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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 methylation-sensitive restriction enzyme-quantitative PCR (MSRE-qPCR) method to detect plasma adenomatous polyposis coli (APC) ) Gene methylation detection in the diagnosis of hepatocellular carcinoma (HCC). Methods: The DNA samples were digested with Hha I and the efficiency of enzyme digestion was evaluated by qPCR. The optimized MSRE-qPCR method was established. The levels of APC methylation in 45 hepatic tissues (20 HCCs and their corresponding matched non-cancerous tissues and 5 normal liver tissues) were detected by MSRE-qPCR. Bisulfite sequencing PCR (BSP) ) Was used to verify the MSRE-qPCR results and compared with the methylation-specific PCR (MSP) method. The APC methylation status of plasma samples from 72 patients with HCC, 37 benign liver diseases and 41 healthy volunteers was detected by MSRE-qPCR. Results: The MSRE-qPCR method was established to quantitatively detect APC methylated fragments down to 1%. The results of MSRE-qPCR and MSP showed that APC gene was hypermethylated in HCC tissue. MSRE-qPCR test results were verified by BSP accuracy, and with MSP test results have a good agreement (Kappa = 0.955, P <0.0001). Plasma APC methylation levels in HCC patients were higher than those in benign liver disease and healthy volunteers (P <0.0001). Plasma APC methylation analysis and serum alpha-fetoprotein (alpha-fetoprotein, AFP) combined detection can improve the diagnostic efficiency of HCC. Conclusion: MSRE-qPCR can quantitatively detect APC methylation level. Plasma APC methylation analysis is of great value for the noninvasive diagnosis of HCC.