人胚肾293细胞中乙酰转移酶p300对二甲基精氨酸二甲胺水解酶2基因表达的调节  

DDAH2 Gene Expression Regulation by Acetyltransferase p300 in Human Embryonic Kidney 293 Cells

作　　者：李英慧[1] 李家亓[1] 孙陆[1] 初国铭[1] 孙志军[2] 

机构地区：[1]中国医科大学基础医学院医学遗传学教研室,辽宁省沈阳市110001 [2]中国医科大学附属盛京医院心内科

出　　处：《中国循环杂志》2013年第1期59-62,共4页Chinese Circulation Journal

基　　金：国家自然科学基金(30900807)

摘　　要：目的:一氧化氮(NO)在高血压发生中具有重要作用,二甲基精氨酸二甲胺水解酶2(DDAH2)可调节NO生物合成。本研究以乙酰转移酶p300为乙酰化作用因素,鉴定人胚肾293(human embryonic kidney 293,HEK293)细胞中乙酰化是否参与调节DDAH2基因的表达。方法:以人胚肾HEK293细胞为研究对象,转染野生型及乙酰转移酶(HAT)结构域缺失的突变型p300表达载体,应用Western blot方法鉴定载体表达情况;real-time PCR方法鉴定野生型及突变型p300对DDAH2 mRNA表达水平的影响;软件分析DDAH2启动子结构;构建DDAH2启动子荧光素酶报告基因载体;双荧光素酶活性检测系统检测野生型及突变型p300对DDAH2启动子活性的影响。结果:Western blot结果显示转染两种p300表达载体的HEK293细胞中p300蛋白表达水平显著升高(P<0.05);实时定量聚合酶链式反应(real-time PCR)结果证明野生型p300可显著升高DDAH2 mRNA表达(P<0.05),而HAT结构域缺失的突变型p300无此作用(P>0.05);软件分析结果显示DDAH2启动子区存在多种潜在的受乙酰化调控的转录因子的结合位点;构建了两个DDAH2启动子荧光素酶报告基因载体,分别命名为pGL530w和pGL171w;双荧光素酶活性检测结果证明基础条件下,pGL530w和pGL171w具有较高的活性(P<0.05),野生型p300可显著升高二者活性,但突变型p300对二者无明显作用(P>0.05)。结论:人胚肾HEK293细胞中p300介导的乙酰化可通过调节DDAH2基因启动子活性,从而参与调节DDAH2基因表达。这一机制可能在肾脏组织NO生物合成中发挥重要作用,从而在高血压发生中发挥一定作用。Objective：Nitric oxide （NO）is important in hypertension development, and dimethylarginine dimethylaminohydrolase 2 （DDAH2） may regulate NO biosynthesis. We want to identify DDAH2 gene expression regulation by acetyltransferase（HAT） p300 in human embryonic kidney 293 （ HEK293 ） cells. Methods ：Wild-type and HAT domain-deleted mutant p300 expression vectors were respectively transfected into HEK293 cells,and the protein expression was assessed by Western blot analysis. The Effects of both wild-type and mutant p300 on DDAH2 gene expression were detected by real-time PCR. DDAH2 promoter structure was analyzed by related softwares. DDAH2 promoter lueiferase reporter vec- tors were constructed and the wild-type and mutant p300 on promoter activities were examined by luciferase assay. Results ： Western blot analysis showed that p300 protein expression in HEK293 cells was significantly increased by both wild- type and mutant p300 vectors transfection. Real-time PCR indicated that wild-type p300 may significantly enhance DDAH2 gene expression, while mutant p300 could not. Software analysis presented that there were many potential binding sites of transcriptionfactors subject to acetylation in DDAH2 promoter, and two DDAH2 promoter luciferase reporter vectors were constructed as pGL530w and pGL171w. Luciferase assay demonstrated that at basal condition,both pGI_530w and pGLlTlw showed high tran- scription activity, and wild-type p300 may greatly increase their activity,but mutant p300 could not. Conclusion：p300-induced acetylation may regulate DDAH2 promoter activity in HEK293 cells and therefore, particpating DDAH2 gene expression regulation. This mechanism may play an important role in NO biosynthesis for hypertension development.

关 键 词：二甲基精氨酸二甲胺水解酶2 一氧化氮 乙酰化 P300 高血压 

分 类 号：R54[医药卫生—心血管疾病]