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目的探讨循环DNA在食管癌异常甲基化检测中的应用价值。方法收集食管癌患者的癌组织、食管远端组织和外周血浆样本及正常体检人群的食管黏膜对照组织,采用450K甲基化芯片初筛食管癌组织中差异甲基化位点,扩大样本采用DNA甲基化时间飞行质谱技术对食管癌组织、对应血浆游离DNA进行甲基化定量检测,验证癌组织与血浆游离DNA甲基化的一致性。结果基于芯片结果获得10个抑癌基因用于后续验证,包括ADAMTS9、AIM2、CASZ1、CDH13、EBF3、ING2、IQGAP2、KLF6、TMEFF2和TRIT1。质谱分析显示,所有位点的平均甲基化水平在食管癌患者血浆游离DNA样品中最高,其次是食管癌组织和正常食管黏膜对照组织。结论基因芯片技术可用于食管癌差异甲基化位点的初筛;但构建的抑癌基因异常甲基化谱在应用前尚需进行大样本、多阶段验证。血浆游离DNA甲基化谱可作为潜在的生物标志物应用于食管癌的诊断。
Objective To investigate the value of circulating DNA in the detection of abnormal methylation in esophageal cancer. Methods The esophageal mucosa of esophageal cancer tissue, esophageal distal and peripheral plasma samples and normal control subjects were collected. The methylated sites in esophageal cancer tissues were sorted by 450K methylated microarray. Methylation time-of-flight mass spectrometry was used to detect methylation of esophageal cancer tissue and corresponding plasma free DNA to verify the consistency of DNA methylation in cancer tissues and plasma. Results Ten tumor suppressor genes were obtained for subsequent validation based on chip results including ADAMTS9, AIM2, CASZ1, CDH13, EBF3, ING2, IQGAP2, KLF6, TMEFF2 and TRIT1. Mass spectrometry analysis showed that the average methylation level of all sites was the highest in plasma free DNA samples of esophageal cancer patients, followed by esophageal cancer tissues and normal esophageal mucosa control tissues. Conclusion Gene chip technology can be used to screen differential methylation sites of esophageal cancer. However, abnormal methylation profiles of tumor suppressor genes need to be verified by large samples and multi-stage before application. Plasma free DNA methylation profile can be used as a potential biomarker in the diagnosis of esophageal cancer.