雄激素通过雄激素受体途径调控小鼠附睾Fkbp5基因的表达
发布时间：2018-07-24 21:15
【摘要】：目的初步研究小鼠附睾Fkbp5(FK506 binding protein 5)基因对雄激素的响应性以及相关的分子机制。方法建立小鼠去势以及雄激素补充模型。采用RT-PCR以及RT-qPCR,检测正常小鼠、去势小鼠以及去势后补充外源雄激素的小鼠附睾中Fkbp5基因的mRNA表达水平。搜索染色体免疫共沉淀-测序(chromatin immunoprecipitation-sequencing,ChIP-seq)数据建立的小鼠附睾全基因组雄激素受体(androgen receptor,AR)结合位点的数据库,寻找与Fkbp5基因相关联的AR结合位点。利用AR抗体进行染色体免疫共沉淀(chromatin immunoprecipitation,ChIP),采用ChIP-PCR以及ChIP-qPCR的方法,检测Fkbp5基因相关联的AR结合位点在体内与AR的结合情况。结果去势后,Fkbp5基因的表达显著降低(P0.05);补加雄激素后,Fkbp5基因的表达又升至正常水平(P0.05)。从小鼠附睾AR结合位点的数据库中鉴定到14个Fkbp5相关联的AR结合位点。ChIP-PCR结果显示,在正常生理状态下,这14个结合位点均与AR结合。ChIP-qPCR结果显示,去势后,这14个AR结合位点的富集倍数均显著下降(P0.05);而补加雄激素后,这些AR结合位点的富集倍数又显著上升至生理水平(P0.05)。结论 Fkbp5在小鼠附睾内的表达受雄激素的正调控,并且是AR的一个直接靶基因。该研究首次证实了Fkbp5的启动子以及上游增强子区域存在雄激素响应元件,为探究雄激素/AR对Fkbp5的调控机制提供了新的视角。
[Abstract]:Objective to study the response of mouse epididymal Fkbp5 (FK506 binding protein 5) gene to androgen and its molecular mechanism. Methods A model of castration and androgen supplementation in mice was established. RT-PCR and RT-qPCR were used to detect the mRNA expression of Fkbp5 gene in epididymis of normal mice, ovariectomized mice and ovariectomized mice supplemented with exogenous androgen. To search the database of androgen receptor AR binding site in mouse epididymis genome based on chromatin immunoprecipitation-sequencing-sequenced data, and to search for the AR binding site associated with Fkbp5 gene. Chromatin immunoprecipitation chip (chip) was carried out with AR antibody. The AR binding sites associated with Fkbp5 gene were detected by ChIP-PCR and ChIP-qPCR in vivo. Results after castration, the expression of Fkbp5 gene decreased significantly (P0.05), and the expression of Fkbp5 gene increased to normal level after androgen supplementation (P0.05). Fourteen AR binding sites associated with Fkbp5 were identified from the mouse epididymal AR binding site database. ChIP-PCR results showed that under normal physiological conditions, these 14 binding sites were all associated with AR. ChIP-qPCR results showed that, after castration, all the 14 binding sites were associated with AR. The enrichment ratio of these 14 AR binding sites decreased significantly (P0.05), and the enrichment multiple of these AR binding sites increased significantly to physiological level after androgen supplementation (P0.05). Conclusion the expression of Fkbp5 in mouse epididymis is positively regulated by androgen and is a direct target gene of AR. This study confirmed for the first time the existence of androgen response elements in the promoter and upstream enhancer of Fkbp5, which provided a new perspective for exploring the regulation mechanism of androgen / AR on Fkbp5.
【作者单位】： 上海大学生命科学学院;上海交通大学医学院附属仁济医院生殖医学科;上海市辅助生殖与优生重点实验室;
【基金】：国家自然科学基金(81571435,81200468)资助
【分类号】：Q492
[Abstract]:Objective to study the response of mouse epididymal Fkbp5 (FK506 binding protein 5) gene to androgen and its molecular mechanism. Methods A model of castration and androgen supplementation in mice was established. RT-PCR and RT-qPCR were used to detect the mRNA expression of Fkbp5 gene in epididymis of normal mice, ovariectomized mice and ovariectomized mice supplemented with exogenous androgen. To search the database of androgen receptor AR binding site in mouse epididymis genome based on chromatin immunoprecipitation-sequencing-sequenced data, and to search for the AR binding site associated with Fkbp5 gene. Chromatin immunoprecipitation chip (chip) was carried out with AR antibody. The AR binding sites associated with Fkbp5 gene were detected by ChIP-PCR and ChIP-qPCR in vivo. Results after castration, the expression of Fkbp5 gene decreased significantly (P0.05), and the expression of Fkbp5 gene increased to normal level after androgen supplementation (P0.05). Fourteen AR binding sites associated with Fkbp5 were identified from the mouse epididymal AR binding site database. ChIP-PCR results showed that under normal physiological conditions, these 14 binding sites were all associated with AR. ChIP-qPCR results showed that, after castration, all the 14 binding sites were associated with AR. The enrichment ratio of these 14 AR binding sites decreased significantly (P0.05), and the enrichment multiple of these AR binding sites increased significantly to physiological level after androgen supplementation (P0.05). Conclusion the expression of Fkbp5 in mouse epididymis is positively regulated by androgen and is a direct target gene of AR. This study confirmed for the first time the existence of androgen response elements in the promoter and upstream enhancer of Fkbp5, which provided a new perspective for exploring the regulation mechanism of androgen / AR on Fkbp5.
【作者单位】： 上海大学生命科学学院;上海交通大学医学院附属仁济医院生殖医学科;上海市辅助生殖与优生重点实验室;
【基金】：国家自然科学基金(81571435,81200468)资助
【分类号】：Q492
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