信号转导与转录激活子1对高迁移率族蛋白1启动子转录的调控作用

Signal transducer and activator of transcription 1 regulates the transcription of high mobility group box-1 promoter

目的:探讨信号转导与转录激活子1(STAT1)对高迁移率族蛋白1(HMGB1)启动子转录的调控作用。方法:在小鼠巨噬细胞RAW264.7中,转染siRNA-STAT1质粒,检测HMGB1转录水平的变化。生物信息学方法预测STAT1与HMGB1基因启动子的结合位点,并据此构建HMGB1基因启动子全长和3个截短的荧光素酶报告基因,将含STAT1的真核表达载体pcDNA3.1-STAT1和上述构建的荧光素酶报告基因共转染人肾293T细胞,检测各组转染细胞的荧光素酶活性。利用染色质免疫共沉淀(ChIP)实验分析STAT1与HMGB1启动子的结合情况。结果:转染siRNA-STAT1质粒可下调HMGB1基因在RAW264.7细胞的转录水平。酶切及测序结果表明,获得的4个不同长度的HMGB1启动子克隆与Genbank DNA序列数据库对比分析序列一致,且插入方向正确。pcDNA3.1-STAT1质粒和HMGB1启动子-1700^-700bp区域共转染荧光素酶活性最强。ChIP结果显示,STAT1直接结合在HMGB1启动子-1700^-700bp区域上。结论:成功构建了HMGB1基因启动子全长及各截短片段荧光素酶报告质粒,并初步确定了STAT1通过直接结合在HMGB1基因启动子1700^-700bp区域,活化HMGB1启动子活性,发挥上调HMGB1启动子转录的作用。

Objective:To investigate the regulatory effect of Signal transducer and activator of transcription 1(STAT1)on the transcription of high mobility group box-1(HMGB1)promoter.Methods:siRNA-STAT1 plasmid was transfected into mouse macrophage cell line RAW264.7 for detecting the changes in transcription level of HMGB1.Bioinformatics approach was used to predict the binding sites of STAT1 to the HMGB1 gene promoter.Based on the prediction,a full-length HMGB1 gene promoter and three truncated luciferase reporter genes were constructed.The eukaryotic expression vector pcDNA3.1-STAT1 containing STAT1 and the constructed luciferase reporter genes abovementioned were co-transfected into human kidney 293T cells.The luciferase activity in each group of transfected cells was measured.The binding of STAT1 to HMGB1 promoter was analyzed by chromatin immunocoprecipitation(ChIP)assay.Results:Transfection of siRNA-STAT1 down-regulated the transcription of HMGB1 gene in RAW264.7 cells.The results of restriction enzyme digestion and sequencing showed that the four resultant HMGB1 promoter clones with different lengths were identical to those in the GenBank DNA sequence database,and with correct orientation.The luciferase activity was strongest with co-transfection of pcDNA3.1-STAT1 to-1700 to-700bp region of HMGB1 promoter.ChIP assay showed that STAT1 directly bound to the-1700 to-700bp region of HMGB1 promoter.Conclusion:A luciferase report plasmid with full-length HMGB1 promoter and truncated luciferase reporter gene fragments was successfully constructed in this study.According to preliminary analysis,STAT1 activates the activity of HMGB1 promoter and up-regulates its transcription by directly binding to the-1700 to-700bp region of HMGB1 promoter.