole-genome amplified DNA, respectively.

  • Fig. 3 Summary of methylation-specific PCR for hepatocellular carcinoma (HCC) cell lines. We examined eight HCC cell lines for methylation status of the 35 genes and found that 26 of the genes (HOXA9, TNFRSF10C, NPY, TIAM1, PDGFRB, IRAK3, SH3BP2, IRF5, HIC2, TAL1, HS3ST2, MLF1, IGF2AS, ADCYAP1, FGF3, WNT2, ADAMTS12, FLT3, PTCH2, GP1BB, RBP1, FZD9, DST, NOTCH3, PLAGL1, and ZP3) were methylated in at least one HCC cell line. Data are color-coded as follows: gray fill indicates the presence of methylation, whereas white fill indicates the absence of methylation.

  • Fig. 4 Summary of methylation-specific PCR results in hepatocellular carcinoma (n=50) and non-neoplastic liver tissue samples (n=18). Out of 26 genes showing methylation in HCC cell lines, promoter methylation was detected in 25 genes in primary tumor tissues. Data are color-coded as follows: gray fill indicates the presence of methylation, while white fill indicates absence of methylation.

  • Fig. 5 Comparison of hypermethylation frequencies for 26 genes. Hepatocellular carcinoma (HCC) cell lines (white column), HCC tissue samples (gray column), and non-neoplastic liver tissue samples (HCN, black column).

  • Fig. 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.


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