The miRNA-mediated Translational De-suppression in Hypoxic Endothelium

描述（由申请人提供）：MicroRNAs （miRNAs）是在转录后水平调节基因表达的非编码小rna。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）内切酶的结合，形成miRNA诱导的沉默复合物（miRISC）。我们利用合成测序（SBS）深度测序研究了缺氧条件下培养血管内皮细胞（ECs）中的mirna，并利用生物信息学方法分析了全基因组水平上的低氧反应mirna。在表达变化较大的mirna中，Let-7s和miR-103/107靶向Ago1。这一结果提示缺氧条件下ECs可能存在“miRNA介导的翻译去抑制”机制。通过高通量筛选和计算机方法获得的这些数据，我们提出了两个特定的目的来验证在缺氧条件下，增加的Let-7s和miR-103/107下调Ago1的假设。这种对Ago1的抑制降低了miriscc介导的miRNA靶向，从而上调了靶向mrna，这些mrna在ec中编码高翻译蛋白，以应对缺氧。Specific Aim 1将研究Ago1调控的miRNA/mRNA在ECs缺氧反应中的靶向作用。我们将使用Ago1交联免疫沉淀测序（CLIP-seq）来分析常氧和缺氧条件下ECs中miriscc相关的mirna及其mRNA靶点。特异性Aim 2将在培养的内皮细胞和小鼠后肢中破译miRNA介导的翻译去抑制的功能后果。具体来说，我们将在体外和体内操纵Let-7s和miR-103/107的表达。在常氧和缺氧条件下，我们将研究miriscc介导的miRNA/mRNA靶向和mRNA编码蛋白。阐明的机制将揭示mirna调控的基因表达在ECs中响应缺氧的机制见解。

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 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.