The miRNA-mediated Translational De-suppression in Hypoxic Endothelium

描述(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。MiRNAs的范围从18到24个核苷酸(一般为22个核苷酸)，通过抑制靶基因的蛋白质翻译或促进其降解来调节基因的表达。针对mRNAs的miRNAs依赖于miRNA/mRNA复合体与ArgAerte(AGO)内切酶的结合而形成miRNA诱导沉默复合体(MiRISC)。我们利用合成测序法(SBS)和生物信息学方法对缺氧培养的血管内皮细胞(ECs)中的miRNAs进行了研究，并在全基因组水平上分析了缺氧反应的miRNAs。在表达变化较大的miRNA中，let-7S和miR-103/107以Ago1为靶点。这一结果表明，在低氧条件下，内皮细胞可能发生了“miRNA介导的翻译去抑制”机制。根据从高通量筛选和电子计算机方法获得的这些数据，我们提出了两个特定的目标来检验在低氧条件下，增加的let-7s和miR-103/107下调Ago1的假设。这种对Ago1的抑制减少了miRISC介导的miRNA靶向，从而上调了靶向mRNAs，后者编码响应低氧的内皮细胞中的高翻译蛋白。特异性目标1将研究Ago1调控的内皮细胞对低氧反应的miRNA/mRNA靶向。我们将使用Ago1交联免疫沉淀测序(CLIP-SEQ)来分析常氧和低氧条件下内皮细胞中miRISC相关的miRNAs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内皮细胞和小鼠后肢中的功能后果。具体地说，我们将在体外和体内操纵let-7s和miR-103/107的表达。将检测在常氧和低氧条件下miRISC介导的miRNA/mRNA靶向和mRNA编码的蛋白质。阐明的机制将揭示miRNA调控内皮细胞对低氧反应的基因表达的机制。

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.