成视网膜细胞瘤结合蛋白4对Sp1介导的HIV长末端重复序列转录作用研究  

Effect of retinoblastoma binding protein 4 （RBBP4）on Sp1-mediated transcription of HIV long terminal repeat in 293 T cells

作　　者：王娟[1] 杨劲[2] 杨宗兴 程林芳[4] 吴南屏[4] Wang Juan Yang Jin Yang Zongxing Cheng Linfang Wu Nanping(Department of Clinical Laboratory, Tongde Hospital of Zhejiang Province, Hangzhou 310012, China （ Wang J Department of Translational Medicine, The Affiliated Hospital of Hangzhou Normal Univers@ , Hangzhou 310015, China（ Yang J Department of Infectious Diseases, Xixi Hospital of Hangzhou, Hangzhou 310023, China（ Yang ZX State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China（ Cheng LF, Wu NP)

机构地区：[1]浙江省立同德医院检验科,杭州310012 [2]杭州师范大学附属医院转化医学平台,310015 [3]杭州市西溪医院感染二科,310023 [4]浙江大学医学院附属第一医院传染病诊治国家重点实验室,杭州310003

出　　处：《中华临床感染病杂志》2017年第1期31-36,共6页Chinese Journal of Clinical Infectious Diseases

基　　金：国家自然科学基金面上项目（81273313）;浙江省医药卫生科技计划项目（2017KY275）志谢：本研究和论文撰写得到了浙江大学医学院附属第一医院传染病诊治国家重点实验室姚航平教授、王英杰教授、靳昌忠助理研究员和卢祥云助理研究员的悉心指导和帮助,在此表示感谢.

摘　　要：目的 研究成视网膜细胞瘤结合蛋白4（RBBP4）对于Sp1介导的HIV长末端重复序列（Long terminal repeat,LTR）的转录作用及其调控机制.方法 利用脂质体共转染的方法,分别将RBBP4表达载体、Sp1表达载体与带荧光素酶标签的HIV启动子载体pHIV-LTR-Luc和Sp1位点突变载体pHIV-LTR-sp1-mut进行转染,用双荧光素酶报告系统检测HIV LTR转录活性.利用染色质免疫共沉淀（ChIP）和凝胶迁移实验（EMSA）进一步研究RBBP4对Sp1在LTR上结合的影响.结果 实验结果显示,在RBBP4的转染剂量为500 ng时,Sp1介导激活的相对萤火虫荧光素酶活性值由62.5下降到16（t=14.52,P〈0.01）.HIV LTR上Sp1结合位点突变后,100、300和500 ng的RBBP4表达载体对HIV LTR介导的萤火虫荧光素酶活性值的影响经双尾配对差异均无统计学意义（t=1.897、2.357和3.162,P值均〉0.05）.ChIP结果显示,当RBBP4在HIV LTR上的结合增加时,Sp1在HIV LTR上的结合量明显增加（t=11.93,P〈0.01）,RBBP4在HIV LTR上结合减少,Sp1在LTR上的结合明显减少（t=11.38,P〈0.01）.EMSA结果也进一步验证了ChIP结果中RBBP4对Sp1结合DNA的影响.结论 RBBP4可抑制Sp1介导HIV LTR的转录.Objective To investigate the effect of retinoblastoma binding protein 4 （RBBP4）in Sp1 -mediated HIV long terminal repeat（LTR）transcription.Methods RBBP4 expression vector and Sp1 expression vector were respectively co-transfected into 293 T cells with HIV promoter pHIV-LTR-Luc or Sp1 site mutated pHIV-LTR-sp1 -mut by liposome transfection,and the transfected cells were examined by dual luciferase reporter assay system.The effect of RBBP4 on the binding of Sp1 to LTR was further studied by chromatin immunoprecipitation （ChIP）and electrophoretic mobility shift assay （EMSA）.Results The relative firefly luciferase activity activated by Sp1 was decreased from 62.5 to 16 at the dose of 500 ng of RBBP4 expression vector （t =14.52,P 〈0.01 ）.When the Sp1 binding sites were mutated,the effects of 100,300 or 500 ng of RBBP4 expression vector on the firefly luciferase activity of HIV LTR were notnbsp;statistically significance （t =1 .897,2.357 and 3.162,all P 〈0.05）.ChIP results showed that when the binding of RBBP4 on HIV LTR increased,the binding of Sp1 on HIV LTR increased significantly （t =11 .93,P 〈0.01 ）,while the reduced binding of RBBP4 on HIV LTR significantly attenuated the binding of Sp1 onto LTR（t =11 .38,P 〈0.01 ）.The effect of RBBP4 on the binding of Sp1 to DNA in ChIP assays was further verified by EMSA assays.Conclusion RBBP4 can inhibit the Sp1 -mediated HIV LTR transcription in 293 T cells.

关 键 词：人类免疫缺陷病毒1 成视网膜细胞瘤结合蛋白4 长末端重复序列 转录 特殊化蛋白1 

分 类 号：R734.2[医药卫生—肿瘤]