TFAP2A对肾小球硬化相关基因ADCK4转录调控机制的研究

Transcriptional regulation of TFAP2A on glomerulosclerosis-related gene ADCK4

目的探索细胞中TFAP2A对局灶节段性肾小球硬化(FSGS)相关基因含aarF结构域的激酶4(ADCK4)的转录调控机制及TFAP2A与ADCK4是否存在特定的结合区域。方法生物信息学分析肾小球硬化基因火山图,ADCK4及TFAP2A表达水平的关系。JASPAR数据库预测ADCK4基因转录起始位点-464 bp/+206 bp区域包含TFAP2A转录因子结合位点;TFAP2A siRNA浓度分别为5、10、15µmol/L,TFAP2A过表达质粒质量浓度分别为50、100、300µg/L,通过双萤光素酶报告基因实验验证TFAP2A对ADCK4基因启动子水平的调控作用。TFAP2A siRNA及TFAP2A过表达质粒转染细胞,实时荧光定量PCR检测TFAP2A、ADCK4 mRNA表达,蛋白免疫印迹实验检测TFAP2A、ADCK4蛋白表达。染色质免疫沉淀试验验证TFAP2A与ADCK4启动子的特定区域结合。结果生物信息学分析显示FSGS肾组织中RNA-Seq RNA表达上调的基因273个,表达下调的基因219个;ADCK4与TFAP2A表达水平呈正相关(P<0.01)。双萤光素酶报告基因实验证明TFAP2A siRNA浓度为10、15µmol/L的ADCK4启动子相对萤光素酶活性增强,TFAP2A过表达质粒质量浓度为100、300µg/L的ADCK4启动子萤光素酶活性降低(P<0.05)。与对照组相比,实验组ADCK4 mRNA和蛋白表达水平升高;过表达实验中,与对照组比较,实验组ADCK4 mRNA和蛋白表达水平降低(P<0.05)。染色质免疫沉淀试验发现TFAP2A能与ADCK4启动子的特定区域结合。结论转录因子TFAP2A负向调控ADCK4基因表达,增加了调控足细胞重要基因的转录因子成员。

Objective To exploring the mechanism of transcriptional regulation of TFAP2A on glomerulosclerosisrelated gene aarF structural domain-containing kinase 4(ADCK4)in cells and the specific binding region between TFAP2A and ADCK4.Methods Bioinformatics was used to analyze volcano map of glomerulosclerosis genes,and the relationship between ADCK4 and TFAP2A expression levels.JASPAR database was used to predict that ADCK4 gene transcription start site-464 bp/+206 bp region containing TFAP2A transcription factor binding site.TFAP2A siRNA concentrations were 5,10 and 15µmol/L,and the mass concentrations of TFAP2A overexpression plasmid were 50,100 and 300µg/L,respectively.The regulatory effect of TFAP2A on the promoter level of the ADCK4 gene was verified by dual-luciferase reporter gene assay.TFAP2A siRNA and TFAP2A overexpression plasmid were used to transfect cells.Real-time fluorescence quantitative PCR was used to detect TFAP2A and ADCK4 mRNA expression.Protein immunoblotting assay was used to detect TFAP2A and ADCK4 protein expression.Chromatin immunoprecipitation assay was used to confirm TFAP2A binding to a specific region of the ADCK4 promoter.Results Bioinformatics analysis showed that 273 genes were up-regulated and 219 genes were down-regulated.The expression levels of ADCK4 and TFAP2A were positively correlated(P<0.01).Dual luciferase reporter gene assay demonstrated that the relative luciferase activity of ADCK4 promoter was enhanced with TFAP2A siRNA concentrations of 10 and 15µmol/L,and that the luciferase activity of ADCK4 promoter was reduced when TFAP2A overexpression plasmid mass concentrations of 100 and 300µg/L(P<0.05).ADCK4 mRNA and protein expression levels were increased in the TFAP2A siRNA group compared with the control siRNA group.ADCK4 mRNA and protein expression levels were decreased in the TFAP2A overexpression plasmid group compared with the pENTER plasmid group(P<0.05).Chromatin immunoprecipitation assay revealed that TFAP2A can bind to specific regions of ADCK4 promoter.Conclusion Negative regulation of ADCK4 gene expression by the transcription factor TFAP2A increases the number of transcription factor members that regulate genes important for podocytes.