MicroRNA Function in Human Embryonic Stem Cells

MicroRNA 在人类胚胎干细胞中的功能

• 批准号: 7686802
• 负责人: Hannele RUOHOLA-BAKER
• 金额: $ 29.64万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-15 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7686802
• 关键词: Adult; Binding; Biological Assay; Cell physiology; Cells; Dicer Enzyme; Drosophila genus; Functional RNA; Future; Gene Targeting; Genes; Genetic Translation; Goals; Informatics; Mediating; Messenger RNA; MicroRNAs; Mus; Phenotype; Plants; Process; RNA Interference; Regenerative Medicine; Regulation; Research; Role; Stem cells; Testing; Tissues; Transplantation; aptamer; base; embryonic stem cell; gain of function; genome-wide; human DICER1 protein; human embryonic stem cell; loss of function; public health relevance; research study; self-renewal; small molecule; tumor

DESCRIPTION (provided by applicant): The long-term goal of this research is to gain a better understanding and control over human embryonic stem cells (hESCs) by identifying and manipulating the key microRNAs required for hESC self-renewal and differentiation. MicroRNAs, small (21-23nt) non-coding RNA molecules that can regulate mRNA translation and stability by binding to the target mRNAs with incomplete complementarity, are required for normal stem cell function in mouse, Drosophila and plants. We have previously shown that microRNAs are required for germline stem cell (GSC) self-renewing divisions in Drosophila. Our preliminary results suggest that microRNAs are also critical for stem cell self-renewing division and differentiation in human ESCs, and the goal of this proposal is to identify the critical microRNAs and their mRNA targets in these cells. Towards this goal, we will analyze the phenotypes observed in hESCs lacking mature microRNAs due to gene knockdown (KD) of microRNA processing enzymes, Dicer or Drosha. We will identify up-regulated mRNAs in these KD-lines by genome wide mRNA profiling and determine whether they are responsible for the Dicer or Drosha phenotypes through loss-of-function (LOF), gain-of-function (GOF) and rescue experiments (Aim1). To identify the critical microRNAs that mediate this regulation we will carry out genome wide QPCR profiling of hESC microRNAs. The function of the hESC enriched microRNAs will be analyzed by LOF, GOF and rescue experiments (Aim2). The two aims will connect: The critical microRNAs should have the capacity to rescue dicer KD phenotypes (Aim 2.2) and may be sufficient to down-regulate the target genes identified in Aim 1.2 (Aim 2.3). This study should result in findings that allow better manipulation and more efficient differentiation of hESC which is of particular importance for regenerative medicine. PUBLIC HEALTH RELEVANCE: This study will be the first analysis of the functions of mature miRNAs in hESCs. Identification of the miRNA function in hESCs holds the future for better control and manipulation of the hESCs in regenerative medicine. This is important due to the troubling fact that embryonic stem cells tend to form tumors when transplanted into adult tissue. microRNAs carry hope as molecules which can control stem cell self-renewing division.

描述（由申请人提供）：本研究的长期目标是通过识别和操作 hESC 自我更新和分化所需的关键 microRNA，更好地了解和控制人类胚胎干细胞 (hESC)。 MicroRNA 是一种小 (21-23nt) 非编码 RNA 分子，可通过与不完全互补的靶 mRNA 结合来调节 mRNA 翻译和稳定性，是小鼠、果蝇和植物中正常干细胞功能所必需的。我们之前已经表明，microRNA 是果蝇生殖干细胞 (GSC) 自我更新分裂所必需的。我们的初步结果表明，microRNA 对于人类 ESC 中的干细胞自我更新分裂和分化也至关重要，该提案的目标是鉴定这些细胞中的关键 microRNA 及其 mRNA 靶标。为了实现这一目标，我们将分析由于 microRNA 加工酶 Dicer 或 Drosha 的基因敲低 (KD) 而缺乏成熟 microRNA 的 hESC 中观察到的表型。我们将通过全基因组 mRNA 分析来识别这些 KD 系中上调的 mRNA，并通过功能丧失 (LOF)、功能获得 (GOF) 和救援实验 (Aim1) 确定它们是否与 Dicer 或 Drosha 表型有关。为了确定介导这种调节的关键 microRNA，我们将对 hESC microRNA 进行全基因组 QPCR 分析。 hESC 富集的 microRNA 的功能将通过 LOF、GOF 和救援实验进行分析 (Aim2)。这两个目标将相互关联：关键的 microRNA 应具有拯救 dicer KD 表型的能力（目标 2.2），并且可能足以下调目标 1.2 中确定的靶基因（目标 2.3）。这项研究应该得出能够更好地操作和更有效地分化 hESC 的结果，这对于再生医学尤其重要。公共健康相关性：本研究将首次分析 hESC 中成熟 miRNA 的功能。 hESC 中 miRNA 功能的鉴定为再生医学中更好地控制和操作 hESC 奠定了基础。这一点很重要，因为胚胎干细胞在移植到成人组织中时往往会形成肿瘤，这一令人不安的事实。 microRNA作为控制干细胞自我更新分裂的分子带来了希望。