Tuberc Respir Dis.  2008 Dec;65(6):495-503.

Identification of DNA Methylation Markers for NSCLC Using Hpall-Mspl Methylation Microarray

• Kwon MH
• Lee GE
• Kwon SJ
• Choi E
• Na MJ
• Cho HM
• Kim YJ
• Sul HJ
• Cho YJ
• Son JW

Abstract

BACKGROUND: Epigenetic alterations in certain genes are now known as at least important as genetic mutation in pathogenesis of cancer. Especially abnormal hypermethylation in or near promoter region of tumor suppressor genes (TSGs) are known to result in gene silencing and loss of gene function eventually. The authors tried to search for new lung cancer-specific TSGs which have CpG islands and HpaII sites, and are thought to be involved in carcinogenesis by epigenetic mechanism.
METHODS
Tumor tissue and corresponding adjacent normal tissue were obtained from 10 patients who diagnosed with non small cell lung cancer (NSCLC) and underwent surgery in Konyang university hospital in 2005. Methylation profiles of promoter region of 21 genes in tumor tissue & non-tumor tissue were examined with HpaII-MspI methylation microarray (Methyl-Scan DNA chip(R), Genomic tree, Inc, South Korea). The rates of hypermethylation were compared in tumor and non-tumor group, and as a normal control, we obtained lung tissue from two young patients with pneumothorax during bullectomies, methylation profiles were examined in the same way.
RESULTS
Among the 21 genes, 10 genes were commonly methylated in tumor, non-tumor, and control group. The 6 genes of APC, AR, RAR-b, HTR1B, EPHA3, and CFTR, among the rest of 11 genes were not methylated in control, and more frequently hypermethylated in tumor tissue than non-tumor tissue.
CONCLUSION
In the present study, HTR1B, EPHA3, and CFTR are suggested as possible novel TSGs of NSCLC by epigenetic mechanism.

Keyword

DNA hypermethylation; Tumor suppressor gene; DNA microarray; Non-small cell lung cancer

MeSH Terms

Carcinoma, Non-Small-Cell Lung
CpG Islands
DNA
DNA Methylation
Epigenomics
Gene Silencing
Genes, Tumor Suppressor
Humans
Lung
Methylation
Oligonucleotide Array Sequence Analysis
Pneumothorax
Promoter Regions, Genetic
Small Cell Lung Carcinoma
DNA

Figure

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  Figure 1 Schematic diagram of methylation microarray. Purified genomic DNA is digested with HpaII and MspI. If the genomic DNA has hypermethylation in CpG islands of promoter region, HpaII is sensitive to methylation status, whereas MspI is still insensitive. Multiplex PCR amplification is accomplished with un-digested and HpaII-, MspI-digested DNA. Each of 9 promoter regions can be amplified simultaneously in a single tube using 9 specific primer pairs, and for 50 promoters and control, 6 sets of PCR are required. During amplification step, fluorescent dyes (Cy3- or Cy5-dUTP) are incorporated into the amplicons. After PCR amplification, all amplicons are mixed and hybridized on Methyl-Scan DNA chip, on which 50 specific promoter regions are spotted. When we observe the hybridization signal of HpaII-amplicon is 2.0-fold greater than MspI-amplicon's signal, we considered 'methylated'.

• 

  Figure 2 Genes commonly methylated in three groups. There were 11 commonly methylated genes; CDH13, CALCA, LAMA5, MTHFR, PIC3CG, S100A2, SHP1, EDN1, H19, MUC2, and LTB4R.

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  Figure 3 Genes of hypermethylation profiles in tumor tissue. Promoter regions of 6 genes of APC, AR, RAR-b, HTR1B, EPHA3, and CFTR were more frequently methylated in tumor tissue than non-tumor tissue and not methylated in control group.

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  Figure 4 Validation of microarray with gel electrophoresis. An example of gel electrophoresis (patient 1). The signal of CFTR was revealed 'negative' in normal tissue (dashed arrow) and 'positive' in tumor tissue (solid arrow) by microarray. And the same results were seen in gel electrophoresis; undigested sample (first lane) only seen in normal tissue (A) and undigested and HpaII-digested sample (second lane) seen with the absence of third lane for tumor tissue (B).

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