胃癌组织及转移组织中DNA甲基化差异的研究

Difference in methylation of genomic DNA between gastric primary cancer and lymph nodes with metastatic gastric cancer

目的研究胃癌原发灶和转移淋巴结组织间基因组DNA甲基化程度的差异以及寻找胃癌淋巴转移抑制基因。方法应用甲基化CpG岛扩增(MCA)方法富集甲基化的基因组DNA序列,以转移淋巴结MCA产物为检测子,原发灶MCA产物为驱赶子,进行代表性差异显示分析(RDA)。将获得的甲基化差异片段克隆、测序,获得碱基序列,用生物信息学进行分析。结果获得19个差异序列,分布于5′端,外显子,内含子及3′端。其中KL59位于9p21,为p16基因的第一外显子内;KL22位于PTPRG基因启动子区。以KL22作探针与驱赶子、检测子及3轮RDA产物杂交,除驱赶子外都有杂交信号。结论胃癌原发灶与转移淋巴结组织间DNA甲基化存在差异,MCA-RDA方法为一种较好的分析方法。

Objective To investigate the difference in methylation of genomic DNA between gastric primary cancer and lymph nodes with metastatic gastric cancer. Methods Methylation CpG island amplification （MCA） was used to enrich the methylated DNA sequences in the cancer tissues and lymph nodes with metastatic gastric cancer resected during operation from 5 patients. Representational difference analysis （RDA） was conducted with the MCA products from the lymph nodes with metastatic gastric cancer as testers and the MCA products of the gastric primary cancer as drivers. The differentially methylated DNA fragments were cloned and sequenced, and underwent similarity analysis by BLAST system. The relationship between the clone sequence and related gene was analyzed by GenBank. Hybridization analysis was performed, using the No 1 ～ 3 round RDA products and the MCA products of the tissues of gastric primary cancer and lymph nodes with metastatic gastric cancer with digoxin-labeled KL22 fragment as probe. Results Nineteen differentially methylated DNA sequences were obtained, distributed at the 5＇-end, exon, intron, and 3＇-end. KL59 fragment was located in the 9q21, the first exon of p16 gene. KL12 fragment was located in the promoter region of phophotyrosine phophatase receptor G （PTPRG） gene. Hybridization signals were obtained for all No 1 ～ 3 round RDA products and all testers, but not for the drivers. Conclusion There is a difference in DNA methylation between the tissues of primary cancer and lymph nodes with metastatic gastric cancer. MCA-RNA is an effective method to study the gene methylation. PTPRG gene may be a candidate gene for metastasis of gastric cancer.