TFAP2A对肾小球硬化相关基因ADCK4转录调控机制的研究

doi:10.11958/20231028

摘要/Abstract

摘要：

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、15 μmol/L，TFAP2A过表达质粒质量浓度分别为50、100、300 μg/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、15 μmol/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、300 μg/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。

关键词: 肾小球硬化症, 局灶节段性, 转录因子AP-2, 启动区, 遗传, 转录调控, 含aarF结构域的激酶4

Abstract:

Objective To exploring the mechanism of transcriptional regulation of TFAP2A on glomerulosclerosis-related 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 15 μmol/L, and the mass concentrations of TFAP2A overexpression plasmid were 50, 100 and 300 μg/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 15 μmol/L, and that the luciferase activity of ADCK4 promoter was reduced when TFAP2A overexpression plasmid mass concentrations of 100 and 300 μg/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.

Key words: glomerulosclerosis, focal segmental, transcription factor AP-2, promoter region, genetic, transcriptional regulation, aarF structural domain-containing kinase 4

中图分类号:

R726.92

图/表 11

表1 siRNA序列

Tab.1 Small interference RNA sequences

基因名称	序列（5'→3'）
TFAP2A siRNA	上游：CCUGCUCACAUCACUAGUATT
TFAP2A siRNA	下游：UACUAGUGAUGUGAGCAGGTT
control siRNA	上游：UUCUCCGAACGUGUCACGU
control siRNA	下游：ACGUGACACGUUCGGAGAATT

表2 qRT-PCR引物及ChIP引物序列

Tab.2 qRT-PCR primers and ChIP primers

基因名称	引物序列（5'→3'）	产物大小/bp
GAPDH	上游：ATGACATCAAGAAGGTGGTG	177
GAPDH	下游：CATACCAGGAAATGAGCTTG	177
ADCK4	上游：TTCCGATTCATGCAGACTGA	182
ADCK4	下游：CTGGACTTCTGCAGGACACA	182
TFAP2A	上游：ATTGACCTACAGTGCCCAGC	118
TFAP2A	下游：TCCATGAAAATGCTTTGGAA	118
ChIP-ADCK4	上游：GTGACGGTGGACAAAGTAAACA	303
ChIP-ADCK4	下游：AATCTGAAGAACACTGGGATAGG	303
ChIP-GAPDH	上游：TACTAGCGGTTTTACGGGCG	166
ChIP-GAPDH	下游：TCGAACAGGAGGAGCAGAGAGCGA	166

图1 生物信息学分析肾小球硬化基因表达火山图及TFAP2A与ADCK4的关系 A：生物信息学分析肾小球硬化基因表达火山图，红色代表表达上调的基因，蓝色代表表达下调的基因；B：生物信息学分析TFAP2A与ADCK4呈正相关。

Fig.1 Bioinformatics analysis of glomerulosclerosis gene expression volcano map

图2 JASPAR数据库预测TFAP2A转录因子结合位点

Fig.2 JASPAR database predicting two TFAP2A transcription factor binding sites

图3 转染不同浓度的TFAP2A siRNA的ADCK4启动子萤光素酶活性比较 A为对照组，B—D分别为5、10、15 μmol/L实验组；a与对照组比较，b与5 μmol/L实验组比较，P<0.05。

Fig.3 Comparison of luciferase activity of TFAP2A siRNA with different concentrations

图4 转染不同浓度的TFAP2A过表达质粒的ADCK4启动子萤光素酶活性比较 A为对照组，B—D分别为50、100、300 μg/L实验组；a与对照组比较，b与50 μg/L实验组比较，P<0.05。

Fig.4 Comparison of luciferase activity of TFAP2A overexpression plasmids with different concentrations

图5 TFAP2A siRNA对TFAP2A、ADCK4 mRNA表达的影响

Fig.5 Effects of TFAP2A siRNA on the expression of TFAP2A and ADCK4 mRNA

图6 TFAP2A过表达对TFAP2A、ADCK4 mRNA表达的影响

Fig.6 Effects of TFAP2A overexpression on TFAP2A and ADCK4 mRNA expression

图7 Western blot检测ADCK4、TFAP2A蛋白的表达 A：对照组；B：实验组；C：过表达对照组；D：过表达实验组。

Fig.7 Expression levels of ADCK4 and TFAP2A protein detected by Western blot assay

表3 siRNA和过表达后TFAP2A蛋白及ADCK4蛋白表达量改变（n=3，$\bar{x}±s$）

Tab.3 Changed expression levels of TFAP2A protein and ADCK4 protein after small interference and overexpression

组别	TFAP2A	t	ADCK4	t
对照组	1.00±0.21	2.936^＊	1.00±0.07	3.040^＊
实验组	0.37±0.03	2.936^＊	1.37±0.01	3.040^＊
过表达对照组	1.00±0.06	2.917^＊	1.00±0.08	9.409^＊
过表达实验组	1.55±0.18	2.917^＊	0.25±0.02	9.409^＊

