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）：微小RNA（miRNA）是在转录后水平调节基因表达的非编码小RNA。范围从18到24 nt（一般为22 nt），miRNA通过抑制靶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依赖于miRNA/mRNA复合物与Argonaute（Ago）核酸内切酶的结合以形成miRNA诱导的沉默复合物（miRISC）。我们使用合成测序（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来分析全基因组范围内的缺氧响应性miRNA。在表达变化较大的miRNAs中，Let-7s和miR-103/107靶向Ago 1。这一结果表明，在缺氧条件下，内皮细胞中可能发生了“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，提出了两个具体的目的来检验在缺氧下，增加的Let-7s和miR-103/107下调Ago 1的假设。Ago 1的这种抑制降低了miRISC介导的miRNA靶向，因此上调了靶向mRNA，其编码响应缺氧的EC中的高度翻译的蛋白质。具体目标1将研究Ago 1调节的miRNA/mRNA靶向在缺氧应答的EC中的作用。我们将使用Ago 1交联免疫沉淀测序（CLIP-seq）来分析常氧和缺氧条件下EC中的miRISC相关miRNA及其mRNA靶点。特异性目的2将解释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。具体而言，我们将在体外和体内操纵Let-7s和miR-103/107的表达。将在常氧和缺氧条件下检查miRISC介导的miRNA/mRNA靶向和mRNA编码的蛋白。阐明的机制将揭示内皮细胞响应缺氧时miRNA调节基因表达的机制见解。