5'-氮杂-2'-脱氧胞苷对结直肠癌细胞株HT-29和LoVo中MGMT基因甲基化状态、mRNA表达及蛋白表达的影响  

Effect of 5'-Aza-2'-deoxycytidine on Methylation Status of MGMT Gene,mRNA Expression and Protein Expression in Cell Lines of HT-29 and LoVo Colorectal Cancer

作　　者：许春伟[1] 葛畅[1] 王鲁平[1] 方园[1] 张玉萍[1] 

机构地区：[1]北京军区总医院病理科,北京100700

出　　处：《解放军医药杂志》2014年第6期33-38,共6页Medical & Pharmaceutical Journal of Chinese People’s Liberation Army

基　　金：首都卫生发展科研专项基金资助项目(2011-5021-02)

摘　　要：目的探讨甲基化酶抑制剂5'-氮杂-2'-脱氧胞苷(5'-Aza-CdR)对结直肠癌(colorectal cancer,CRC)细胞株HT-29和LoVo中MGMT基因甲基化水平、mRNA及蛋白表达的影响。方法用0.5、1.0、1.5μmol/L浓度的5'-Aza-CdR处理CRC细胞株HT-29和LoVo。应用MethyLight方法、实时荧光定量PCR方法及蛋白印迹试验(Westernblot)检测药物处理前后HT-29和LoVo细胞中MGMT基因的甲基化状态、mRNA和蛋白表达情况。结果 MethyLight检测HT-29和LoVo细胞中MGMT蛋白在药物作用后异常甲基化得到逆转。实时荧光定量PCR检测5'-Aza-CdR浓度为0.5、1.0、1.5μmol/L组HT-29细胞株和LoVo细胞株MGMT基因mRNA表达水平均较对照组上调,Western blot检测5'-Aza-CdR浓度为0.5、1.0、1.5μmol/L组MGMT蛋白表达水平均较对照组上调,且均具有药物剂量依赖性(P<0.05,P<0.01)。结论 CRC细胞株HT-29和LoVo中MGMT基因启动子甲基化可能是导致该基因表达下调甚至失活的主要原因。5'-Aza-CdR能够逆转CRC细胞株HT-29和LoVo中MGMT基因的甲基化状态,并能恢复mRNA及蛋白重新表达。Objective To investigate the effects of 5＇-Aza-2＇-deoxycytidine（ 5＇-Aza-CdR） of a methylation inhibitor on methylation status of MGMT gene,the mRNA expression and protein expression in cell lines of HT-29 and LoVo colorectal cancer（ CRC）. Methods Cell lines of HT-29 and LoVo CRC were treated with 0. 5,1. 0 and 1. 5 μmol /L concentrations of 5＇-Aza-CdR respectively. Values of methylation status of MGMT gene,the mRNA expression and protein expression in cell lines of HT-29 and LoVo colorectal cancer were determined by methods of MethyLight,SYBR Green PCR and Western blot respectively. Results MethyLight detection showed that the abnormal methylation of protein expression in cell lines of HT-29 and LoVo colorectal cancer deteriorated after medication. The levels of mRNA expression in cell lines of HT-29 and LoVo colorectal cancer at 0. 5,1. 0 and 1. 5 μmol /L groups of 5＇-Aza-CdR were increased compared with those in control group by SYBR Green PCR method; the levels of protein expression at 0. 5,1. 0and 1. 5 μmol /L concentrations of 5＇-Aza-CdR were also increased compared with those in control group by Western blot method,and medicamentous dose dependence was also found（ P 〈 0. 05,P 〈 0. 01）. Conclusion The methylation of promoter region may be a main cause of expression regulation and inactivation of MGMT gene in cell lines of HT-29 and LoVo CRC. 5＇-Aza-CdR may effectively reactivate the gene transcription through a demethylation role and recover the mRNA expression and protein expression.

关 键 词：结直肠肿瘤 DNA甲基化 5’-氮杂-2’-脱氧胞苷 MGMT基因 

分 类 号：R735.34[医药卫生—肿瘤]