生物钟核受体NR1D1调控子宫内膜基质细胞蜕膜化过程的作用机制

Nuclear receptor NR1D1 is an important component of circadian clock system. Female mice with the deletion of NR1D1 exhibit the phenotype of infertility. In addition, the decidualization of uterus endometrial stromal cells is a key step to embryo implantation. Previous studies showed that NR1D1 is involved in the regulation of the decidualization of endometrial stromal cells, but the specific mechanism remains unclear. Here, we hypothesize that the circadian clock gene Nr1d1 controls the decidualization of endometrial stromal cells by regulating Clock-controlled genes (Ccgs). Endometrial stromal cells are isolated and cultured in vitro from pregnant 4.5 d and 6.5 d Per2-dLuc transgenic mice and WT mice. The cells will be further treated by agonist/antagonist of NR1D1 or CRISPR/Cas9 gene editing. Screening the downstream target genes of NR1D1 and investigating the roles of NR1D1 will be carried out by using RNA sequencing, qPCR, WB, and IHC technologies. Furthermore, the analysis of promoter activity and ChIP will be adopted to determine the Ccgs of NR1D1 regulating decidualization of endometrial stromal cells. In conclusion, the project implementation will uncover a new mechanism of NR1D1 regulating the decidualization at the molecular level, providing us the theoretical basis and experimental theory of searching a new target for the treatment of infertility and abortion from the point of circadian clock.

核受体NR1D1是生物钟系统的重要组成部分，其基因缺失可导致雌性小鼠不孕。子宫内膜蜕膜化是胚胎植入的关键，有证据表明NR1D1参与调控子宫内膜基质细胞蜕膜化的生理过程，但其具体作用机制尚不清楚。本项目以“生物钟核受体NR1D1通过下游钟控基因调控子宫内膜基质细胞蜕膜化”为理论假说，拟采用WT小鼠和Per2-dLuc转基因小鼠模型，分离培养妊娠4.5d和6.5d小鼠子宫内膜基质细胞并结合NR1D1激动剂、拮抗剂处理和CRISPR/Cas9基因编辑，利用RNA测序、qPCR、WB、IHC等技术明确NR1D1对子宫内膜基质细胞蜕膜化的影响并筛选NR1D1下游钟控基因，进一步通过启动子活性分析、ChIP方法确定NR1D1调控与子宫内膜基质细胞蜕膜化相关的钟控基因，从分子水平揭示NR1D1调控子宫内膜基质细胞蜕膜化过程的作用机制，为从生物钟角度寻找治疗不孕和流产等疾病的新靶点提供理论基础和实验依据。

生物钟是生物体为适应光照、温度等环境因子昼夜节律性变化而逐渐形成的周期性内源计时系统。生物钟作为生命体中一种重要的调控系统，对机体的分子、细胞、生理、生化等各层次的生理功能均起到重要的调节作用。已经有研究表明在大鼠、小鼠以及人类的子宫内膜基质细胞和睾丸间质细胞中，生物钟基因的表达均存在24 h节律性，但其具体作用尚未完全揭示清楚。我们通过体外和体内实验，利用RT-qPCR、WB、ELISA、双荧光素酶报告以及EMSA等实验技术揭示了玉米赤霉烯酮和草甘膦通过抑制生物钟系统影响小鼠睾丸间质细胞睾酮分泌降低的分子机制；此外，本研究揭示了小鼠子宫内膜基质细胞在妊娠第4天时生物钟调控前列腺素E2合成的分子机制。.本研究的主要结论：.1. 赤霉烯酮扰乱小鼠睾丸间质细胞的生物钟并抑制睾酮合成；.2. 雌二醇分泌增加可通过Bmal1上调Ptgs2的转录，最终促进妊娠4天小鼠前列腺素E2的合成；.3. 草甘膦暴露可通过上调NR1D1抑制小鼠睾丸间质细胞StAR的表达来降低睾酮合成。