转录因子KLF7对IL-6基因的转录调控分析  

Transcriptional regulation analysis of IL-6 gene by transcription factor KLF7

作　　者：王宁[1,2,3] 尤欣 徐海冬 娄明 娄钰琦 闫晓红 李辉[1,2,3] WANG Ning;YOU Xin;XU Haidong;LOU Ming;LOU Yuqi;YAN Xiaohong;LI Hui(School of Animal Sciences and Technology,Northeast Agricultural University,Harbin 150030,China;Key Laboratory of Animal Genetics,Breeding and Reproduction in Heilongjiang Province,Harbin 150030,China;Key Laboratory of Chicken Genetics and Breeding,Ministry of Agriculture and Rural Affairs,Harbin 150030,China)

机构地区：[1]东北农业大学动物科学技术学院,哈尔滨150030 [2]黑龙江省普通高等学校动物遗传育种与繁殖重点实验室,哈尔滨150030 [3]农业农村部鸡遗传育种重点实验室,哈尔滨150030

出　　处：《东北农业大学学报》2020年第10期45-55,共11页Journal of Northeast Agricultural University

基　　金：国家自然科学基金项目(31872346)。

摘　　要：选取小鼠IL-6基因转录起始位点上游2000 bp序列作为启动子序列,采用在线生物信息学软件分析转录因子结合位点;利用PCR扩增小鼠IL-6基因启动子及其5'端截短突变体片段,构建相应的荧光素酶报告基因重组质粒,转染3T3-L1细胞后检测报告基因活性;采用共转染分析转录因子KLF7对IL-6基因启动子活性的影响;利用siRNA技术探究敲低KLF7对细胞内源性IL-6基因表达的影响。生物信息学分析显示,小鼠IL-6基因启动子区存在5个Sp1/KLFs结合位点;报告基因分析和定点突变分析发现,KLF7通过IL-6基因启动子区-98至-89 bp处(CCCCACCCAC)发挥调控IL-6基因启动子活性的作用;Real-time RT-PCR和Western blot分析显示,敲低KLF7抑制细胞内源性IL-6基因表达。文章首次发现KLF7直接调控IL-6基因表达,研究结果对揭示KLF7在脂肪生成中的作用机制具有重要意义。The 2000-bp sequence upstream of transcription start site of mouse IL-6 gene was selected as IL-6 promoter region.The transcription factor binding sites were analyzed using the online bioinformatics software.The mouse IL-6 promoter region and its 5'end truncated promoter fragments were amplified by PCR,cloned into pGL3 basic vector to yield their corresponding promoter luciferase reporter constructs.These promoter reporter constructs were individually transfected into 3T3-L1 cells,and luciferase reporter activity was assayed using a Dual-Luciferase Reporter Assay kit.The promoter reporters with mutation of KLF7 binding sites were generated by DNA synthesis,and cotransfection was performed to investigate the effect of KLF7 on IL-6 gene promoter activity.In addition,effect of knockdown KLF7 by siRNA on the endogenous IL-6 expression in 3T3-L1 cells was investigated.The results showed that IL-6 gene promoter region contained five putative Sp1/KLFs binding sites.Reporter gene and mutation analyses showed that KLF7 inhibited IL-6 pomoter activity,and mutation of site 1(CCCCACCCAC)(-98 to-89 bp)abolished the inhibition of KLF7 on IL-6 promoter activity.Real-time RT-PCR and Western blot showed that knockdown KLF7 reduced the endogenous IL-6 gene expression.The present study for the first time demontrated that KLF7 directly regulated IL-6 gene and the findings would contribute to understand the role and molecular mechanism of KLF7 in adipogenesis.

关 键 词：鼠 KLF7 IL-6 启动子 转录调控 

分 类 号：S831.2[农业科学—畜牧学]