5-Aza-dC和TSA对肝细胞癌细胞株中3-OST-2甲基化和基因表达的影响  

Effects of 5-Aza-dC and TSA on methylation and expression of 3-OST-2 gene in human hepatocelluar carcinoma cells

作　　者：姚树哲[1] 陈海燕[1] 张晓莹[1] 盛燕[1] 周庚寅[1] 张翠娟[1] 

机构地区：[1]山东大学医学院病理学教研室,济南250012

出　　处：《临床与实验病理学杂志》2013年第4期356-360,共5页Chinese Journal of Clinical and Experimental Pathology

基　　金：国家自然科学基金面上项目(81272277);国家自然科学基金青年科学基金(30801108)

摘　　要：目的探讨5-Aza-dC和TSA对肝细胞癌(hepatocellular carcinoma,HCC)细胞中3-OST-2甲基化水平、基因表达以及对核转录因子UHRF1(ubiquitin-like with PHD and ring finger domains 1,也称为ICBP90/NP95)表达的影响。方法采用甲基化特异性PCR(methylation specific PCR,MSP)法检测药物作用前后HCC细胞株中3-OST-2甲基化的状态改变;采用实时荧光定量RT-PCR法检测3-OST-2和UHRF1 mRNA的表达变化;采用细胞爬片免疫组化法检测UHRF1蛋白表达。结果 3-OST-2在HCC细胞株中呈完全甲基化状态,5-Aza-dC或TSA均能部分逆转3-OST-2甲基化,其mRNA表达分别增加2.7倍和4.9倍,两种药物联合处理后,3-OST-2甲基化被完全逆转,其mRNA表达增加9.1倍。5-Aza-dC或TSA均能降低UHRF1 mRNA和蛋白的表达,TSA比5-Aza-dC疗效更好(P<0.01),两种药物联用具有协同作用(P<0.01)。结论启动子甲基化和组蛋白修饰共同导致3-OST-2基因表达降低。5-Aza-dC和TSA单独作用均能部分逆转3-OST-2甲基化,增加其mRNA表达,抑制核蛋白UHRF1表达可能是其中机制之一。Purpose To investigate the effects of 5-Aza-dC and TSA on promoter methylation and gene expression of 3-OST-2 as well as the expression of nuclear transcription factor UHRF1. Methods The changes of promoter methylation of 3-OST-2 before and after the treatment of 5-Aza-dC and/or TSA were detected by methylation specific PCR （MSP）. The changes of mRNA and protein expres- sion of 3-OST-2 or UHRF1 were determined by real time RT-PCR and immunoeytoehemistry, respectively. Results 3-OST-2 was completely methylated in hepatocellular carcinoma cells and was partially reversed by 5-Aza-dC or TSA alone, consistently, mRNA was increased by 2. 7-fold and 4. 9-fold, respectively. After the treatment of the inhibitors in combination, 3-OST-2 methylation was com- pletely reversed and mRNA was significantly increased by 9. 1-fold. UHRF1 mRNA and protein expression were both inhibited by 5- Aza-dC or TSA, and TSA was more efficient than 5-Aza-dC （P 〈 0. 01 ）. There was synergistic effect between the two inhibitors（P 〈 0. 01 ）. Conclusions Promoter methylation and histone modifications both cause the decrease of 3-OST-2 expression. Either 5-Aza-dC or TSA can reverse 3-OST-2 methylation and increase its expression, and the inhibition of UHRFl may be one of the mechanisms.

关 键 词：肝细胞肿瘤 5-Aza—dC TSA 甲基化 抑癌基因 

分 类 号：R735.7[医药卫生—肿瘤]