转录因子AP2A对肾病相关基因Gas6的调控研究
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南京医科大学第一附属医院

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国家自然科学基金项目(面上项目,重点项目,重大项目)


The regulation of the transcription factor AP-2a to human nephrosis-related gene Gas6
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the First Affiliated Hospital of Nanjing Medical University

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    摘要:

    目的:探究肾脏病相关生长抑制特异性基因6(growth arrest specific 6,Gas6)与肾脏疾病、凋亡的相关关系。研究AP-2a转录因子(transcription factor AP-2 alpha,TFAP2A)对基因Gas6的影响及其机制。方法:构建阿霉素肾病(adriamycinnephropathy,AN)细胞模型,检测该模型中Gas6的表达变化;用Gas6蛋白刺激MPC5细胞,检测肾病相关指标的表达变化。用Gas6蛋白干预HEK-293T细胞,检测细胞凋亡率或凋亡相关指标的表达变化。构建Gas6启动子片段的荧光素酶基因报告重组质粒,检测Gas6基因启动子片段在HEK-293T细胞中的活性,预测Gas6启动子区的功能性转录因子结合位点并验证,在HEK-293T细胞中敲低或过表达TFAP2A后检测Gas6启动子片段活性、mRNA表达、蛋白表达的变化。结果: Gas6在阿霉素肾病细胞模型中高表达,且过量的Gas6可诱导MPC5细胞中肾病相关指标Nephrin和Podocin与阿霉素肾病细胞模型趋势相同。与对照组相比,Gas6蛋白组的细胞凋亡明显减少。成功构建有活性的Gas6基因启动子片段荧光素酶基因报告重组质粒,且在此片段内含有TFAP2A的结合位点。TFAP2A可在启动子、mRNA及蛋白水平对Gas6正向调控。结论:Gas6对肾病综合征的发生有促进作用。Gas6在HEK-293T细胞中抗凋亡。TFAP2A对Gas6正向转录调控。

    Abstract:

    Objective: To explore the correlation between nephrosis-related growth arrest specific 6(Gas6) and nephrotic syndrome and apoptosis. The effect of AP-2 transcription factor a(TFAP2A) on Gas6 was investigated. Methods:The cell model of adriamycinnephropathy(AN) was constructed to detect the expression of Gas6, and MPC5 cells were stimulated with Gas6 protein to measure the expression of nephropathy related indicators. HEK-293T cells were intervened with Gas6 protein to measure apoptosis rate or the expression of apoptosis-related indicators. The luciferase gene reporter recombinant plasmid of Gas6 promoter was constructed to measure the activity of Gas6 promoter in HEK-293T cells. And the potential transcriptional binding sites were predicted and verified. The effects of knockdown or overexpression of TFAP2A on Gas6 gene expression were measured at the promoter, mRNA, and protein levels. Results: Gas6 was highly expressed in AN cell model, and excessive Gas6 may induce the nephropathy-related indicators in MPC5 cells the same trend as that of AN cell model. Apoptosis was significantly reduced in the Gas6 protein group compared to control in HEK-293T cells.The active luciferase reporter plasmid of human Gas6 promoter fragment was successfully constructed, and the binding sites of TFAP2A were included. TFAP2A positively regulates Gas6 at the promoter, mRNA, and protein levels. Conclusion: Gas6 promotes the development of nephrotic syndrome. Gas6 is antiapoptotic in HEK-293T cells. TFAP2A positively promotes the expression of human nephrosis-related gene Gas6.

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  • 收稿日期:2022-09-28
  • 最后修改日期:2022-11-01
  • 录用日期:2023-08-09
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