The miRNA-mediated Translational De-suppression in Hypoxic Endothelium

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。范围为18 - 24 nt（22 nt在 一般而言，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）深度测序来研究缺氧条件下培养的血管内皮细胞（EC）中的miRNA。然后使用生物信息学方法在全基因组范围内分析缺氧反应miRNAs。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，“miRNA介导的翻译去抑制”机制可能发生在缺氧条件下的EC中。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，包括血管内皮生长因子。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miRISC相关的miRNA及其mRNA靶点。特异性目的2将解释在培养的EC和小鼠后肢中miRNA介导的翻译去抑制的功能后果。具体而言，我们将在体外和体内操纵Let-7s和miR-103/107的表达。将检查常氧和缺氧条件下miRISC介导的miRNA/mRNA靶向、mRNA编码蛋白和血管生成。阐明的机制将揭示微RNA调控的基因表达在EC响应缺氧的机制的见解。

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. Bioinformatics approaches were then used 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 an "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, including vascular endothelial growth factor. 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, mRNA-encoded proteins, and angiogenesis 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.