B7-02: Identification of differentially methylated CpG Islands in the early stage of human pulmonary adenocarcinoma

Abstract

In the development of cancer, a series of tumor suppressor genes is inactivated by point mutation and chromosomal deletion. Aberrant methylation of CpG islands has recently been shown to serve as an alternative way of inactivating such genes in cancer. Methylation of cytosine residues in CpG dinucleotides is an extremely important epigenetic modification of the eukaryotic genome that affects various cell processes. Methylation is known to play an important role in neoplasia by inactivating tumor suppressor genes such as Rb, p16, and estrogen receptor. In this study we used CpG island amplification and suppression subtractive hybridization to identify the aberrant methylation of CpG islands in A/J mouse lung adenoma, in order to clarify whether a hypermethylated homologue gene exists in human pulmonary adenocarcinoma. NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone)-induced A/J mouse lung adenoma and normal tissue were used in this study. The histology of A/J mouse adenoma mimics the early stage of human pulmonary adenocarcinoma. The genomic DNA was extracted using standard procedures. The PCR-based methylated CpG island amplification (MCA) technique was used for detection of methylated CpG islands. Suppression subtractive hybridization (SSH) and differential screening (DS) were used to identify the differentially methylated sequence in A/J mouse lung adenoma tissue. For the genes selected after SSH and DS analysis, quantitative Real-time PCR was used to check their expression level in A/J mouse lung adenoma tissue and normal tissue. Using homoloGene software, human homologue genes were then detected. Real-time PCR was also performed to check the expression level of these genes in human lung adenocarcinoma and normal lung tissue. Bisulfite genomic sequencing was done to confirm the methylation status of these genes in the promotor area. After MCA-SSH and DS, 117 clones from a total of 1000 were selected for sequence analysis. Four genes were identified in the DNA fragments from 117 clones from A/J mouse lung tumor tissue. These were KIF21A (kinesin family member 21A), Samd14 (sterile alpha motif domain containing 14), EG436235 (similar to regulator of G-protein signal 3) and Vwa1 (von Willebrand factor A domain containing 1). Among these four genes, two gene fragments had a genomic region that met the criteria for a CpG island. RT-PCR showed differences in expression level between A/J mouse normal lung tissue and lung adenoma tissue for the KIF21A (p<0.005), Samd14 (p=0.005) and EG436235 (p<0.01) genes. Further study of the human homologues of KIF21A (p<0.01) and Samd14 (p<0.002) showed that they were expressed in normal lung tissue but markedly down-regulated in lung adenocarcinoma tissue. Bisulfite sequencing revealed that the promtor region of Samd14 in human lung adenocarcinoma was methylated more frequently than that of its normal counterpart. The expression level of the KIF21A (location: 12q12) and Samd14 (location: 17q21.33) genes is down regulated in human pulmonary adenocarcinoma tissue. The methylation frequency of the Samd14 gene in the promotor region is higher in human pulmonary adenocarcinoma than in normal lung tissue. These data suggest that promotor methylation of these genes is a specific event in pulmonary adenocarcinogenesis, and that their down-regulation may be functionally associated with malignant progression of lung adenocarcinoma