Post-Transcriptional Regulation of Embryo Implantation

项目摘要 成功建立妊娠需要子宫经历几次适时的细胞变化， 让胚胎着床。因此，即使胚泡发育正常， 导致着床失败或早期胚胎流产。子宫内膜为着床做好准备， 两步首先，子宫内膜上皮增生，失去极性，并分化，使胚胎 连接。其次，底层基质细胞增殖并分化为蜕膜细胞， 胚胎植入这两个过程是协调细胞类型的具体反应类固醇 雌激素和孕激素然而，我们缺乏对下游反应的完整了解， 这些激素，阻碍了我们开发新策略以防止早孕丢失的能力。解决 这一知识空白，这一建议集中在子宫内膜生理学的一个新领域，选择性mRNA剪接。 具体来说，这项提议将检验剪接因子SF 3B 1介导孕酮的中心假设。 驱动的选择性剪接，对子宫容受性和蜕膜化至关重要。这个想法是建立在 以下是初步数据。首先，高通量siRNA筛选显示SF 3B 1是一种特异性的表达载体。 人类子宫内膜间质细胞脱落所需的。第二，SF 3B 1的敲低在体外受损 蜕膜化超过敲除其他八个剪接因子。第三，SF 3B 1特异性治疗 抑制剂普拉地辛B抑制人子宫内膜间质细胞蜕膜化 体内子宫内膜蜕膜化。第四，用普拉地仑B治疗损害胚胎着床， 小鼠的蜕膜化。第五，SF 3B 1蛋白在着床期子宫内膜间质细胞中升高， 小鼠最后，在人工蜕膜化过程中，间质细胞中SF 3B 1蛋白而非mRNA表达升高， 孕激素稳定SF 3B 1蛋白，但不是mRNA在原代人子宫内膜间质细胞。 这里提出的工作将建立在这些强有力的初步数据的基础上，并通过追求 以下具体目的：（目的1）确定SF 3B 1在子宫容受性和蜕膜化中的功能;（目的2） 鉴定子宫内膜中孕酮诱导的SF 3B 1依赖性选择性剪接变异体;（目的3） 确定孕酮调节SF 3B 1的机制。在基础科学的层面上，这个项目 将确定SF 3B 1驱动的mRNA剪接的机制，这对孕酮至关重要， 驱动子宫内膜蜕膜化。翻译的意义，这项工作将确定新的转录变体 这可能会导致反复流产。从长远来看，这些知识可以用来开发新的 诊断或预防早孕流产的策略。这项工作将有助于推动《千年宣言》的主题2。 NICHD 2020战略计划，旨在“确定可能导致或有助于早期 妊娠丢失”。

PROJECT SUMMARY Successful establishment of pregnancy requires the uterus to undergo several well-timed cellular changes to allow the embryo to implant. Thus, even when the blastocyst develops normally, impaired uterine function can lead to implantation failure or early embryo miscarriage. The uterine endometrium prepares for implantation in two steps. First, the endometrial epithelium proliferates, loses polarity, and differentiates, allowing the embryo to attach. Second, the underlying stromal cells proliferate and differentiate into decidual cells, allowing the embryo to implant. These two processes are coordinated by cell-type specific responses to the steroid hormones estrogen and progesterone. However, we lack a complete picture of the downstream responses to these hormones, hampering our ability to develop new strategies to prevent early pregnancy loss. To address this knowledge gap, this proposal focuses on a new area in endometrial physiology, alternative mRNA splicing. Specifically, this proposal will test the central hypothesis that the splicing factor SF3B1 mediates progesterone- driven alternative splicing that is essential for uterine receptivity and decidualization. This idea is founded on the following pieces of preliminary data. First, a high-throughput siRNA screen revealed that SF3B1 was required for human endometrial stromal cell decidualization. Second, knock down of SF3B1 impaired in vitro decidualization more than knock down of eight other splicing factors. Third, treatment with the SF3B1-specific inhibitor Pladienolide B inhibited human endometrial stromal cell decidualization in vitro and murine endometrial decidualization in vivo. Fourth, treatment with Pladienolide B impaired embryo implantation and decidualization in mice. Fifth, SF3B1 protein is elevated in endometrial stromal cells during peri-implantation in mice. Finally, SF3B1 protein but not mRNA in stromal cells was elevated during artificial decidualization in mice, and progesterone stabilized SF3B1 protein but not mRNA in primary human endometrial stromal cells. The work proposed here will build on these strong preliminary data and test the hypothesis by pursuing the following specific aims: (Aim 1) Define the functions of SF3B1 in uterine receptivity and decidualization; (Aim 2) Identify progesterone-induced, SF3B1-dependent alternative splice variants in the endometrium; (Aim 3) Determine the mechanism by which progesterone regulates SF3B1. At the level of basic science, this project will identify the mechanisms that underlie SF3B1-driven mRNA splicing, which is crucial for progesterone- driven endometrial decidualization. Of translational significance, this work will identify novel transcript variants that may contribute to recurrent pregnancy loss. In the long term, such knowledge can be used to develop new strategies to diagnose or prevent early pregnancy loss. Together, this work will help advance Theme 2 of the NICHD 2020 Strategic Plan, which aims to "identify biological factors that can lead or contribute to early pregnancy loss".