膀胱癌的DNA甲基化为对象的基础和应用表观遗传学研究

项目名称： 膀胱癌的DNA甲基化为对象的基础和应用表观遗传学研究

项目编号： No.30872963

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 轻工业、手工业

项目作者： 余坚

作者单位： 上海市肿瘤研究所

项目金额： 33万元

中文摘要： 本项目运用Methylcap-Seq建立了膀胱癌细胞系5637、T24和2例正常人膀胱粘膜全基因组甲基化谱式，在膀胱癌细胞系中得到了包括promotor、5UTR、mir、Enchancer、exon等在内的4256个异常甲基化位点和401个异常去甲基化位点。对162个promotor位点的筛选，确定了8个在膀胱癌与正常对照中甲基化差异最大的基因：ECEL1、KcNV1、LMX1A、PROX1A、SLA620、TAL1、TMEM26和VAX1，为首次在膀胱移行上皮癌中发现的异常甲基化位点。运用MSP方法对上述8个位点在膀胱癌病人术前尿液212例、术后尿液21例、其他尿路疾病患者尿液41例、疑是膀胱癌门诊膀胱镜患者尿液48例，同年龄正常人尿液149例中验证，最终得到了由VAX1、KCNV1、CFTR、PROX1、TAL1组成的尿沉淀膀胱癌甲基化检测标志物，在尿液中对膀胱癌的检测敏感性为85.36%，特异性为90.0%。同时该甲基化谱式经横向队列（157例原发，55例复发）比较分析和纵向随访（145例原发中37例复发）分析，VAX1和LMX1a的甲基化与膀胱癌的复发有较显著的相关性。

中文关键词： 膀胱癌;尿液;DNA甲基化;甲基化组学;肿瘤生物标志物

英文摘要： In this study，we use MBD methylCap/seq to profile the methylation status of two BCa cell lines (T24 and 5637) and two normal bladder mucosal tissues on genome-wide level. As a result, compared with normal bladder mucosal tissues, in BCa cell lines we found 4256 hypermethylated regions and 401 hypomethylated regions including promoter,5'UTR, mir, enchancer and exon. The subsequent MSP screening of 162 promoters in clinical urine samples identified eight genes (ECEL1、KCNV1、LMX1A、PROX1A、SLA620、TAL1、TMEM26, and VAX1), which are most qualified to discriminate BCa from normal controls. And all of those genes were first reported to be hypermethylated in Bca. We validated those 8 genes by MSP in urine samples of 212 pre-surgery BCa patients, 21 post-surgery Bca patients, 41 patients with noncancerous urinary lesions, 48 outpatient cases, who were highly suspected for BCa and about to urdergo a cystoscope examination, and 149 healthy controls. A set of 5 gene targets including VAX1, KCNV1, CFTR, PPOX1 and TAL1, was proved to be the best combination of methylation markers for Bca diagnosis, which can achieve a sensitivity of 85.36% and a specificity of 90.0%. In addition, the methylation of VAX1 and LMX1A were found to be valuable in the prediction of tumor recurrence, supported by the analysis of the methylation data of those genes in 157 cases of primary Bca patients and 55 cases of recurrent Bca patients; and the conclusion was also validated by the following up of 145 primary Bca cases (no recurrence: 108; recurrence: 37).

英文关键词： Bladder cancer;urine;DNA methylation;methylome;tumor biomarker