摘要
目的研究细胞周期分裂蛋白42(cdc42)的启动子区CpG岛是否作为调节因子参与基因表达的调控。方法选择消化道肿瘤早期相关基因cdc42为研究对象,构建氯霉素乙酰基转移酶3-增强子(pCAT3-Enhancer)重组体:内含cdc42基因启动子区第一个CpG岛的序列及一段不含CpG岛序列,非甲基化修饰的重组体和pCAT3-Enhancer空载体转化至甲基化酶缺陷大肠杆菌中(ER1793),经甲基化酶修饰后的重组体及空载体转化至可持续甲基化状态的大肠杆菌中(JM109),阳性克隆转染至Eca109中,提取蛋白,通过薄层层析检测氯霉素乙酰基转移酶(CAT)活性。结果转染至细胞株后,甲基化空载体CAT的活性显著低于非甲基化的;任意DNA-pCAT3-Enhancer重组体甲基化及非甲基化CAT的活性无差异;cdc42启动子区CpG岛-pCAT3-Enhancer重组体,甲基化CAT的活性显著低于非甲基化CAT的活性,差异有统计学意义(P<0.05)。结论通过食管癌细胞株Eca109实验,cdc42启动子区CpG岛的甲基化修饰后,CAT的活性降低,非甲基化CAT仍保持较高活性。说明cdc42启动子区CpG岛甲基化程度的改变具有调节下游基因表达的作用。
Objective To study whether the CpG island in the promoter of cdc42 can be a regulator participated in the regulation of gene expression. The digestive tract cancer early related gene cdc42 choosed as object,explored the change of their promoter CpG island methylation affect on the downstream report gene expression in esophageal cancer(EC) cell line Eca109,then got the relation of methylation and gene expression. Methods DNA cloning and thin layer chromatography(TLC) was used to detect the activity of chloramphenicol acetyl transferase(CAT). Used PCR to amplify cdc42 gene promoter CpG island,and a sequence has not CpG islands,divided two sequences into two groups,one group was methylated,another group kept the original state. Constructed the recombinants one contained the methylated CpG island-pCAT3-Enhancer,another contained the unmethylated CpG island-pCAT3-Enhancer,transfected into the Eca109,detected the activity of CAT. Results After transfecting into ECa cell line,the activity of methylated empty vector CAT was significantly lower than that of unmethylated vector,and there was no difference between methylated and unmethylated random DNA-pCAT3-Enhancer recombinant CAT. The CAT activity of recombinant which has methylated cdc42 promoter region CpG island-pCAT3-Enhancer was significantly lower than unmethylated. The difference was statistically significant( P <0.05). Conclusion In EC cell line Eca109,methylated CpG island of cdc 42 reduces the CAT expression in the downstream,however,the unmethylated can maintain the high activity of CAT. This indicates that the change in the degree of methylation of CpG island in the promoter region of cdc42 has the effect of regulate the expression of downstream genes.
作者
刘玲
陈艳
李卉
张法煌
李依珂
伊丽达娜.斯提瓦尔地
喻亮
李惠武
Liu Ling;Chen Yan;Li Hui(Basic Medical College,Xinjiang Medical University,Urumqi 830011;Central Laboratory,Xinjiang Medical University,Urumqi 830011)
出处
《安徽医科大学学报》
CAS
北大核心
2019年第5期691-695,共5页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:81460419
81060172)