EEN-B1及其启动子甲基化在外阴鳞状细胞癌中的表达及意义

目的：检测EEN—B1在外阴鳞癌组织及外阴鳞癌细胞株中的启动子甲基化及蛋白表达情况，探讨其启动子异常甲基化在外阴鳞癌发病过程中的作用。方法：采用重亚硫酸盐测序PCR（bisulfite genomic se—quencing PCR，BSP）联合TA克隆测序法，以正常外阴组织作对照，检测外阴鳞癌组织中EEN—B1启动子区CpG岛的甲基化状态，以甲基化酶抑制剂5-aza-2-deoxycitydine（5-aza—dC）作用于外阴鳞癌细胞A-431，检测作用前后甲基化率变化情况；Westernblot方法检测外阴鳞癌及对照组织中EEN—B1蛋白表达情况，检测5-aza—dC处理前后A-431中该蛋白表达情况。结果：在癌组织中EEN—B1启动子的甲基化率为54．66％，与对照组织的12．16％相比，差异具有统计学意义；而在外阴鳞癌组织中EEN—B1蛋白明显下调；经1×10 -7mol／L5-aza—dC处理72小时后，A-431细胞系中EEN—B1启动子的甲基化率由67．05％下降至26．82％（P〈0．05），而该蛋白表达明显上调。结论：外阴鳞癌细胞株A-431及癌组织中EEN—B1基因表达与启动子区甲基化状态有关，启动子区的高甲基化可能导致了外阴鳞癌组织及细胞系中EEN—B1基因的表达沉默。

Methylation of promoter in EEN -B1 gene and its impact on expression in vulvar squa- mous cell carcinomas

Objective：To explore the methylation status and expression of EEN B1, and the possible effect be tween promoter aberrant methylation and the pathogenesis of vulvar squamous cell carcinomas cancer. Methods：We detected the methylation status of EEN B1 promoter CpG island（CGI） in vulvar cancer cell line A 431, normal vulvar tissues and vulvar squamous cell carcinomas tissues by using bisulfite genomic sequencing PCR（BSP） com bined with TA clone for sequencing. The methylation rate of EEN B1 was compared before and after treatment of the inhibitor of DNA methyltransferase （ 5 aza 2 deoxycitydine, 5 aza dC ） , and Western blot were used to explore protein expression of the EEN B1 protein. Results：The average methylation rate of EEN B1 promoter CGI was 54.66% in vulvar squamous cell carcinomas tissues, 12.16% in normal vulvar tissues. The methylation rate of vulvar squamous cell carcinomas tissues was higher than that of normal vulvar tissues （P 〈 0.05）. After the treat ment of 5 aza dC（ 1 x 10 7 mol/L） ,72h,the methylation rate in A 431 was decreased from 67.05% to 26.82% （P 〈 0.05）, but the protein expression of EEN B1 increased. Concluslon：This promoter hypemlethylation is corre lated with EEN B1 gene expression in vulvar cancer cell line A 431 and vulvar cancer tissues, and plays a key role in EEN B1 silencing. Aberrant hypermethylation of EEN B1 could probably become early diagnosis index and treatment target of vulvar squamous cell cancer.