Small RNAs in skeletogenesis

骨骼发生中的小RNA

• 批准号: 7187622
• 负责人: TATSUYA KOBAYASHI
• 金额: $ 22.1万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7187622
• 关键词: Binding; Biochemical; Biological Process; Bone Development; Cartilage; Cell Proliferation; Cells; Chondrocytes; Chromatin; Class; Condition; Data; Databases; Development; Disease; Disruption; Endoribonucleases; Exons; Family; Functional RNA; Gene Expression; Gene Expression Regulation; Gene Targeting; Generations; Genes; Genetic Recombination; Goals; In Situ Hybridization; Informatics; Knowledge; Length; Mammalian Cell; Mammals; Methods; MicroRNAs; Mus; Mutant Strains Mice; Nucleotides; Organism; Osteogenesis; Pancreatic ribonuclease; Pattern; Physiological; Plants; Play; Polymerase Chain Reaction; Population; Regulation; Ribonuclease III; Ribonucleases; Role; Screening procedure; Skeletal Development; Skeletal system; Small Interfering RNA; Small RNA; Staging; System; Tissues; Transgenic Mice; Translating; Validation; Zebrafish; base; cell type; fetal; fly; gene cloning; gene therapy; human DICER1 protein; in vivo; mouse genome; novel therapeutics; postnatal; recombinase; research study; therapeutic target

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.

描述（由申请人提供）：小RNA（sRNA）构成了一个长达30个核苷酸的调节性非编码RNA家族。两大类内源性小RNA，microRNA（miRNA）和内源性小干扰RNA（siRNA）分别通过结合靶mRNA和引导染色质沉默机制来负调控基因表达。这些sRNA已被证明在各种生物体如植物、蠕虫、苍蝇和斑马鱼的多种生物过程中发挥关键作用。然而，尽管存在大量的miRNAs，关于sRNAs在哺乳动物中的作用的知识是有限的，更不用说在哺乳动物骨骼组织中了。本项目的目标是首先确定sRNA是否在体内骨骼组织中发挥重要的生理作用，其次确定sRNA在骨骼组织中的表达模式和水平。在目的I中，我们将使用Cre-/oxP重组系统在小鼠软骨中删除编码Dicer的基因，Dicer是产生成熟miRNA和siRNA所必需的RNA酶。将分析缺失Dicer的小鼠的骨骼异常以确定miRNA和siRNA缺失的影响。该实验应建立Dicer依赖性sRNA在骨骼发育过程中的生理作用。在目标II中，我们将分析miRNAs的表达模式和水平，miRNAs是哺乳动物中sRNAs的主要群体。我们将使用miRNA阵列分析进行筛选，定量PCR和原位杂交用于验证和进一步表征从筛选中选择的每个miRNA。Aim II的实验将为软骨中miRNA表达提供一个基线数据库，这将有助于形成关于特定miRNA及其靶基因在骨骼发育中作用的假设。了解sRNA在骨骼组织中的作用不仅可以揭示基因表达的另一个调节水平，还可以为疾病条件下的遗传干预提供新的治疗靶点。