5'-氮杂-2'-脱氧胞苷对结直肠癌细胞株HT-29和LoVo中MLH-1基因甲基化状态、mRNA及蛋白表达的影响  

Effect of 5'-Aza-2'-deoxycytidine on mRNA and protein expression and methylation of MLH-1 gene in HT-29 and LoVo colorectal cancer

作　　者：葛畅[1] 许春伟[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,北京100700

出　　处：《皖南医学院学报》2014年第6期478-482,共5页Journal of Wannan Medical College

基　　金：首都卫生发展科研专项基金项目(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-2'-deoxycytidine)对结直肠癌细胞株HT-29和Lo Vo中MLH-1基因甲基化水平、mRNA及蛋白表达的影响。方法:用不同浓度5'-氮杂-2'-脱氧胞苷处理结直肠癌细胞株HT-29和Lo Vo。应用Taq Man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Lo Vo细胞中MLH-1基因的甲基化状态、mRNA和蛋白表达。结果:Methylight检测HT-29细胞株中MLH-1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、1.5μmol/L 5'-氮杂-2'-脱氧胞苷处理后MLH-1基因mRNA和蛋白表达上调,实时荧光定量PCR检测对照组和不同浓度实验组在HT-29细胞株相对表达量分别为1.000±0.000、1.171±0.126、1.356±0.085和1.422±0.090;在Lo Vo细胞株相对表达量分别为1.000±0.000、1.117±0.094、1.273±0.062和1.285±0.070。Western blot检测MLH-1蛋白对照组和不同浓度实验组在HT-29细胞株相对表达量分别为0.114±0.033、0.365±0.040、0.831±0.041和0.849±0.051;Lo Vo细胞株相对表达量分别为0.602±0.020、0.621±0.028、0.934±0.029和1.057±0.045。以上作用均呈时间、剂量依赖性,MLH-1基因mRNA在HT-29细胞株表达(F=8.918,P=0.010)和Lo Vo细胞株表达(F=8.351,P=0.011)具有统计学意义。MLH-1蛋白表达在HT-29细胞株表达(F=226.825,P=0.000)和Lo Vo细胞株表达(F=153.642,P=0.000)亦具有统计学意义。结论:结直肠癌细胞株HT-29和Lo Vo中MLH-1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'-氮杂-2'-脱氧胞苷能够较成功的逆转结直肠癌细胞株HT-29和Lo Vo中MLH-1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,a methylation inhibitor,on the methylation level,mRNA and protein expression of MLH-1 gene in HT-29 and colorectal cancer cell lines（LoVo）.Methods：HT-29 and LoVo were treated with different dosage of 5′-Aza-2′-deoxycyti-dine.MLH-1gene DNA,mRNA and protein,and determined by Methylight,SYBR Green PCR and Western blot respectively.Results：Methylight detection showed that the MLH-1 gene methylation was reversed by 5′-Aza-2′-deoxycytidine.Moreover,the expression levels of MLH-1 gene mRNA treated with 5′-Aza-2′-deoxycytidine were up-regulated in HT-29 colorectal cell line（1.000 ＆#177;0.000,1.171 ＆#177;0.126,1.356 ＆#177;0.085 and 1.422 ＆#177;0.090,respectively） as well as in LoVo cell lines（1.000 ＆#177;0.000,1.117 ＆#177;0.094,1.273 ＆#177;0.062 and 1.285 ＆#177;0.070,respectively）.Western blot indicated that 5′-Aza-2′-de-oxycytidine could recover the MLH-1 protein expression in HT-29 colorectal cell line（0.114 ＆#177;0.033,0.365 ＆#177;0.040,0.831 ＆#177;0.041 and 0.849 ＆#177;0.051, respectively）,and LoVo cell line（0.602 ＆#177;0.020,0.621 ＆#177;0.028,0.934 ＆#177;0.029 and 1.057 ＆#177;0.045,respectively）.Statistical significance was observed concerning the MLH-1 gene mRNA effects within certain dosage and time-dependent in HT-29 colorectal cancer line（F=8.918,P=0.010） and LoVo（F=8.351,P=0.011）,as well as the MLH-1 protein effects within certain extent dose and time-dependent in HT-29 （F=226.825,P=0.000） and LoVo （F=153.642,P=0.000）.Conclusion：The methylation of promoter region may be primary responsibility for transcriptional inactivation of MLH-1 gene in HT-29 and LoVo colorectal cancer cell lines,yet 5′-Aza-2′-deoxycytidine may effectively reactivate the gene transcription through demethylation .

关 键 词：DNA甲基化 5’-氮杂-2’-脱氧胞苷 HT-29 LOVO MLH-1基因 

分 类 号：R735.3[医药卫生—肿瘤]