NF-κB p65调控IRF-8基因启动子活性及其启动子结合元件的初步鉴定  

The regulatory effects of NF-kB p65 on IRF-8 gene promoter activity and initial identification of its binding elements

作　　者：王文博 钱宝梅 罗灿 彭明玉 张婧[1] 赵聃[1] 王迎伟[1] 邱文[1] 季明德[2] WANG Wenbo;QIAN Baomei;LUO Can;PENG Mingyu;ZHANG Jing;ZHAO Dan;WANG Yingwei;QIU Wen;JI Mingde(Department of Immunology,Nanjing Medical University,Nanjing 211166;Department of Laboratory Medicine,Jiangsu Provincial Hospital of Chinese Medicine,Nanjing 210006,China)

机构地区：[1]南京医科大学免疫学系,江苏南京211166 [2]江苏省中医院检验科,江苏南京210006

出　　处：《南京医科大学学报（自然科学版）》2020年第5期638-644,共7页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金(31470853,81603358,31770934,81971468)。

摘　　要：目的:探讨大鼠核因子-κB(nuclear factor-κB,NF-κB)p65亚基在细胞内过表达和其活性变化对干扰素调节因子-8(interferon regulatory factor-8,IRF-8)基因启动的影响,并初步筛查IRF-8启动子上可能的p65结合元件。方法:采用聚合酶链式反应(polymerase chain reaction,PCR)技术扩增大鼠p65基因蛋白编码区(complete sequence coding,CDS)序列,将其插入到空载p IRES2-EGFP质粒中,构建大鼠野生型(wild type,WT)p65过表达质粒(pIRES2-p65 WT)。在此基础上,将p65第535位丝氨酸(serine,S)突变为天冬氨酸(aspartic,D)或丙氨酸(alanine,A),分别构建p65持续活化突变型质粒(pIRES2-p65 S535D)和p65显性负性突变型质粒(pIRES2-p65 S535A)。之后应用生物信息学软件预测IRF-8基因启动子区p65的结合元件,并据此构建IRF-8启动子全长(full-length,FL)和3个截短的荧光素酶报告质粒,即pGL3-IRF-8-FL(-1892^+174 nt)、pGL3-IRF-8-1(-1360^+174 nt)、pGL3-IRF-8-2(-752^+174 nt)和pGL3-IRF-8-3(-68^+174 nt)。将上述质粒行不同组合共转染人胚肾293T(human embryonic kidney 293T,HEK-293T)细胞,Western blot和荧光素酶实验分别检查p65的表达和IRF-8的启动子活性,并分析IRF-8启动子区可能的p65结合元件。结果:菌液PCR及测序证实上述质粒构建成功。分别将pIRES2-p65 WT、pIRES2-p65S535D、pIRES2-p65 S535A和p GL3-IRF-8-FL共转染HEK-293T,发现过表达pIRES2-p65 WT或pIRES2-p65 S535D均可明显增加IRF-8启动子活性,且以pIRES2-p65 S535D更为显著;而过表达pIRES2-p65 S535A后,IRF-8启动子活性无明显变化。将pGL3-IRF-8-FL、pGL3-IRF-8-1~3和pIRES2-p65 S535D共转染HEK-293T后发现,pGL3-IRF-8-3的启动子活性显著低于pGL3-IRF-8-FL、pGL3-IRF-8-1和p GL3-IRF-8-2,提示大鼠IRF-8启动子-752~68 nt区域可能存在p65结合元件。结论:在HEK-293T细胞内过表达野生型或持续活化突变型p65可显著促进IRF-8基因的启动,且p65与IRF-8启动子的结合元件可能位于-752^-68 nt部位。Objective:This study aims to investigate the effect of rat nuclear factor-κB(NF-κB)p65 subunit over-expression and its activity changes on the gene promoter activity of interferon regulatory factor-8(IRF-8),and initially screen the possible p65-binding elements within IRF-8 promoter.Methods:To construct the rat wild type p65 over-expression plasmid(pIRES2-p65 WT),complete sequence coding(CDS)of rat p65 was amplified by polymerase chain reaction(PCR)and cloned into pIRES2-EGFP.Then,S535 D and S535 A mutation was done respectively based on the wild type p65 over-expression plasmid to construct p65 constitutively active mutant(p IRES2-p65 S535 D)and p65 dominant negative mutant(pIRES2-p65 S535 A).The potential p65-binding elements within IRF-8 promoter were predicted by using bioinformatics software.Based on the predicted results,luciferase reporter plasmids of full-length(FL)and three truncated IRF-8 gene promoter were constructed,namely pGL3-IRF-8-FL(-1892^+174 nt),pGL3-IRF-8-1(-1360^+174 nt),pGL3-IRF-8-2(-752^+174 nt),pGL3-IRF-8-3(-68^+174 nt).The above-mentioned plasmids were co-transfected into human embryonic kidney 293 T(HEK-293 T)cells in different groups.Then,the expression level of p65 was detected by Western blot,and the promoter activity of IRF-8 was detected by luciferase experiment to screen the p65-binding elements.Results:It was verified that above-mentioned plasmid was constructed correctly by PCR analysis and nucleotide sequencing.The plasmids of pIRES2-p65 WT,pIRES2-p65 S535 D,pIRES2-p65 S535 A were respectively transfected into HEK-293 T cells together with pGL3-IRF-8-FL.The luciferase results showed that the activity of IRF-8 promoter was markedly increased in response to pIRES2-p65 WT and pIRES2-p65 S535 D,especially the later.However,there was no significant change of IRF-8 promoter activity after over-expression of pIRES2-p65 S535 A.The plasmids of pGL3-IRF-8-FL or pGL3-IRF-8-1~3 and pIRES2-p65 were co-transfected into HEK-293 T cells,and the result displayed that the activity of pGL3-IRF-8

关 键 词：核因子-κB p65(NF-κB p65) 干扰素调节因子-8(IRF-8) 启动子 结合元件 

分 类 号：H329.25[语言文字—法语]