胰腺癌细胞系BxPC3及胰腺癌组织Ras相关区域家族1A启动子CpG岛的甲基化状态

Aberrant methylation of Ras association domain family 1A promoter CpG island in pancreatic cancer cell line BxPC3 and tissues

目的 检测Ras相关区域家族1A（Ras association domain family 1A,RASSF1A）在胰腺癌细胞株BxPC3及胰腺癌组织中的甲基化状态,探讨其启动子异常甲基化在胰腺癌发病机制中的可能作用.方法 采用结合重亚硫酸盐的限制性内切酶法（combined bisulfite restriction analysis,COBRA）检测胰腺癌细胞株BxPC3、5例正常胰腺组织、13对胰腺癌及相应癌旁正常胰腺组织中RASSF1A启动子CpG岛的甲基化状态,计算其甲基化率.以甲基化酶抑制剂5-Aza-dC（5-Aza-2-deoxycitydine）处理BxPC3,观察处理前后甲基化率变化情况及RASSF1A mRNA表达变化.结果 在BxPC3细胞株中,RASSF1A启动子的CpG岛甲基化率为62.90%;正常胰腺、癌旁及癌组织中平均分别为9.14%、53.79%和55.82%.与正常胰腺组织相比,胰腺癌旁及癌组织的RASSF1A启动子甲基化率明显增高（P值〈0.01）,而癌旁及癌组织之间无明显差异（P〉0.05）.BxPC3经5-Aza-dC处理后,RASSF1A的CpG岛甲基化率显著下降至42.50%（P〈0.05）,同时RASSF1A mRNA表达增强.结论 RASSF1A启动子CpG岛异常甲基化是胰腺癌发生发展中的早期事件,可能参与胰腺癌的发病过程.

Objective To determine the methylation status and expression of Ras association domain family 1A （RASSF1A）, and the possible effect between promoter aberrant methylation and pathogenesis of pancreatic cancer. Methods The methylation status of RASSF1A promoter CpG island （CGI） pancreatic cancer cell line BxPC3 was detected in 5 cases of normal pancreatic tissue and 13 pairs of pancreatic cancer tissues （tumor and peri-tumor） by using COBRA （combined bisulfite restriction analysis） and the methylation rate was calculated. The RASSF1A mRNA expression of BxPC3 was compared between pre- and post-treatment of the inhibitor of DNA methyltransferase （5-Aza-2-deoxycitydine, 5-Aza-dC）. Results The average methylation rate of RASSF1A promoter CGI was 62.90% in BXPC3, 9.14% in normal pancreas, 53.79% in peri-tumors （TP）, and 55.82% in tumors. The methylation rates in port-tumors and tumors were significantly increased when compared with that of normal pancreas （P 〈 0.01）, while there was no significant difference between in peri-tumors and tumors （P 〉 0.05）. After 5-Aza-dC treatting BxPC3 cells, the methylation rates decreased to 42.5% （P 〈 0. 05） and RASSF1A mRNA expression was enhanced. Conclusions Aberrant hypermethylation of RASSF1A promoter CGI could be considered as an early event in the process of pancreatic cancer and participates in the pathogenesis of pancreatic cancer.