Small RNAs in skeletogenesis

描述（由申请人提供）：小rna （sRNAs）构成了一个长度为30个核苷酸的调节非编码rna家族。两大类内源性小rna， microRNAs （miRNAs）和内源性小干扰rna （sirna）分别通过结合靶mrna和指导染色质沉默机制负性调节基因表达。这些sRNAs已被证明在各种生物（如植物、蠕虫、苍蝇和斑马鱼）的各种生物过程中发挥关键作用。然而，尽管存在大量的mirna，关于srna在哺乳动物中的作用的知识，更不用说在哺乳动物骨骼组织中的作用，是有限的。本项目的目标是首先确定sRNAs是否在体内骨组织中发挥重要的生理作用，其次确定sRNAs在骨组织中的表达模式和水平。在Aim I中，我们将使用Cre-/oxP重组系统删除小鼠软骨中编码Dicer的基因，Dicer是生成成熟miRNA和siRNA所必需的rna酶。将分析缺失Dicer的小鼠骨骼异常，以确定mirna和sirna缺失的影响。本实验旨在确定dicer依赖性sRNAs在骨骼发育过程中的生理作用。在Aim II中，我们将分析哺乳动物中主要的sRNAs群体mirna的表达模式和水平。我们将使用miRNA阵列分析进行筛选，定量PCR和原位杂交对筛选中选择的每个miRNA进行验证和进一步表征。Aim II的实验应提供软骨中miRNA表达的基线数据库，这将有助于形成关于特定miRNA及其靶基因在骨骼发育中的作用的假设。了解sRNAs在骨骼组织中的作用不仅可以揭示基因表达调控的另一个层面，而且可能为疾病的遗传干预提供新的治疗靶点。

DESCRIPTION (provided by applicant): Small RNAs (sRNAs) constitute a family of regulatory non-coding RNAs of up to 30 nucleotides in length. Two major classes of endogenous small RNAs, microRNAs (miRNAs) and endogenously occurring small interfering RNAs (siRNAs) negatively regulate gene expression by binding target mRNAs and by guiding chromatin silencing machineries, respectively. These sRNAs have been demonstrated to play critical roles in diverse biological processes in various organisms such as plants, worms, flies, and zebrafish. However, despite the abundant presence of miRNAs, knowledge about roles of sRNAs in mammals, let alone in mammalian skeletal tissues, is limited. The goals of this proposed project are first, to determine whether sRNAs play physiologically important roles in skeletal tissues in vivo, and second, to determine expression patterns and levels of sRNAs in skeletal tissues. In Aim I, we will delete the gene encoding Dicer, an RNase essential for generation of mature miRNA and siRNA, in mouse cartilage using the Cre-/oxP recombination system. Skeletal abnormalities of mice missing Dicer will be analyzed to define the effect of loss of miRNAs and siRNAs. This experiment should establish physiological roles of Dicer-dependent sRNAs during skeletal development. In Aim II, we will analyze expression patterns and levels of miRNAs, the predominant population of sRNAs in mammals. We will use miRNA array analysis for screening, quantitative PCR and in situ hybridization for validation and further characterization of each miRNA selected from the screening. Experiments in Aim II should provide a baseline database for miRNA expression in cartilage, which would help to form hypotheses about roles of specific miRNAs and their target genes in skeletal development. Understanding roles of sRNAs in skeletal tissues would not only reveal another level of regulation of gene expression, but also may provide novel therapeutic targets for genetic intervention in disease conditions.