实时荧光定量PCR和甲基化特异性PCR检测结直肠癌APC、MLH1基因甲基化状态

Detection of APC and MLH1 Gene Hypermethylation with Real-time Fluorescent Quantitative PCR and Methylation-specific PCR in Colorectal Cancer

背景:启动子区甲基化致肿瘤抑制基因失活在结直肠癌的发生、发展中起重要作用,检测肿瘤相关基因的甲基化状态,可能为寻找新的结直肠癌诊断、预后相关标记物提供依据。目的:比较实时荧光定量PCR(FQ-PCR)与甲基化特异性PCR(MSP)检测基因甲基化状态的差异。方法:以FQ-PCR检测66例结直肠癌组织和20例癌旁组织中与结直肠癌的发生、发展相关的抑癌基因APC和错配修复基因MLH1的甲基化状态,同时以MSP检测结直肠癌组织中APC基因的甲基化状态。结果:根据FQ-PCR结果,结直肠癌组织中APC、MLH1基因甲基化阳性率显著高于癌旁组织(48.5%和54.5%对0%和0%,P=0.000)。FQ-PCR和MSP可检出的甲基化阳性对照DNA最低浓度分别为0.015 ng/μl和1.5 ng/μl,两者对APC基因甲基化状态的检测结果差异有统计学意义(P<0.05)。结论:在DNA甲基化的检测手段中,FQ-PCR的敏感度优于MSP。

Background： Inactivation of tumor suppressor genes by promotor methylation plays an important role in the development and progression of eolorectal cancer. Detection of hypermethylation of tumorigenesis-related genes might provide promising biomarkers for the diagnosis and prognosis of colorectal cancer. Aims： To compare the value of real- time fluorescent quantitative PCR （FQ-PCR） and methylation-specific PCR （MSP） for detecting the gene methylation. Methods： Hypermethylations of tumor suppressor gene APC and mismatch repair gene MLH1, which were related with the development and progression of colorectal cancer, were detected in 66 colorectal cancer specimens and 20 adjacent tissue specimens by FQ-PCR. Meanwhile, hypermethylation of APC in colorectal cancer specimens was also detected by MSP. Results： APC hypermethylation and MLH1 hypermethylation were detected in 32 and 36 eases of coloreetal cancer tissues respectively by FQ-PCR, with the positivity rates significantly higher than those in adjacent tissues （48.5% and 54.5% vs. 0% and 0%, P=0.000）. The minimum concentrations of methylated DNA that could be detected by FQ-PCR and MSP were 0.015 ng/μl and 1.5 ng/μl, respectively, and the difference of FQ-PCR and MSP for detecting APC hypermethylation was statistically significant （P〈0.05）. Conclusions： The sensitivity of FQ-PCR in detecting gene methylation is superior to that nf MSP