摘要
目的:建立一种可检测微量DNA标本中DNA甲基化的甲基化敏感性限制性内切酶-定量PCR(methylation-sensitive restriction enzymes-based quantitative PCR,MSRE-qPCR)方法,并运用该技术探讨血浆Ras相关区域家族蛋白1A(Ras association domain family1A,RASSF1A)基因甲基化检测在肝细胞癌(hepatocellutar carcinoma,HCC)非侵入性诊断中的价值。方法:用MSRE HhaⅠ消化DNA样品,再用qPCR技术分析酶切结果,建立检测RASSF1A基因甲基化的MSRE-qPCR方法。以45例肝组织(20对HCC患者手术切除肿瘤标本及匹配非癌组织和5例正常肝)为材料,测试该方法的应用价值;运用亚硫酸氢盐测序PCR(bisulf ite sequencing PCR,BSP)技术进行进一步验证,并与甲基化特异性PCR(methylation specif ic PCR,MSP)方法相比较。再运用该技术检测150例血浆标本(包括72例HCC患者、37例肝硬化或慢性肝炎等良性病变患者和41例健康对照)的RASSF1A基因甲基化状态,并分析其与HCC患者临床病理参数的关系。结果:MSRE-qPCR法可定量检测低至1%以下的RASSF1A甲基化片段。20例HCC组织中有14例(70%)发生RASSF1A高甲基化,对应非癌组织中RASSF1A甲基化阳性率为25%,而5例正常肝组织均为阴性。MSRE-qPCR结果经BSP验证无误,且与MSP检测结果具有较好的一致性。HCC患者血浆RASSF1A甲基化阳性率(47/72,65.3%)显著高于健康对照(1/41,2.4%)和肝良性病变组(3/37,8.1%),差异均有统计学意义(P<0.0001)。联合检测血浆RASSF1A甲基化与血清AFP可显著提高HCC诊断效率。结论:建立的MSRE-qPCR方法要求样本少、操作简便、成本低廉,可定量检测RASSF1A基因甲基化水平。血浆RASSF1A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 enzymes-based quantitative PCR （MSRE-qPCR） method for methylation analysis of Ras-association domain family 1A （RASSF1A） gene, and to further assess the clinical value of plasma methylation analysis using this method for noninvasive diagnosis of hepatocellutar carcinoma （HCC）. Methods： MRSE HhaⅠ was used to digest genomic DNA samples, and the digestion result was evaluated by using qPCR. Then the MSRE-qPCR method for methylation analysis of RASSF1A gene was established. The efficacy of this method was tested in 45 liver tissues （20 surgically resected HCC specimens and the matched non-cancerous tissues, as well as 5 normal liver tissues）, and which was further validated by using bisulfite sequencing PCR （BSP）. The results of MRSE-qPCR were compared with those of methylation-specific PCR （MSP） assay. The established MSRE-qPCR method was used to detect the RASSF1A methylation levels in 150 plasma samples from 72 cases of HCC, 37 cases of benign liver diseases such as liver cirrhosis and chronic hepatitis, and 41 normal controls, and the associations of RASSF1A methylation level with the clinicopathological parameters of patients with HCC were evaluated. Results： The established MSRE-qPCR method could detect as low as 1% methylated target sites in given DNA samples. Of the 20 HCC tissues, 14 （70%） were hypermethylated in the target CpG of RASSF1A promoter. Five matched non-cancerous tissues were also found to be methylated, whereas no methylated RASSF1A sequence was detected in 5 normal liver tissues. The result of MSRE-qPCR was accurate and comparable with that of MSP. The positive rate of RASSF1A methylation in plasma samples from patients with HCC was 65.3% （47/72）, which was significantly higher than those from patients with benign liver diseases （3/37, 8.1%） and normal controls （1/41, 2.4%） （P0.000 1）. Combined analysis of plasma RASSF1A methylation and serum AFP revealed an increased diagnostic efficacy for HCC. Conclusion： The established MSRE-qPCR is a simple, accurate and economical method for quantitative analysis of RASSF1A DNA methylation in clinical samples. Methylation analysis of RASSF1A gene in plasma is a valuable method for noninvasive diagnosis of HCC.
出处
《肿瘤》
CAS
CSCD
北大核心
2011年第8期742-747,共6页
Tumor
基金
江苏省自然科学基金资助项目(编号:BK2008114)
