The miRNA-mediated Translational De-suppression in Hypoxic Endothelium

缺氧内皮细胞中 miRNA 介导的翻译去抑制

• 批准号: 8716847
• 负责人: John YJ Shyy
• 金额: $ 22.8万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-21 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8716847
• 关键词: miRNA; mediated; Translational; De; suppression

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are non-coding small RNAs that regulate gene expression at the post- transcriptional level. Ranging from 18 to 24 nt (22 nt in general), miRNAs regulate gene expression by suppressing protein translation of target mRNAs or enhancing their degradation. miRNAs targeting mRNAs depends on the association of the miRNA/mRNA complex with Argonaute (Ago) endonuclease to form the miRNA-induced silencing complex (miRISC). We used sequencing by synthesis (SBS) deep sequencing to study the miRNAs in cultured vascular endothelial cells (ECs) exposed to hypoxia and bioinformatics approaches to analyze the hypoxia-responsive miRNAs at the genome-wide scale. Among miRNAs with greatly changed expression, Let-7s and miR-103/107 target Ago1. This result suggests that a "miRNA- mediated translational de-suppression" mechanism may occur in ECs under hypoxia. With these data acquired from high-throughput screening and in silico approaches, two specific aims are proposed to test the hypothesis that under hypoxia, the increased Let-7s and miR-103/107 down-regulate Ago1. Such a suppression of Ago1 decreases miRISC-mediated miRNA targeting and hence up-regulates targeted mRNAs, which encode highly translated proteins in ECs responding to hypoxia. Specific Aim 1 will study the Ago1- regulated miRNA/mRNA targeting in ECs responding to hypoxia. We will use Ago1 cross-linking immunoprecipitation sequencing (CLIP-seq) to profile the miRISC-associated miRNAs and their mRNA targets in ECs under normoxia and hypoxia. Specific Aim 2 will decipher the functional consequences of the miRNA- mediated translational de-suppression in cultured ECs and mouse hindlimb. Specifically, we will manipulate the expression of Let-7s and miR-103/107 in vitro and in vivo. The miRISC-mediated miRNA/mRNA targeting and mRNA-encoded proteins under normoxia and hypoxia will be examined. The elucidated mechanism will reveal mechanistic insights underlying the miRNA-regulated gene expression in ECs in response to hypoxia.

描述（由申请人提供）：MicroRNA（miRNA）是在转录后水平调节基因表达的非编码小RNA。 miRNA 的长度范围为 18 至 24 nt（一般为 22 nt），通过抑制目标 mRNA 的蛋白质翻译或增强其降解来调节基因表达。靶向 mRNA 的 miRNA 取决于 miRNA/mRNA 复合物与 Argonaute (Ago) 核酸内切酶的结合，形成 miRNA 诱导的沉默复合物 (miRISC)。我们使用边合成边测序 (SBS) 深度测序来研究暴露于缺氧的培养血管内皮细胞 (EC) 中的 miRNA，并使用生物信息学方法在全基因组范围内分析缺氧响应性 miRNA。在表达发生巨大变化的 miRNA 中，Let-7s 和 miR-103/107 靶向 Ago1。这一结果表明缺氧条件下 EC 中可能会发生“miRNA 介导的翻译去抑制”机制。利用从高通量筛选和计算机模拟方法获得的这些数据，提出了两个具体目标来检验以下假设：在缺氧条件下，增加的 Let-7s 和 miR-103/107 下调 Ago1。这种对 Ago1 的抑制会减少 miRISC 介导的 miRNA 靶向，从而上调靶向 mRNA，这些 mRNA 在响应缺氧的 EC 中编码高度翻译的蛋白质。具体目标 1 将研究 Ago1 调节的 miRNA/mRNA 靶向内皮细胞对缺氧的反应。我们将使用 Ago1 交联免疫沉淀测序 (CLIP-seq) 来分析常氧和缺氧下 EC 中 miRISC 相关 miRNA 及其 mRNA 靶标。具体目标 2 将破译 miRNA 介导的翻译去抑制在培养的 EC 和小鼠后肢中的功能后果。具体来说，我们将在体外和体内操纵 Let-7s 和 miR-103/107 的表达。将检查常氧和缺氧下 miRISC 介导的 miRNA/mRNA 靶向和 mRNA 编码蛋白。阐明的机制将揭示 EC 响应缺氧时 miRNA 调节基因表达的机制。