多基因甲基化联合检测在云南地区肺癌早期诊断中的应用研究

发布时间：2018-08-19 09:03

【摘要】：背景与目的：肺癌是复杂性疾病,由遗传和环境因素共同作用所致。DNA甲基化体现了机体内部遗传和外界环境的交互作用,是一种早期事件,先于肺癌表型出现之前,在早期诊断中具有潜在的应用价值。同时,大部分肺癌患者在临床确诊之时已到中晚期,寻找一种有效的生物标志物迫在眉睫。而液体活检技术在肿瘤早期诊断、预后,以及精准医疗中占有越来越重要的地位。另外,云南多个地区(如宣威)是世界著名的肺癌高发区。因此,本文旨在通过对该地区肺癌患者样本进行研究,探讨血浆游离DNA(cell-free DNA,cfDNA)中多基因甲基化联合检测在肺癌早期诊断中的意义,并为云南肺癌发生机理提供参考依据。方法：本文研究对象来源于云南地区,其中,23例样本取自于肺癌患者肿瘤组织,42例样本为肺癌患者血浆,此外,10例健康血浆样本作为对照。对于所有样本,均使用试剂盒提取DNA/cfDNA与重亚硫酸盐处理,之后采用巢式甲基化特异性PCR(nested methylation-specific PCR, nMSP)法进行甲基化检测。首先根据文献报道,在肺癌组织样本中检测了8个基因(p16、DLECI、 CDH1、 DAPK、RUNX3、APC、WIF1及MGMT)的甲基化情况,之后筛选出阳性率较高的基因,用于在血浆cfDNA中进行检测。由于9例患者既包括血浆,也含有组织,也对甲基化情况进行一致性分析。此外,部分血浆样本的甲基化及非甲基化产物,采用亚硫酸氢盐测序法进行验证。最后,分析甲基化与患者临床病理信息之间的关系。结果：在组织样本中,除MGMT以外的其余7个基因,其甲基化阳性率在39%-74%之间,并且联合检测时所有样本至少1个基因发生甲基化的检出率为96%,至少2个基因共同甲基化的检出率为91%。其中,p16、DLEC、CDH1、 DAPK、RUNX3这5个基因的甲基化率较高(≥48%),因而在血浆cfDNA中进行检测。结果发现,cfDNA中5个基因的甲基化率在14%-76%之间,并且联合检测时所有样本至少1个或2个基因发生甲基化的检出率分别为95%和71%。健康对照实验中,这5个基因的甲基化均无检出。此外,9例配对血浆和组织样本中的甲基化情况基本一致。结合临床数据统计分析显示,CDH1基因甲基化与肺癌病理分型、临床分期和远处转移有关,而W1F1基因甲基化与肺癌临床病理分型有关。最后,在组织或血浆DNA甲基化情况中,CDH1、RUNX3和W1F1基因易受患者年龄、性别或吸烟等因素的影响。主要结论：(1)云南地区肺癌发生发展与DNA甲基化密切相关,多个基因的甲基化联合检测比单基因更具优势。(2)肺癌样本血浆cfDNA中p16、DLECI、 CDH1、 DAPK、RUNX3等5个基因的甲基化检出具有较高的敏感性和特异性,其组合可进一步研究,有望开发成为云南肺癌早期诊断的表观遗传生物标志物。(3)CDH1基因的甲基化与肺癌临床多个病理特征相关,在肺癌的预后判断中具有一定的应用价值。
[Abstract]:Background & objective: lung cancer is a complex disease. DNA methylation caused by genetic and environmental factors reflects the interaction between internal genetics and external environment. It is an early event before the appearance of lung cancer phenotype. It has potential application value in early diagnosis. At the same time, most lung cancer patients have reached the middle and late stage of clinical diagnosis, so it is urgent to find an effective biomarker. Fluid biopsy plays a more and more important role in early diagnosis, prognosis and accurate medicine. In addition, many areas of Yunnan Province (such as Xuanwei) are the world famous lung cancer high incidence area. Therefore, the purpose of this study was to investigate the significance of polygenic methylation in plasma free DNA (cell-free DNA cf DNA) in the early diagnosis of lung cancer, and to provide a reference for the pathogenesis of lung cancer in Yunnan Province. Methods: in this study, 23 samples were taken from tumor tissue of lung cancer patients, 42 samples were plasma samples from lung cancer patients, and 10 healthy plasma samples were taken as control. For all samples, DNA/cfDNA was extracted by kit and treated with sulfite, and then methylation was detected by nested methylation specific PCR (nested methylation-specific PCR, nMSP). Firstly, the methylation of eight genes (p16DLECI, CDH1, DAPKUX3RUNX3APCWIF1 and MGMT) were detected in lung cancer tissue samples according to literature reports, and then the genes with high positive rate were screened for detection in plasma cfDNA. Since 9 patients included both plasma and tissue, a consistent analysis of methylation was carried out. In addition, the methylation and demethylation products of some plasma samples were verified by bisulfite sequencing. Finally, the relationship between methylation and clinicopathological information was analyzed. Results: in tissue samples, the methylation positive rate of 7 genes except MGMT ranged from 39% to 74%. The detection rate of methylation of at least one gene in all samples was 96, and that of common methylation of at least two genes was 91. The methylation rate (鈮，

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