TFAP2A对肾小球硬化相关基因NPHS2的转录调控作用观察

Effect of TFAP2A on transcriptional regulation of glomerular sclerosis-related gene NPHS2

目的观察转录因子AP-2α(TFAP2A)对肾小球硬化相关基因NPHS2的转录调控作用。方法基于GEO数据库及GEO Profiles数据集,比较肾透明细胞癌组织与正常肾组织中NPHS2、TFAP2A的表达差异。构建含NPHS2基因5′侧翼区域重组报告质粒,预测NPHS2基因的-519 bp~+20 bp启动子区包含转录因子TFAP2A的结合位点。培养HEK-293T细胞,对照siRNA组转染control siRNA,实验组分别转染TFAP2A siRNA(浓度分别为5、10、15 pmol/µL)和TFAP2A过表达质粒(质量浓度分别为50、100、300 ng/mL),通过双萤光素酶报告基因实验验证TFAP2A对NPHS2基因启动子水平的调控作用。将HEK-293T细胞分为对照组、干扰组及过表达组,分别转染con‐trol siRNA、TFAP2A siRNA及TFAP2A过表达质粒;采用荧光定量PCR检测TFAP2A、NPHS2 mRNA,采用Western blotting法检测NPHS2蛋白。结果肾透明细胞癌组织中TFAP2A、NPHS2表达低于正常肾组织。成功构建含NPHS2基因5′侧翼区域重组报告质粒,NPHS2基因转录起始位点上游-519 bp~+20 bp区域包含两个TFAP2A转录因子结合位点。双萤光素酶报告基因实验结果显示,与对照siRNA组相比,转染5、10、15 pmol/µL TFAP2A siRNA的实验组细胞NPHS2启动子荧光素酶活性均增强,转染300 ng/mL TFAP2A过表达质粒的实验组细胞NPHS2启动子荧光素酶活性降低(P均<0.05)。干扰组NPHS2 mRNA及蛋白表达高于对照组,过表达组NPHS2 mRNA及蛋白表达低于对照组(P均<0.05)。结论NPHS2启动子-519 bp~+20 bp区域存在潜在的TFAP2A转录因子结合位点;TFAP2A可下调NPHS2的启动子活性、mRNA及蛋白表达。

Objective To observe the transcriptional regulatory effect of transcription factor AP-2α(TFAP2A)on the glomerulosclerosis-related gene NPHS2.Methods Based on the GEO database and GEO Profiles data set,the expression differences of NPHS2 and TFAP2A between the renal clear cell carcinoma tissue and normal renal tissue were compared.A recombinant reporter plasmid containing the 5′flanking region of the NPHS2 gene was constructed,and the-519 bp to+20 bp promoter region of the NPHS2 gene containing the binding site for the transcription factor TFAP2A was predicted.HEK-293T cells were cultured,cells in the control siRNA group were transfected with control siRNA,and the experimental group with 5,10,and 15 pmol/μL TFAP2A siRNA and 50,100,and 300 ng/mL TFAP2A overexpression plasmid.The dual-luciferase reporter gene experiment was used to verify the regulatory effect of TFAP2A on the promoter level of the NPHS2 gene.HEK-293T cells were divided into the control group,interference group and overexpression group,which were transfected with control siRNA,TFAP2A siRNA,and TFAP2A overexpression plasmid,respectively.Fluorescence quantitative PCR was used to detect TFAP2A and NPHS2 mRNA,and Western blotting was used to detect NPHS2 protein.Results The expression levels of TFAP2A and NPHS2 in the renal clear cell carcinoma tissues were lower than those in the normal renal tissues.The recombinant reporter plasmid containing the 5'flanking region of NPHS2 gene was successfully constructed.The-519 bp to+20 bp region upstream of NPHS2 gene transcription start site con‐tained two TFAP2A transcription factor binding sites.The results of dual-luciferase reporter gene experiment showed that compared with the control siRNA group,the luciferase activity at the NPHS2 promoter in cells of the experimental group transfected with 5,10,and 15 pmol/μL TFAP2A siRNA was enhanced,and the luciferase activity of the NPHS2 promoter in cells in the experimental group transfected with 300 ng/mL TFAP2A overexpression plasmid decreased(all P<0.05).The expression levels of NPHS2 mRNA and protein in the interference group were higher than those of the control group,and the expression levels of NPHS2 mRNA and protein in the overexpression group were lower than those of the control group(all P<0.05).Conclusions There are potential TFAP2A transcription factor binding sites in the-519 bp to+20 bp region of the NPHS2 promoter.TFAP2A can down-regulate the promoter activity,mRNA and protein expression of NPHS2.