基于报告基因系统的肝细胞核因子4α活性检测体系的建立

目的 建立一个基于荧光素酶报告基因系统的肝细胞核因子4α（HNF4α）活性检测系统，筛选可调控HNF4α活性的小分子化合物。方法 采用亲和层析法纯化HNF4α蛋白，行蛋白热迁移实验检测相关DNA片段及小分子化合物与HNF4α蛋白的直接相互作用。分别构建含有3个拷贝和9个拷贝的Ninjurin 1（NINJ1）基因启动子区HNF4α反应元件的荧光素酶报告基因质粒pGL3-NINJ1-3p和pGL3-NINJ1-9p，转染肝癌细胞，采用荧光素酶报告基因法检测肝癌细胞内HNF4α转录活性的改变，qPCR检测小分子化合物木犀草素或阿尔维林处理后肝癌细胞中HNF4α及下游基因的表达情况，荧光素酶报告基因法检测小分子化合物处理后细胞内HNF4α转录活性的改变。结果 蛋白热迁移实验证实，NINJ1基因启动子区HNF4α的DNA结合片段可与HNF4α蛋白结合。在过表达HNF4α的肝癌细胞中，pGL3-NINJ1-3p和pGL3-NINJ1-9p均可检测到肝癌细胞中HNF4α活性的改变，且pGL3-NINJ1-9p较pGL3-NINJ1-3p的检测灵敏度更高（P<0.01）。木犀草素和阿尔维林与HNF4α蛋白存在直接相互作用，分别下调和上调HNF4α靶基因；pGL3-NINJ1-9p可检测到木犀草素和阿尔维林对HNF4α转录活性的影响。结论 利用报告基因载体pGL3-NINJ1-9p成功建立了HNF4α活性的检测系统，为筛选调控HNF4α活性的小分子化合物等提供了基础工具。
Objective To establish a luciferase reporter gene system for detecting the activity of hepatocyte nuclear factor 4α (HNF4α), so as to screen the small molecule compounds regulating the activity of HNF4α. Methods HNF4α was purified by affinity chromatography. The direct interaction of DNA fragment or small molecule compounds to the HNF4α 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 HNF4α response element in the Ninjurin 1 (NINJ1) promoter, was transfected into hepatoma carcinoma cells. The transcriptional activity of HNF4α was detected by dual-luciferase reporter gene assay. The expressions of HNF4α 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 HNF4α transcriptional activity of cells treated with luteolin or alverine were estimated by luciferase reporter gene assay. Results Protein thermal shift confirmed that the HNF4α response element in NINJ1 promoter bound to HNF4α protein. In the hepatoma carcinoma cells with overexpression of HNF4α, both pGL3-NINJ1-3p and pGL3-NINJ1-9p could detect the alteration of the transcriptional activity of HNF4α, and pGL3-NINJ1-9p was more sensitive than pGL3-NINJ1-3p (P<0.01). Luteolin and alverine, both directly interacting with HNF4α, down-regulated and up-regulated the expression of HNF4α target genes, respectively. Moreover, pGL3-NINJ1-9p could validate the effect of luteolin or alverine on the transcriptional activity of HNF4α. Conclusion We successfully establish a detection system for HNF4α 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 HNF4α activity