图8 TFAP2A与ADCK4启动子区域直接结合 A：Marker；B：IgG抗体；C：全染色体为模板的对照（Input）；D：TFAP2A抗体。

Fig.8 TFAP2A binded directly to the ADCK4 promoter region

参考文献 20

[1]	SUN L, ZHANG X, WANG Z. ADCK4 gene polymorphism aggravates renal damage caused by focal segmental glomerulosclerosis with COL4A3 mutation[J]. Biosci Rep, 2021, 41(1):BSR20203248. doi:10.1042/BSR20203248.
[2]	NAKAGAWA N, KIMURA T, SAKATE R, et al. Demographics and treatment of patients with primary nephrotic syndrome in Japan using a national registry of clinical personal records[J]. Sci Rep, 2023, 13(1):14771. doi:10.1038/s41598-023-41909-5.
[3]	HODSON E M, SINHA A, COOPER T E. Interventions for focal segmental glomerulosclerosis in adults[J]. Cochrane Database Syst Rev, 2022, 2(2):CD003233. doi:10.1002/14651858.CD003233.pub3.
[4]	DE VRIESE A S, WETZELS J F, GLASSOCK R J, et al. Therapeutic trials in adult FSGS:lessons learned and the road forward[J]. Nat Rev Nephrol, 2021, 17(9):619-630. doi:10.1038/s41581-021-00427-1.
[5]	FANG Z, ZHANG C, JIN Y, et al. Adult-onset focal segmental glomerulosclerosis with steroid-dependent nephrotic syndrome caused by a novel TBC1D8B variant:a case report and literature review[J]. Am J Kidney Dis, 2023, 81(2):240-244. doi:10.1053/j.ajkd.2022.06.012.
[6]	ZHAI S B, ZHANG L, SUN B C, et al. Early-onset COQ8B(ADCK4) glomerulopathy in a child with isolated proteinuria:a case report and literature review[J]. BMC Nephrol, 2020, 21(1):406. doi:10.1186/s12882-020-02038-7.
[7]	MORRIS A D, FLOYD L, WOYWODT A, et al. Rituximab in the treatment of primary FSGS:time for its use in routine clinical practice?[J]. Clin Kidney J, 2023, 16(8):1199-1205. doi:10.1093/ckj/sfad122.
[8]	MITCHELL P J, WANG C, TJIAN R. Positive and negative regulation of transcription in vitro:enhancer-binding protein AP-2 is inhibited by SV40 T antigen[J]. Cell, 1987, 50(6):847-861. doi:10.1016/0092-8674(87)90512-5.
[9]	CHAMBERS B E, GERLACH G F, CLARK E G, et al. Tfap2a is a novel gatekeeper of nephron differentiation during kidney development[J]. Development, 2019, 146(13):dev172387. doi:10.1242/dev.172387.
[10]	CHAMBERS B E, CLARK E G, GATZ A E, et al. Kctd15 regulates nephron segment development by repressing Tfap2a activity[J]. Development, 2020, 147(23):dev191973. doi:10.1242/dev.191973.
[11]	MIAO Z, BALZER M S, MA Z, et al. Single cell regulatory landscape of the mouse kidney highlights cellular differentiation programs and disease targets[J]. Nat Commun, 2021, 12(1):2277. doi:10.1038/s41467-021-22266-1.
[12]	MUTO Y, WILSON P C, LEDRU N, et al. Single cell transcriptional and chromatin accessibility profiling redefine cellular heterogeneity in the adult human kidney[J]. Nat Commun, 2021, 12(1):2190. doi:10.1038/s41467-021-22368-w.
[13]	UFFING A, PEREZ-SAEZ M J, MAZZALI M, et al. Recurrence of FSGS after kidney transplantation in adults[J]. Clin J Am Soc Nephrol, 2020, 15(2):247-256. doi:10.2215/CJN.08970719.
[14]	DOCTOR G T, GALE D P, CHAN M M. Genomics in the kidney clinic[J]. Clin Med(Lond), 2023, 23(3):246-249. doi:10.7861/clinmed.2023-RM2.
[15]	MORELLE J, MARECHAL C, YU Z, et al. AQP1 promoter variant,water transport,and outcomes in peritoneal dialysis[J]. N Engl J Med, 2021, 385(17):1570-1580. doi:10.1056/NEJMoa2034279.
[16]	LI P, JIANG M, LI K, et al. Glutathione peroxidase 4-regulated neutrophil ferroptosis induces systemic autoimmunity[J]. Nat Immunol, 2021, 22(9):1107-1117. doi:10.1038/s41590-021-00993-3.
[17]	CHEN X, LIU S, CHEN J, et al. Transcription factor Kruppel-like factor 5 positively regulates the expression of AarF domain containing kinase 4[J]. Mol Biol Rep, 2020, 47(11):8419-8427. doi:10.1007/s11033-020-05882-w.
[18]	HUA P, BADAT M, HANSSEN L L P, et al. Defining genome architecture at base-pair resolution[J]. Nature, 2021, 595(7865):125-129. doi:10.1038/s41586-021-03639-4.
[19]	LI X, QIAN X, WANG B, et al. Programmable base editing of mutated TERT promoter inhibits brain tumour growth[J]. Nat Cell Biol, 2020, 22(3):282-288. doi:10.1038/s41556-020-0471-6.
[20]	MATHARU N, RATTANASOPHA S, TAMURA S, et al. CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency[J]. Science, 2019, 363(6424):eaau0629. doi:10.1126/science.aau0629.