论文部分内容阅读
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Ninjurin1(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 detection system of hepatocyte nuclear factor 4α (HNF4α) activity based on the luciferase reporter gene system and screen small molecule compounds that can regulate the activity of HNF4α. Methods The HNF4α protein was purified by affinity chromatography and the direct interaction between DNA fragments and small molecule compounds and HNF4α protein was detected by heat transfer assay. The luciferase reporter plasmids pGL3-NINJ1-3p and pGL3-NINJ1-9p containing 3 copies and 9 copies of the HNF4α response element in the promoter region of Ninjurin1 (NINJ1) were constructed and transfected into hepatoma cells respectively. Luciferase The reporter gene method was used to detect the transcriptional activity of HNF4α in hepatocellular carcinoma cells. QPCR was used to detect the expression of HNF4α and its downstream genes in hepatocellular carcinoma cells treated with small molecule compounds luteolin or alverine. Luciferase reporter assay was used to detect small molecule compounds Change of intracellular HNF4α transcriptional activity. Results The results of heat transfer experiments confirmed that the DNA binding fragment of HNF4α in the promoter region of NINJ1 gene could bind to HNF4α protein. In HNF4α-overexpressing hepatocellular carcinoma cells, the changes of HNF4α activity in hepatoma cells were detected by both pGL3-NINJ1-3p and pGL3-NINJ1-9p, and the detection sensitivity of pGL3-NINJ1-9p was higher than that of pGL3-NINJ1-3p P <0.01). Luteolin and alverine directly interacted with HNF4α protein to down-regulate and up-regulate the target gene of HNF4α respectively; pGL3-NINJ1-9p could detect the transcriptional activity of HNF4α by luteolin and alverine. Conclusion The detection system of HNF4α activity was successfully established by using reporter gene vector pGL3-NINJ1-9p, which provided a basic tool for screening small molecule compounds that regulate the activity of HNF4α.