Egr-1对APP基因表达的调控作用  

Regulatory Effects of Egr-1 on APP Gene Expression

作　　者：马琳 周瑜[2] 李永玲[2] 柴娟 贾中发 郭克[2] 孙涛 崔建奇 

机构地区：[1]宁夏颅脑疾病重点实验室,省部共建国家重点实验室培育基地,银川750004 [2]宁夏医科大学基础医学院,银川750004

出　　处：《中国细胞生物学学报》2015年第1期39-46,共8页Chinese Journal of Cell Biology

基　　金：国家重点基础研究发展计划(973计划)(批准号:2012CB722408);国家自然科学基金(批准号:81260197)资助的课题~~

摘　　要：该研究构建了含有APP启动子的荧光素酶报告质粒以及缺失突变的报告质粒,将该质粒与Egr-1真核表达载体p CDNA3-Egr-1共转染到HEK293与U87MG细胞中进行荧光素酶活性测定,以确定Egr-1对APP基因表达的调控作用。通过染色质免疫共沉淀实验(chromatin immunoprecipitation assay,Ch IP)和凝胶电泳迁移实验(electrophoretic mobility shift assay,EMSA)确定Egr-1蛋白在APP启动子上的结合部位,同时利用组蛋白去乙酰化酶抑制剂TSA(trichostatin A)和butyrate诱导内源性Egr-1的表达以及Egr-1抑制剂suramin来作用于细胞,通过蛋白免疫印迹分析(Western blot)检测Egr-1对APP蛋白表达的作用。结果显示,Egr-1蛋白在APP基因启动子上5′UTR区域有特异性的结合部位,去除该结合部位则Egr-1失去对APP基因启动子的调控作用,Ch IP和EMSA的结果也证实了该结合位点;通过HDAC抑制剂和suramin干扰内源性Egr-1的表达则显著影响了Egr-1对APP基因表达的调控作用。The luciferase reporter plasmids which contain APP gene promoter and mutant APP gene promoter were constructed. The Egr-1 eukaryotic expression vector pCDNA3-Egr-1 and the constructed reporter plasmids were co-transfected into HEK293 and U87MG cells to analyze the effects of Egr- 1 on APP promoter activities. The chromatin immunoprecipitation assay （CHIP） and electrophoretic mobility shift assay （EMSA） were performed to determine the binding sites of Egr-1 on 5＇UTR in APP promoter. Cells were treated with histone deacetylase （HDAC） inhibitor trichostatin A （TSA） and butyrate as well as Egr-1 inhibitor suramin and Western blot was performed to evaluate the changes of endogenous Egr-1 expression and the effects of Egr-1 on APP promoter activities were verified. The results of luciferase assay demonstrated that Egr-1 could up-regulate APP gene expression, and Egr-1 could bind to the specific binding sites in the 5＇UTR of APP promoter. The up-regulatory effects of Egr-1 on APP promoter would be eliminated when these binding sites were deleted. ChIP and EMSA results confirmed that Egr-1 could bind to these sites. In addition, the effects of Egr- 1 on APP promoter activities were dramatically influenced if the endogenous expression of Egr- 1 was disturbed by HDAC inhibitor and suramin.

关 键 词：EGR-1 淀粉样前体蛋白 阿尔茨海默病 基因表达调控 

分 类 号：R749.16[医药卫生—神经病学与精神病学]