5’-氮杂-2’-脱氧胞苷对HT-29和LoVo结直肠癌细胞株中Runx3基因甲基化状态,mRNA表达及蛋白表达的影响

摘要

Objective To investigate the effects of 5’‐Aza‐2’‐deoxycytidine (5’‐Aza‐CdR) ,a methylation inhibi‐tor ,on the mRNA expression ,protein expression of Runx3 gene in HT‐29 and LoVo Colorectal cancer cell lines . Method HT‐29 and LoVo Colorectal cancer cell lines was treated with different dosages of 5’‐Aza‐CdR .Runx3 gene DNA ,mRNA and protein were determined by Methylight ,SYBR Green PCR and western blot (WB) test respec‐tively .Result Methylight HT‐29 and LoVo cells and abnormal methylation of Runx3 protein in drug action were re‐versed .Real‐time fluorescent quantitative PCR and WB test showed that Runx3 gene mRNA and protein expressed again with 0 .5 ,1 .0 ,1 .5 μm 5’‐Aza‐CdR ,( P<0 .05) ,there were statistical significantly differences .Conclusion The methylation of promoter region is a main cause for transcriptional inactivation of Runx3 gene in HT‐29 and Lo‐Vo Colorectal cancer cell lines .5’‐Aza‐CdR might effectively reactivate the gene transcription through a demethyla‐tion role .%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Runx3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Runx3基因的甲基化状态、mRNA和蛋白表达。结果 TaqMan探针为基础的实时定量PCR法检测HT‐29和LoVo细胞中Runx3蛋白在药物作用后异常甲基化得到逆转；实时荧光定量PCR和Western Blot检测到0．5，1．0，1．5μM 5’‐Aza‐CdR处理后Runx3基因mRNA和蛋白均重新表达，具有统计学意义（P均＜0．05）。结论结直肠癌细胞株 HT‐29和LoVo中Runx3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能够较成功的逆转结直肠癌细胞株HT‐29和LoVo中Runx3基因的甲基化状态，并能恢复mRNA及蛋白重新表达。