基于基因芯片检测胃癌中p16（Ink4a）基因5'-CpG岛甲基化变化的方法

Microarray-based method for detecting methylation changes of p16(Ink4a) gene 5'-CpG islands in gastric carcinomas.

作者信息

Hou Peng, Shen Jia-Yao, Ji Mei-Ju, He Nong-Yue, Lu Zu-Hong

机构信息

Chien-Shiung Wu Laboratory, Department of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, Jiangsu Province, China.

出版信息

World J Gastroenterol. 2004 Dec 15;10(24):3553-8. doi: 10.3748/wjg.v10.i24.3553.

DOI:10.3748/wjg.v10.i24.3553

PMID:15534905

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4611991/

Abstract

AIM

Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation of the CpG sites of the tumor suppressor gene is closely associated with carcinogenesis. However, large-scale analysis of candidate genes has so far been hampered by the lack of high-throughput approach for analyzing DNA methylation. The aim of this study was to describe a microarray-based method for detecting changes of DNA methylation in cancer.

METHODS

This method used bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. Therefore, the amplified product might contain a pool of DNA fragments with altered nucleotide sequences due to differential methylation status. Nine sets of oligonucleotide probes were designed to fabricate a DNA microarray to detect the methylation changes of p16 gene CpG islands in gastric carcinomas. The results were further validated by methylation-specific PCR (MSP).

RESULTS

The experimental results showed that the microarray assay could successfully detect methylation changes of p16 gene in 18 gastric tumor samples. Moreover, it could also potentially increase the frequency of detecting p16 methylation from tumor samples than MSP.

CONCLUSION

Microarray assay could be applied as a useful tool for mapping methylation changes in multiple CpG loci and for generating epigenetic profiles in cancer.

摘要

目的

CpG位点的异常DNA甲基化是癌症中最早且最常见的改变之一。多项研究表明，肿瘤抑制基因CpG位点的异常甲基化与致癌作用密切相关。然而，迄今为止，由于缺乏用于分析DNA甲基化的高通量方法，对候选基因的大规模分析受到了阻碍。本研究的目的是描述一种基于微阵列的方法来检测癌症中DNA甲基化的变化。

方法

该方法使用亚硫酸氢盐修饰的DNA作为PCR扩增的模板，导致感兴趣的CpG岛内未甲基化的胞嘧啶而非甲基化的胞嘧啶转化为胸腺嘧啶。因此，由于甲基化状态不同，扩增产物可能包含一组核苷酸序列改变的DNA片段。设计了九组寡核苷酸探针来制作DNA微阵列，以检测胃癌中p16基因CpG岛的甲基化变化。结果通过甲基化特异性PCR（MSP）进一步验证。