Establishment of a reporter gene system for activity detection of hepatocyte nuclear factor 4alpha

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	Establishment of a reporter gene system for activity detection of hepatocyte nuclear factor 4alpha
	Sun Bingjie 3; Chen Shijie 1; Deng Longfei 3; Ding Chenhong 2; Chen Fei 2; Luo Cheng1 ; Xie Weifen 2; Zhang Xin 3
刊名	Academic Journal of Second Military Medical University
	2017
卷号	38 期号:9 页码:1112-1118
关键词	Ninjurin 1 hepatocyte nuclear factor 4alpha Ninjurin 1 reporter gene system small molecular compound
ISSN号	0258-879X
其他题名	基于报告基因系统的肝细胞核因子4alpha活性检测体系的建立
文献子类	Article
英文摘要	Objective To establish a luciferase reporter gene system for detecting the activity of hepatocyte nuclear factor 4alpha (HNF4alpha), so as to screen the small molecule compounds regulating the activity of HNF4alpha. Methods HNF4alpha was purified by affinity chromatography. The direct interaction of DNA fragment or small molecule compounds to the HNF4alpha was determined by protein thermal shift assay. The constructing recombinant plasmid pGL3-NINJ1-3p or pGL3-NINJ1-9p, which contained three copies or nine copies of the HNF4alpha response element in the Ninjurin 1 (NINJ1) promoter, was transfected into hepatoma carcinoma cells. The transcriptional activity of HNF4alpha was detected by dual-luciferase reporter gene assay. The expressions of HNF4alpha and its down-stream genes were analyzed in hepatoma carcinoma cells treated with small molecular compound luteolin or alverine by real-time quantitative PCR. The changes of HNF4alpha transcriptional activity of cells treated with luteolin or alverine were estimated by luciferase reporter gene assay. Results Protein thermal shift confirmed that the HNF4alpha response element in NINJ1 promoter bound to HNF4alpha protein. In the hepatoma carcinoma cells with overexpression of HNF4alpha, both pGL3-NINJ1-3p and pGL3-NINJ1-9p could detect the alteration of the transcriptional activity of HNF4alpha, and pGL3-NINJ1-9p was more sensitive than pGL3-NINJ1-3p (P<0.01). Luteolin and alverine, both directly interacting with HNF4alpha, down-regulated and up-regulated the expression of HNF4alpha target genes, respectively. Moreover, pGL3-NINJ1-9p could validate the effect of luteolin or alverine on the transcriptional activity of HNF4alpha. Conclusion We successfully establish a detection system for HNF4alpha activity in hepatoma carcinoma cells by the reporter gene vector pGL3-NINJ1-9p. This system is a tool for screening small molecule compounds that regulate HNF4alpha activity.
资助项目	上海市科学技术委员会生物医药领域科技支撑项目[00000000]
WOS研究方向	General & Internal Medicine (provided by Clarivate Analytics)
语种	中文
CSCD记录号	CSCD:6077114
内容类型	期刊论文
源URL	[http://119.78.100.183/handle/2S10ELR8/269194]
专题	药物发现与设计中心
作者单位	1.Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.; 2.Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China. 3.School of Biotechnology, East China University of Science and Technology, Shanghai 200237, China.;
推荐引用方式 GB/T 7714	Sun Bingjie,Chen Shijie,Deng Longfei,et al. Establishment of a reporter gene system for activity detection of hepatocyte nuclear factor 4alpha[J]. Academic Journal of Second Military Medical University,2017,38(9):1112-1118.
APA	Sun Bingjie.,Chen Shijie.,Deng Longfei.,Ding Chenhong.,Chen Fei.,...&Zhang Xin.(2017).Establishment of a reporter gene system for activity detection of hepatocyte nuclear factor 4alpha.Academic Journal of Second Military Medical University,38(9),1112-1118.
MLA	Sun Bingjie,et al."Establishment of a reporter gene system for activity detection of hepatocyte nuclear factor 4alpha".Academic Journal of Second Military Medical University 38.9(2017):1112-1118.