高分辨熔解曲线法检测哈萨克族食管癌患者癌组织中FHIT、CDKN2A启动子区甲基化水平及临床意义

 建立高分辨熔解曲线法(HRM)定量检测哈萨克族食管癌患者癌组织中FHIT、CDKN2A甲基化水平,并分析甲基化水平与食管癌病理参数的相关性。选取30例食管癌癌患者癌组织及30例癌旁组织,提取DNA,进行甲基化修饰。将标准品DNA(甲基化DNA与非甲基化DNA相互的掺入)稀释成0,5%,25%,50%,75%,100%甲基化DNA,并进行重复性和灵敏性评价。应用HRM定量检测癌组织及癌旁组织甲基化水平,探讨FHIT、CDKN2A基因启动子区甲基化水平与食管癌发生发展的关系。100%,80%,50%,30%,10%,0甲基化标准品的高分辨率熔解曲线从右向左依次排列,待测基因在标准曲线上的位置即表示其甲基化程度。HRM最低检测限为1%,明显高于MSP结果最低检测限10%。在30例食管癌癌患者癌组织中,检测出存在FHIT甲基化的为36.7%。其中8例甲基化程度为0—5%,3例为5%—10%。CDKN2A甲基化的为100%,其中7例甲基化程度为0—5%,11例为5%—10%,12例为10%—25%。与正常组织比较,抑癌基因的甲基化与癌症的发生无明显相关性。进一步讨论甲基化与食管癌的病理级别以及甲基化与分化程度的关系,二者均无相关性。通过HRM技术成功建立了定量检测哈萨克族食管癌组织中FHIT、CDKN2A甲基化程度的方法,FHIT、CDKN2A基因启动子甲基化与哈萨克族食管癌的发生发展无明显相关性,但多个抑癌基因的甲基化有望成为其早期发生发展的分子指标。

Determination of FHIT and CDKN2A Promoter Methylation Levels in Hazak's Esophageal Cancer Tissue Based on Methylation-sensitive High Resolution Melting Analysis and Its Clinical Significance

The promoter methylation of tumor suppressor gene is one of the most important mechanisms of tumorigenesis. The method of Methylation-Sensitive High-Resolution Melting-curve (MS-HRM) analysis was used to analyze methylation differences at the FHIT, CDKN2A locus in DNA samples from individuals with Esophageal Cancer (EC) (n=30) and adjacent normal tissues (n=30). After extracting and modifying DNA by methylating-agents, the standard DNA samples were mixed with the completely methylated DNA to make the dilute methyled DNAs of 0%, 5%, 25%, 50%, 75%, and 100%, respectively. And then their sensitivity and repeatability were evaluated. Using HRM, the methylation level was detected. And the relation between the methylation levels of FHIT and CDKN2A promoters and the development of EC is discussed. The high-resolution melting-curves of 100%, 80%, 50%, 30%, 10%, and 0% methylated DNA are decreasingly arranged from right to left, the locations on the standard curve denote the methylation level. The lowest detection limit of HRM is 1%, and it is more sensitive than the 10% detection limit of MSP. With the MS-HRM assay, 36.7% of the FHIT promoters (11/30) are methylated. Among them, there are eight cases with methylation level between 0%—5%,and three cases with methylation level between 5%—10%. 100% of the CDKN2A promoters (30/30) are methylated. There are seven cases with methylation level between 0%—5%, 11 cases with methylation level between 5%—10%, and 12 cases with methylation level between 10%—25%. Compared with the adjacent normal tissues, there is no significant relationship between the methylation and the development of the Hazak's EC. There is no significant relationship either between the methylation and the TNM or between methylation and differentiation of two genes in the Hazak's esophageal cancer. It is found that the MS-HRM is a simple, rapid, and robust method for screening methylation differences at the FHIT, CDKN2A locus; furthermore the more suppressor genes will be screened and found as the early diagnostic markers.