雄激素通過雄激素受體途徑調(diào)控小鼠附睪Fkbp5基因的表達
發(fā)布時間：2018-07-24 21:15
【摘要】：目的初步研究小鼠附睪Fkbp5(FK506 binding protein 5)基因?qū)π奂に氐捻憫砸约跋嚓P的分子機制。方法建立小鼠去勢以及雄激素補充模型。采用RT-PCR以及RT-qPCR,檢測正常小鼠、去勢小鼠以及去勢后補充外源雄激素的小鼠附睪中Fkbp5基因的mRNA表達水平。搜索染色體免疫共沉淀-測序(chromatin immunoprecipitation-sequencing,ChIP-seq)數(shù)據(jù)建立的小鼠附睪全基因組雄激素受體(androgen receptor,AR)結合位點的數(shù)據(jù)庫,尋找與Fkbp5基因相關聯(lián)的AR結合位點。利用AR抗體進行染色體免疫共沉淀(chromatin immunoprecipitation,ChIP),采用ChIP-PCR以及ChIP-qPCR的方法,檢測Fkbp5基因相關聯(lián)的AR結合位點在體內(nèi)與AR的結合情況。結果去勢后,Fkbp5基因的表達顯著降低(P0.05);補加雄激素后,Fkbp5基因的表達又升至正常水平(P0.05)。從小鼠附睪AR結合位點的數(shù)據(jù)庫中鑒定到14個Fkbp5相關聯(lián)的AR結合位點。ChIP-PCR結果顯示,在正常生理狀態(tài)下,這14個結合位點均與AR結合。ChIP-qPCR結果顯示,去勢后,這14個AR結合位點的富集倍數(shù)均顯著下降(P0.05);而補加雄激素后,這些AR結合位點的富集倍數(shù)又顯著上升至生理水平(P0.05)。結論 Fkbp5在小鼠附睪內(nèi)的表達受雄激素的正調(diào)控,并且是AR的一個直接靶基因。該研究首次證實了Fkbp5的啟動子以及上游增強子區(qū)域存在雄激素響應元件,為探究雄激素/AR對Fkbp5的調(diào)控機制提供了新的視角。
[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.
【作者單位】： 上海大學生命科學學院;上海交通大學醫(yī)學院附屬仁濟醫(yī)院生殖醫(yī)學科;上海市輔助生殖與優(yōu)生重點實驗室;
【基金】：國家自然科學基金(81571435,81200468)資助
【分類號】：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.
【作者單位】： 上海大學生命科學學院;上海交通大學醫(yī)學院附屬仁濟醫(yī)院生殖醫(yī)學科;上海市輔助生殖與優(yōu)生重點實驗室;
【基金】：國家自然科學基金(81571435,81200468)資助
【分類號】：Q492
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8 Santen R ,陸仁康 ,王t，
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