低氧调控p53RFP基因启动子活性的初步研究  

Preliminary study of regulative function of hypoxia to the p53RFP gene promoter

作　　者：裴静娴[1] 莫沛[1] 王月刚[2] PEI Jingxian;MO Pei;WANG Yuegang(Department of Cardiology,the Second Affiliated Hospital of Guangzhou Medical University,Guangzhou 510260,China;Department of Cardiology,Nanfang Hosiptal,Southern Medical University,Guangzhou 510515,China)

机构地区：[1]广州医科大学附属第二医院心血管内科,广州510260 [2]南方医科大学南方医院心血管内科,广州510515

出　　处：《广州医药》2023年第1期25-29,共5页Guangzhou Medical Journal

基　　金：国家自然科学基金青年基金资助项目(30800462)。

摘　　要：目的观察低氧对p53RFP基因表达及启动子活性的影响,探索低氧反应元件(HRE)是否参与了低氧对p53RFP基因启动子活性的调控。方法将HEK293细胞置于低氧条件下培养,实时定量PCR及Western blot检测p53RFP mRNA及蛋白表达水平;通过定点突变技术构建HRE突变型p53RFP启动子双荧光素酶报告基因载体并转染HEK293细胞,分别置于常氧和低氧条件下培养,双荧光素酶报告基因检测系统检测启动子活性。结果低氧处理不同时间点p53RFP mRNA表达水平均显著增加,差异有统计学意义(F=96.493,P<0.001);低氧组p53RFP及HIF-1α蛋白表达量均呈时间依赖性递增。成功构建了HRE突变型p53RFP启动子双荧光素酶报告基因载体,双荧光素酶报告基因检测显示,与常氧组相比,低氧条件下野生型p53RFP基因启动子活性增加(t=-19.504,P<0.001),HRE单突变或双突变后启动子活性均较野生型降低(F=160.891,P<0.001)。结论低氧可诱导p53RFP基因表达上调;成功构建了HRE突变型p53RFP双荧光素酶报告基因载体,HRE位点可能在低氧调控p53RFP基因启动子活性中发挥关键作用。Objective To investigate the effect of hypoxia on p53RFP gene expression and p53RFP promoter activity,and explore whether hypoxia responsive element(HRE)is involved in the regulation of p53RFP gene promoter activity by hypoxia.Methods HEK293 cells were cultured under normoxia and hypoxia conditions,p53RFP expressions at mRNA and protein levels were detected by real-time PCR and Western blot respectively.The HRE mutant p53RFP promoter dual luciferase reporter gene vector was constructed by site-directed mutagenesis technology and transfected into HEK293 cells under normoxia and hypoxia conditions.The promoter activities were detected by dual luciferase reporter system.Results Compared with the normoxia group,the expression of p53RFP mRNA in the hypoxia group increased significantly at different time points,and the difference was statistically significant(F=96.493,P<0.001);the expression of p53RFP and HIF-1αprotein in the hypoxia group increased in a time-dependent manner.The luciferase reporter vectors containing p53RFP with mutant HRE were successfully constructed.The dual luciferase reporter gene assay showed that the activity of the wild-type p53RFP gene promoter was significantly increased under hypoxic conditions compared to normoxic condition(t=-19.504,P<0.001),and the promoter activity of p53RFP with HRE single mutation or double mutation were both significantly lower than that of wild type under hypoxic condition(F=160.891,P<0.001).Conclusions p53RFP gene expression was induced by hypoxia;the p53RFP promoter with mutant HRE dual luciferase reporter gene vectors were successfully constructed,and the HRE locus may play a key role in the hypoxia regulation of p53RFP gene promoter activity.

关 键 词：低氧 p53RFP 启动子 低氧反应元件 

分 类 号：R346[医药卫生—基础医学]