NF-κB结合元件缺失对NOX1启动子转录活性的影响

Effect of NF-κB binding element deletion on transcriptional regulation of NOX1

目的:构建含NADPH氧化酶1(NOX1)近端启动子区的pGL3萤光素酶报告基因载体及缺失NF-κB结合元件的相应载体,分别测定其相应活性,探讨NF-κB结合元件缺失对NOX1启动子区转录活性的影响。方法:采用PCR法克隆NOX1启动子区序列(1 415 bp),将目的片段与pGL3萤光素酶载体分别双酶切、纯化后进行连接(pGL3-NOX1-1415),测序鉴定;应用Alibaba 2.1软件分析NOX1近端启动子区,获取NF-κB结合元件;重叠PCR法将含该元件的启动子区域(88 bp)缺失,并构建相应载体(pGL3-NOX1-1327)。将两载体分别与pRL-TK内参质粒瞬时转染进入A549细胞,采用TNF-α(10μg/L)刺激细胞24 h,萤光酶标仪检测A549细胞的萤光素酶活性。结果:测序鉴定结果提示pGL3-NOX1-1415及NF-κB结合元件缺失的pGL3-NOX1-1327载体构建成功;细胞实验显示,TNF-α刺激后,转染pGL3-NOX1-1415的A549细胞萤光素酶活性明显强于对照组(P<0.05),而转染NF-κB结合元件缺失的pGL3-NOX1-1327的细胞萤光素酶活性明显低于转染pGL3-NOX1-1415组(P<0.05)。结论:TNF-α诱导A549细胞NOX1基因活化与NF-κB密切相关,NF-κB参与了TNF-α诱导的NOX1基因启动子的转录调控。

AIM： To investigate the effect of NF-κB binding element deletion on transcriptional regulation of（ NOX1）. METHODS： pGL3-Basic vector inserted with the NOX1 proximal promoter,and the same vector inserted with the NOX1 proximal promoter in the absence of the positive NF-κB binding element,were constructed. After cloning,digestion and purification,NOX1 proximal promoter（ ≈1 415 bp） was inserted into the multicloning site of the pGL3-Basic vector and then sequenced（ pGL3-NOX1-1415）. NF-κB binding elements in the NOX1 promoters were predicted by Alibaba2. 1 software. The positive element was deleted by overlapping PCR. The deletion mutant was inserted into the pGL3 vector in the same way（ pGL3-NOX1-1327）. The plasmids were transfected into A549 cells,and then the cells were stimulated with TNF-α. The luciferase activity was monitored on MD SpectraMax M5 enzyme-labeled instrument. RESULTS： The sequences of pGL3-NOX1-1415 and pGL3-NOX1-1327 were identified to be correct. Compared with control group,the luciferase activity was significantly higher in the cells transfected with pGL3-NOX1-1327（ P〈0.05）,but it was significantly lower than that in the cells transfected with pGL3-NOX1-1415（ P 〈0.05）. CONCLUSION： NF-κB plays an essential role in the transcriptional regulation of NOX1 in TNF-α-induced A549 cells. Activated NF-κB binds to specific elements in NOX1 promoter regions to control the transcription.