5'-Aza-CdR对HT-29和LoVo结直肠癌细胞株中CDX2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, mRNA and protein expression of CDX2 gene in colorectal cancer HT-29 and LoVo cells

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]中国人民解放军北京军区总医院病理科,北京市100700

出　　处：《世界华人消化杂志》2014年第10期1423-1430,共8页World Chinese Journal of Digestology

基　　金：首都卫生发展科研专项基金资助项目;No.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-2'-deoxycytidine,5'-Aza-CdR)对结直肠癌细胞株HT-29和LoVo中CDX2基因甲基化水平、mRNA及蛋白表达的影响.方法:用不同浓度5'-Aza-CdR处理结直肠癌细胞株HT-29和LoVo.应用TaqMan探针为基础的实时定量PCR(Methylight)方法、SYBR Green PCR方法及蛋白印迹实验(Western blot)检测药物处理前后HT-29和LoVo细胞中CDX2基因的甲基化状态、mRNA和蛋白表达.结果:Methylight检测HT-29和LoVo细胞中C D X2蛋白在药物作用异常甲基化未得到逆转;实时荧光定量PCR和Western blot检测到0.5、1.0、1.5μmol/L 5'-Aza-CdR处理后C D X2基因m R N A和蛋白重新表达,实时荧光定量PCR检测对照组和不同浓度实验组在HT-29细胞株mRNA相对表达量分别为1.000±0.000、0.973±0.024、1.014±0.019和1.094±0.020;在LoVo细胞株相对表达量分别为1.000±0.000、0.966±0.038、1.050±0.029和1.007±0.019.Western blot检测CDX2蛋白在对照组和不同浓度实验组中HT-29细胞株相对表达量分别为0.454±0.049、0.501±0.041、0.340±0.050和0.531±0.046;LoVo细胞株相对表达量分别为0.527±0.037、0.415±0.037、0.432±0.040和0.626±0.046.以上作用无时间、剂量依赖性,但CDX2基因mRNA在HT-29细胞株表达(F=25.146,P=0.000)和LoVo细胞株表达(F=5.470,P=0.024)具有统计学差异,C D X2蛋白表达在HT-29细胞株表达(F=9.700,P=0.005)和LoVo细胞株表达(F=17.701,P=0.001)亦具有统计学差异.结论:结直肠癌细胞株HT-29和LoVo中CDX2基因mRNA和蛋白表达不受中DNA甲基化的表观遗传修饰的调控.AIM： To investigate the effect of 5＇-Aza-2＇-deoxycytidine （5＇-Aza-CdR）, a methylation inhibitor, on the methylation, mRNA and protein expression of the CDX2 gene in colorectal ancer cell lines HT-29 and LoVo. METHODS： HT-29 and LoVo cells were treated with different dosages of 5＇-Aza-CdR. After treatment, CDX2 gene methylation was determined by Methylight assay, and CDX2 mRNA and protein expression was detected by real- time PCR and Western blot, respectively.RESULTS： Methylight assay showed that CDX2 gene methylation was not reversed by 5＇-Aza- CdR. The expre＇ssion levels of CDX2 mRNA were increased in both HT-29 （1.000 ± 0.000, 0.973 ± 0.024, 1.014 ± 0.019 and 1.094 ± 0.020, respectively） and LoVo cells （1.000 ± 0.000, 0.966 ± 0.038, 1.050 ± 0.029 and 1.007 ± 0.019, respectively） cells treated with 5＇-Aza-CdR for different durations. Western blot analysis indicated that 5＇-Aza-CdR treatment could recover the CDX2 protein expression in both HT-29 （0.454 ± 0.049, 0.501 ± 0.041, 0.340 ± 0.050 and 0.531 ± 0.046, respectively） and LoVo （0.527 ± 0.037, 0.415 ± 0.037, 0.432 ± 0.040 and 0.626 ± 0.046, respectively） cells. The effect of 5＇-Aza-CdR on CDX2 mRNA and protein expression was not dose- or time-dependent, but the expression levels of CDX2 mRNA and protein differed significantly in HT-29 （mRNA： F = 25.146, P = 0.000; protein： F = 9.700, P = 0.005） and LoVo cells （mRNA： F = 5.470, P = 0.024; protein： F = 17.701, P = 0.001）. CONCLUSION： CDX2 mRNA and protein expression is not affected by DNA methylation in HT-29 and LoVo cells.

关 键 词：DNA甲基化 5’-氮杂-2’-脱氧胞苷 HT-29 LOVO CDX2基因 

分 类 号：R735.3[医药卫生—肿瘤]