转录因子RUNX2对IPO8基因启动子的调节作用  

Regulation of Human IPO8 Promoter by Transcription Factor RUNX2

作　　者：熊建军[1] 龚帧[1] 周小鸥[1] 王庭[1] 刘建云 李卫东 

机构地区：[1]九江学院基础医学院药理学教研室,江西九江332000 [2]江西省系统生物医学重点实验室,江西九江332000

出　　处：《中国医科大学学报》2014年第7期585-588,共4页Journal of China Medical University

基　　金：国家自然科学基金(81060075;81260140);江西省教育厅科学研究项目(GJJ12683)

摘　　要：目的研究转录因子RUNX2对IPO8基因启动子的调控作用。方法基于PCR技术,分别突变和剔除IPO8基因启动子片段P-732/+134上RUNX2结合位点(-497^-502 bp),改造后的启动子片段定向插入荧光素酶表达载体PGL-3 Basic。重组质粒分别转染Saos-2与He La细胞,检测报告基因荧光素酶活性。应用染色质免疫共沉淀(Ch IP)证实RUNX2与IPO8启动子在Saos-2细胞内结合。慢病毒介导RUNX2在Saos-2细胞的靶向沉默,定量PCR检测相应组别中IPO8 m RNA的表达。结果成功突变和剔除IPO8启动子上RUNX2结合位点,破坏RUNX2结合位点导致IPO8启动子活性在Saos-2细胞中显著下降(P<0.05)。转录因子RUNX2在Saos-2细胞与IPO8启动子有效结合,下调RUNX2的表达导致IPO8转录水平下降(P<0.05)。结论转录因子RUNX2正性调控IPO8基因的转录。Objective To investigate the regulation effects of transcription factor RUNX2 on human importin 8 （IPO8） promoter.Methods Mutation or deletion of the RUNX2-binding site （-497 to-502 bp） within the IPO8 promoter region was performed by PCR technology.The modified IPO8 promoters were subsequently inserted into luciferase report vector PGL-3 Basic.The recombinant plasmids were transfected into human Saos-2 and HeLa cells respectively and the reporter gene luciferase activities were measured by dual luciferase reporter system.Furthermore,ChIP assay was performed to confirm the binding of RUNX2 to IPO8 promoter in Saos-2 cells.In addition,lentivirus-mediated siRNA down-regulated RUNX2 expression in Saos-2 cells and expression of IPO8 mRNA was determined by real-time PCR.Results The RUNX2 binding site was mutated and deleted within IPO8 promoter.Disruption of RUNX2 binding site led to significant decrease of IPO8 promoter activity in Saos-2 cells （P ＜ 0.05）.ChIP assay demonstrated that RUNX2 binds to IPO8 promoter.Down-regulation of RUNX2 attenuated the transcription level of IPO8 gene （P ＜ 0.05）.Conclusion Transcription factor RUNX2 positively regulates the activity of IPO8 promoter.

关 键 词：IPO8 RUNX2 启动子 荧光素酶 

分 类 号：R34[医药卫生—基础医学]