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

ZHANG Xiaotian; ZHOU Guoping

doi:10.3969/j.issn.1002-266X.2023.32.005

Abstract：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 ex-pression 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.