Post-Transcriptional Regulation of Embryo Implantation

胚胎植入的转录后调控

• 批准号: 10682386
• 负责人: Ramakrishna Kommagani
• 金额: $ 41.58万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-11 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10682386
• 关键词: Address; Alternative Splicing; Area; Basic Science; Biochemical; Biological Assay; Biological Factors; Biopsy; Cell Differentiation process; Cell Proliferation; Cells; Chromatin; Conceptions; Data; Decidual Cell; Decidual Cell Reactions; Diagnosis; Embryo; Endometrial; Endometrial Stromal Cell; Endometrium; Epithelium; Estrogens; Genes; Genomics; Gonadal Steroid Hormones; Homologous Gene; Hormones; Human; Impairment; Implant; In Vitro; Knockout Mice; Knowledge; Lead; LoxP-flanked allele; Measures; Mediating; Messenger RNA; Molecular; Monitor; Mus; National Institute of Child Health and Human Development; Pathway interactions; Physiology; Post-Transcriptional Regulation; Pregnancy; Pregnancy loss; Process; Progesterone; Progesterone Receptors; Proliferating; Proteins; RNA Splicing; Recurrence; Reverse Transcriptase Polymerase Chain Reaction; Role; Small Interfering RNA; Spontaneous abortion; Strategic Planning; Stromal Cells; Testing; Transcript; Ubiquitin; Uterus; Variant; Woman; Work; blastocyst; cell type; chromatin immunoprecipitation; conditional knockout; data integration; early pregnancy loss; failure Implantation; human tissue; implantation; in vivo; inhibitor; insight; knock-down; natural Blastocyst Implantation; non-genomic; novel; prevent; response; steroid hormone; transcriptome sequencing; uterine receptivity

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".

项目摘要 成功建立怀孕需要子宫经过几种恰当的细胞变化 允许胚胎植入。因此，即使胚泡正常发展，子宫功能也可以 导致植入失败或早期胚胎流产。子宫子宫内膜准备植入 两个步骤。首先，子宫内膜上皮增殖，失去极性和区分，允许胚胎 连接。其次，下面的基质细胞增殖并分化为candual细胞，使得 植入胚胎。这两个过程通过细胞类型对类固醇的特异性反应进行了协调 激素雌激素和孕酮。但是，我们缺乏下游回应的完整图片 这些激素阻碍了我们制定新策略以防止早期妊娠丧失的能力。解决 该提议的这一知识差距着重于子宫内膜生理，替代mRNA剪接的新领域。 具体而言，该提案将检验中心假设，即剪接因子SF3B1介导孕酮 - 驱动的替代剪接对于子宫的接受和斜视是必不可少的。这个想法建立在 以下初步数据。首先，高通量siRNA屏幕显示SF3B1是 人类子宫内膜基质细胞脱义所必需的。其次，击倒SF3B1体外受损 降解不仅仅是击倒其他八个剪接因子。第三，用SF3B1特异性处理 抑制剂pladienolide B抑制人子宫内膜基质细胞在体外和鼠中抑制 体内子宫内膜分解。第四，用pladienolide b治疗胚胎植入和 小鼠的分解。第五，SF3B1蛋白在子宫内膜基质细胞中升高在植入术中 老鼠。最后，在人工deDializatizatization中，SF3B1蛋白但没有基质细胞中的mRNA升高 小鼠和孕酮稳定了SF3B1蛋白，而不是原发性人子宫内膜基质细胞中的mRNA。 这里提出的工作将基于这些强大的初步数据，并通过追求该假设来检验该假设 以下特定目的：（目标1）定义了SF3B1在子宫接受和斜视中的功能； （目标2） 鉴定子宫内膜中孕激素诱导的SF3B1依赖性替代剪接变体； （目标3） 确定孕酮调节SF3B1的机制。在基础科学层面，这个项目 将确定SF3B1驱动的mRNA剪接的机制，这对于孕酮至关重要 驱动的子宫内膜分解。具有翻译意义，这项工作将确定新颖的成绩单变体 这可能导致复发性妊娠丧失。从长远来看，这些知识可用于开发新的知识 诊断或预防早期妊娠损失的策略。这项工作将有助于推进主题2 NICHD 2020年战略计划，旨在“确定可以导致或有助于早期促成的生物学因素 怀孕丧失”。