Ribeiro-Filho Leopoldo Alves, Franks Joseph, Sasaki Masahiro, Shiina Hiroaki, Li Long-Cheng, Nojima Dana, Arap Sami, Carroll Peter, Enokida Hideki, Nakagawa Masayuki, Yonezawa Suguru, Dahiya Rajvir
Department of Urology, San Francisco Veterans Affairs Medical Center and University of California, 94121, USA.
Mol Carcinog. 2002 Aug;34(4):187-98. doi: 10.1002/mc.10064.
Several studies have shown that E-cadherin expression is lost during malignant transformation. We hypothesized that CpG methylation in the promoter region may inactivate the expression of the E-cadherin gene in human bladder cancer. Normal and bladder cancer samples from 51 patients were compared in terms of E-cadherin gene expression and methylation status by immunohistochemistry, methylation-specific polymerase chain reaction (MSP), and bisulfite genome-sequencing techniques. Ten different CpG sites (nt 863, 865, 873, 879, 887, 892, 901, 918, 920, and 940) in the promoter region were studied. Thirty-five of 51 (69%) bladder cancer samples lacked E-cadherin expression, whereas only six of 51 (12%) normal bladder samples lacked E-cadherin immunoreactivity. MSP analysis of bladder cancer samples suggested that 43 of 51 (84%) showed methylation of the promoter region, whereas only 12 of 51 (24%) normal bladder samples showed hypermethylation. Sodium bisulfite genome-sequencing analysis revealed that of 10 CpG sites, two sites (nt 892 and nt 940) showed 100% methylation in all the cancer samples analyzed. Other CpG sites were partially methylated (47-91%). Normal tissue showed only 12% methylation (range, 1-33%) on various CpG sites. Also supporting these data, E-cadherin-negative bladder cancer cell lines restored expression of the E-cadherin gene after treatment with the demethylating agent 5-aza-2'-deoxycytidine. The present study showed that CpG hypermethylation was an important mechanism of E-cadherin gene inactivation in bladder cancer and also that specific CpG sites consistently presented higher methylation levels than others. These findings may provide a better strategy for the diagnosis and management of bladder cancer.
多项研究表明，在恶性转化过程中E-钙黏蛋白表达缺失。我们推测，启动子区域的CpG甲基化可能使人类膀胱癌中E-钙黏蛋白基因的表达失活。通过免疫组织化学、甲基化特异性聚合酶链反应（MSP）和亚硫酸氢盐基因组测序技术，对51例患者的正常和膀胱癌样本进行了E-钙黏蛋白基因表达和甲基化状态的比较。研究了启动子区域的10个不同的CpG位点（核苷酸863、865、873、879、887、892、901、918、920和940）。51例膀胱癌样本中有35例（69%）缺乏E-钙黏蛋白表达，而51例正常膀胱样本中只有6例（12%）缺乏E-钙黏蛋白免疫反应性。膀胱癌样本的MSP分析表明，51例中有43例（84%）显示启动子区域甲基化，而51例正常膀胱样本中只有12例（24%）显示高甲基化。亚硫酸氢盐基因组测序分析显示，在分析的所有癌症样本中，10个CpG位点中的两个位点（核苷酸892和核苷酸940）显示100%甲基化。其他CpG位点部分甲基化（47-91%）。正常组织在各个CpG位点仅显示12%的甲基化（范围为1-33%）。用去甲基化剂5-氮杂-2'-脱氧胞苷处理后，E-钙黏蛋白阴性的膀胱癌细胞系恢复了E-钙黏蛋白基因的表达，这也支持了这些数据。本研究表明，CpG高甲基化是膀胱癌中E-钙黏蛋白基因失活的重要机制，并且特定CpG位点的甲基化水平始终高于其他位点。这些发现可能为膀胱癌的诊断和治疗提供更好的策略。
