抑癌基因APC启动子甲基化检测及其在肺癌诊断中的应用

目的　建立抑癌基因APC启动子部位甲基化的检测方法 ,并对肺癌临床样品进行初步检测研究。方法 :对肺癌组织提取的DNA及转甲基后的脐带血DNA进行化学修饰 ,以甲基化特异性引物进行基因扩增 (methylationspecificPCR ,MSP)和甲基化序列分析 (methylationsequencing ,MS)。结果 :经转甲基的人脐带血DNA样品MSP检测为阳性 ,其序列与预期相符 ,未经转甲基样品为阴性。 17例肺癌患者的肿瘤及其正常肺组织APC甲基化检测阳性率分别为 4 7% (8/ 17)和 18% (3/ 17) ,1例肺部良性肿瘤及其正常肺组织的检测结果均为阴性 ;对MSP检测为阴性的 9例肺癌患者肿瘤及其正常肺组织进行MS检测分析 ,有 4例APC启动子部位存在CpG甲基化。结论 :利用MSP和MS两种甲基化检测分析手段 ,对 17例临床确诊肺癌组织的检测总阳性率可达 71% (12 / 17)。MSP操作简便 ,价格低廉 ,可适合于大规模肿瘤患者样本的筛查 ;而对于MSP检测为阴性的临床样品 ,可以利用MS进行进一步的确认 ,为临床肺癌诊断提供更准确的信息。

Detection of promoter hypermethylation of the adenomatous polyposis coli promoter 1A and It's clinical significance

Promoter hypermethylation is a major mechanism for gene silencing and offers a promising starting point for developing molecular biomarkers.To determine aberrant methylation of the adenomatous polyposis coli (APC) gene promoter 1A with respect to its prevalence and the clinical pathological significance in non-small cell lung cancer (NSCLC),we developed the positive control for methAPC methylation specific PCR (MSP) and methylation sequencing (MS),examined the promoter methylation status of the APC gene using both of the two methods in 17 NSCLC patients and one case of lung benign tumor.The frequencies of methylation in NSCLCs and corresponding non-neoplastic lung tissues were:47% (8 of 17) and 18% (3 of 17) for MSP.There were four cases existed methylation in APC gene promoter 1A among the nine MSP negative cases by MS.We approved the total positive detection of methAPC to 71% by using the two methods of MSP and MS.MS may be much more sensitive than MSP regarding to the poly sites of methAPC and the templates which were influenced by heterozygosity change of methAPC for MSP amplification.Our findings suggest that promoter hypermethylation can be detected in the majority of lung cancer patients with the combined detection methods for methAPC.This approach needs to be evaluated in large early detection and surveillance studies of lung cancer.