Post-Transcriptional Regulation of Embryo Implantation

胚胎植入的转录后调控

基本信息

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
项目类别：
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

项目摘要

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".

项目概要成功怀孕需要子宫经历几次适时的细胞变化让胚胎着床。因此，即使囊胚发育正常，子宫功能也会受损导致着床失败或早期胚胎流产。子宫内膜为植入做好准备两步。首先，子宫内膜上皮增殖、失去极性并分化，使胚胎附加。其次，底层基质细胞增殖并分化为蜕膜细胞，从而允许胚胎植入。这两个过程由细胞类型对类固醇的特异性反应来协调激素雌激素和黄体酮。然而，我们缺乏下游反应的完整情况这些激素阻碍了我们制定新策略来预防早孕流产的能力。致地址为了弥补这一知识差距，本提案重点关注子宫内膜生理学的一个新领域，即选择性 mRNA 剪接。具体来说，该提案将检验剪接因子 SF3B1 介导黄体酮的中心假设—— 驱动的选择性剪接对于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 SF3B1 人子宫内膜基质细胞蜕膜化所需的。二、体外敲低SF3B1受损蜕膜化不仅仅是敲除其他八个剪接因子。三、用SF3B1特异性治疗抑制剂 Pladienolide B 在体外和小鼠体内抑制人子宫内膜基质细胞蜕膜化体内子宫内膜蜕膜化。第四，用 Pladienolide B 治疗受损的胚胎植入和小鼠的蜕膜化。第五，子宫内膜基质细胞中的 SF3B1 蛋白在围植入期间升高。老鼠。最后，在人工蜕膜化过程中，基质细胞中的 SF3B1 蛋白升高，但 mRNA 没有升高。小鼠中，黄体酮可以稳定 SF3B1 蛋白，但不能稳定原代人子宫内膜基质细胞中的 mRNA。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，并通过追求以下内容来检验假设：具体目标如下：（目标1）明确SF3B1在子宫容受性和蜕膜化中的功能；（目标2）鉴定子宫内膜中黄体酮诱导的、SF3B1 依赖性的选择性剪接变异体；（目标 3）确定黄体酮调节 SF3B1 的机制。在基础科学层面，本项目将确定 SF3B1 驱动的 mRNA 剪接的机制，这对于黄体酮至关重要带动子宫内膜蜕膜化。具有翻译意义，这项工作将鉴定新的转录本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期妊娠流产的策略。总之，这项工作将有助于推进主题 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”。