腺瘤性结肠息肉病基因甲基化在肝细胞癌分子诊断中的价值

Clinical value of methylation of plasma adenomatous polyposis coli gene in the molecular diagnosis of hepatocellular carcinoma

目的:建立甲基化敏感性限制性内切酶-定量PCR(methylation-sensitive restriction enzyme-quantitative PCR,MSRE-qPCR)方法,并应用该方法探讨血浆腺瘤性结肠息肉病(adenomatous polyposis coli,APC)基因甲基化检测在肝细胞癌(hepatocellular carcinoma,HCC)诊断中的应用价值。方法:用HhaⅠ消化DNA样品,qPCR技术评估酶切效率,建立优化的MSRE-qPCR方法。用MSRE-qPCR法检测45例肝组织(20例HCC及其相应匹配的非癌组织和5例正常肝组织)中APC甲基化水平;应用亚硫酸氢盐测序PCR(bisulfi te sequencing PCR,BSP)对MSRE-qPCR检测结果进行验证,并与甲基化特异性PCR(methylation-specifi c PCR,MSP)检测方法比较。运用MSRE-qPCR技术检测72例HCC、37例肝良性病变和41例健康志愿者血浆标本的APC甲基化状态。结果:建立的MSRE-qPCR方法可定量检测低至1%的APC甲基化片段。MSRE-qPCR和MSP检测结果均显示,HCC组织中APC基因发生高频率甲基化。MSRE-qPCR检测结果经BSP验证准确无误,且与MSP检测结果具有较好的一致性(Kappa=0.955,P<0.0001)。HCC患者血浆APC甲基化水平高于肝良性病变及健康志愿者(P<0.0001)。血浆APC甲基化分析与血清甲胎蛋白(alpha-fetoprotein,AFP)联合检测可提高HCC诊断效率。结论:MSRE-qPCR可定量检测APC甲基化水平。血浆APC甲基化分析对于HCC的非侵入性诊断具有重要价值。

Objective： To establish a method of methylation-sensitive restriction enzymes- quantitative PCR （MSRE-qPCR） for methylation analysis of adenomatous polyposis coli （APC） gene, and to further assess the clinical value of plasma methylation detection by using this method in diagnosis of hepatocellular carcinoma （HCC）. Methods： Hha I was used to digest genomic DNA, and the digestion efficiency was evaluated by using qPCR technique. Then the optimized MSRE-qPCR method was established. The methylation levels of APC in 45 liver tissues （20 surgically resected HCC specimens and the matched non-cancerous tissues, as well as 5 normal liver tissues） were detected by MSRE-qPCR, and then further validated by using bisulfite sequencing PCR （BSP）. The results of MSRE-qPCR were compared with those of methylation-specific PCR （MSP） assay. MSRE-qPCR method was used to detect the APC methylation status of plasma samples from 72 cases of HCC, 37 cases of benign liver diseases and 41 healthy volunteers. Results： The established MSRE-qPCR method could detect as low as 1% methylated target sites in given DNA samples. The results of MSRE-qPCR and MSP showed that APC gene was hypermethylated in HCC tissues. The result of MSRE-qPCR was verified by BSP, and it was comparable with that of MSP （Kappa=0.955, P〈0.000 1）. Methylation level of plasma APC in patients with HCC was significant higher than those in patients with benign liver diseases and the healthy volunteers （P〈0.000 1）.Combined analysis of plasma APC methylation and serum alpha-fetoprotein （AFP） revealed an increased diagnostic efficacy for HCC. Conclusion： MSRE-qPCR is a method for quantitative analysis ofAPC methylation level. Methylation analysis of plasma APCis a valuable method for the noninvasive diagnosis of HCC.