缺氧/辐射双敏感性启动子的构建及其转录调控特性

A hypoxia/radiation dual-sensitive promoter and its transcriptional activity

目的 :构建缺氧 /辐射双敏感性启动子 ,阐明其转录调控特性 .方法 :通过基因重组将缺氧反应元件 (HREs)序列插入早期生长反应基因 1 (Egr 1 )启动子的上游 ,构成缺氧 /辐射双敏感性HRE Egr启动子及其报告载体pHEL ,转染肺癌A5 4 9细胞 .在 2 ,4 ,6 ,8,1 0Gy照射 (γ 射线 )后及合并缺氧(氧浓度 1 ,5 ,1 0 ,2 5 ,5 0mL/L)时 ,检测转染细胞内报告基因荧光素酶 (luciferase ,Luc)表达活性 ,以及在 6Gy照射合并氧浓度 1 0mL/L时Luc表达的动态变化 .结果 :成功构建了HRE Egr启动子及其报告载体 ,转染A5 4 9细胞 ,发现常氧下HRE Egr启动子照射后表达活性迅速升高 ,呈辐射剂量依赖性 ,与Egr 1启动子相一致 .缺氧时 ,Egr 1启动子辐射诱导活性明显下降 (P <0 .0 5 ) ,而HRE Egr启动子活性则显著升高(P <0 .0 1 ) ,同时后者活性持续时间 (32h)明显长于前者 (1 6h) .结论 :HRE Egr启动子具有辐射 /缺氧双重敏感性 。

AIM: To construct a hypoxia/radiation dual sensitive promoter and to elucidate its transcriptional characteristics. METHODS: An chimeric promoter HRE Egr was generated by inserting the hypoxia response elements (HREs) from human vascular endothelium growth factor 5′ flanking sequence into the upstream of human early growth response gene 1 (Egr 1) promoter, which was transfected into human lung adenocarcinoma A549 cells by liposome. The expression of luciferase in transfected cells, exposed to 2, 4, 6, 8, 10 Gy irradiation combined with or without hypoxia (1, 5, 10, 25,5 0 mL/L oxygen concentrations), was detected. The dynamic changes of luciferase expression in transfected cells were observed following 6 Gy irradiation and 10 mL/L oxygen concentration. RESULTS: A hypoxia/radiation dual sensitive promoter HRE Egr and its reporter vector were successfully constructed in the present study. HRE Egr promoter transcriptional activity in the transfected cells demonstrated significant increment following irradiation, which displayed obvious dose dependent alteration. The luciferase expression in HRE Egr transfected cells exposed to radiations combined with oxygen concentration below 2.5% showed markedly enhancement compared with that of Egr 1 promoter transfected cells, which demonstrated remarkable decrease under the same conditions as above. The prolongation in radio inducible activity of HRE Egr promoter was seen in transfected cells exposed to hypoxia. CONCLUSION: The HRE Egr promoter generated in the present study possesses hypoxia/radiation dual sensitive identity, the transcriptional activity of which can be enhanced by hypoxia.