Role of microRNAs in skeletal development

microRNA 在骨骼发育中的作用

• 批准号: 8466927
• 负责人: TATSUYA KOBAYASHI
• 金额: $ 35.86万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8466927
• 关键词: Affect; Animals; Antibodies; Base Pairing; Binding; Biogenesis; Biological Assay; Cartilage; Cell Cycle Deregulation; Cell Cycle Progression; Cell Differentiation process; Cell Extracts; Cell Proliferation; Cell Proliferation Regulation; Chondrocytes; Complex; Defect; Digoxigenin; Epiphysial cartilage; Flow Cytometry; Functional RNA; Gene Expression; Gene Expression Regulation; Genes; Goals; Human; Immunoprecipitation; In Vitro; Incubated; Intervention; Knockout Mice; Knowledge; Label; Lead; Life; Malignant Neoplasms; Mental disorders; MicroRNAs; Microprocessor; Molecular; Mus; Nature; Organism; Pathology; Phenotype; Physiological; Physiology; Play; Process; RNA; RNA Processing; RNA-Induced Silencing Complex; Regulation; Research; Role; Skeletal Development; Skeletal system; System; Therapeutic Intervention; Transcript; Vertebrates; base; gene discovery; human DICER1 protein; human disease; new therapeutic target; public health relevance; reconstitution; skeletal disorder; skeletal tissue; skeletogenesis

DESCRIPTION (provided by applicant): Small non-coding microRNAs (miRNAs) regulate gene expression mainly at the posttranscriptional level. miRNAs play fundamentally important roles in various types of living organisms. Defects in miRNA-dependent gene regulation have been implicated in human diseases such as cancer and mental disorders. However, knowledge about functions of miRNAs in the mammalian skeletal system is limited. The goal of this research is to establish the roles of miRNAs during skeletal development in mice. miRNAs are generated through multiple processing steps; primary transcripts of miRNAs are first processed by the microprocessor containing Drosha and DGCR8 as critical components, and then by Dicer. Mature miRNAs are then loaded onto the RNA-induced silencing complex (RISC), that contains Argonaute (Ago) as the core component, and bind to their target transcripts through partial base-pairing. Our findings in Dicer- deficient mouse growth plates lead to our central hypothesis that chondrocytic miRNAs regulate cell proliferation and differentiation. However, because Dicer is known to process RNA species other than miRNAs, it is not clear to what extent the loss of miRNAs contribute to the phenotype of Dicer-deficient chondrocytes. In Aim I, we will confirm the role of miRNAs in regulation of chondrocyte proliferation and differentiation by generating and analyzing conditional knockout mice missing DGCR8 and Drosha to eliminate miRNAs independently of Dicer. In Aim II, to understand the mechanisms by which miRNAs regulate chondrocyte proliferation and differentiation, first we will experimentally identify miRNA-regulated transcripts in chondrocytes by profiling Ago2- associated RNAs. Second, proliferation defect of miRNA-deficient chondrocytes will be characterized by flow cytometry. In Aim III, we will investigate the physiological role of chondrocyte-specific miRNAs, miR-140 and miR-140*, in skeletogenesis. The Mir140 gene, encoding these miRNAs, is evolutionarily conserved in vertebrates, suggesting its important role in the skeletal system. Mice missing the Mir140 gene will be generated and characterized. Further, target transcripts of Mir140 miRNAs will be experimentally identified by the labeled miRNA pull-down assay (LAMP). Digoxigenin-labeled pre- Mir140 miRNA will be mixed with chondrocytes to reconstitute RISCs in vitro. Mir140 miRNA-bound transcripts will be immunopurified using anti-digoxigenin antibody. Defining roles of miRNAs and identifying their targets in chondrocytes may lead to identification of novel therapeutic targets for skeletal disease intervention.

描述（由申请人提供）：小型非编码microRNA（miRNA）主要在转录后水平调节基因表达。 miRNA在各种类型的生物体中起根本重要的作用。 miRNA依赖性基因调节的缺陷与癌症和精神疾病等人类疾病有关。但是，关于miRNA在哺乳动物骨骼系统中功能的知识是有限的。这项研究的目的是确定小鼠骨骼发育中miRNA的作用。 miRNA是通过多个处理步骤生成的； miRNA的主要转录本首先是由包含Drosha和DGCR8作为关键组件的微处理器处理，然后由DICER处理。然后将成熟的miRNA加载到RNA诱导的沉默复合物（RISC）上，该复合物包含Argonaute（AGO）作为核心成分，并通过部分碱基对结合其目标转录本。我们在丁香无缺乏的小鼠生长板中的发现导致了我们的中心假设，即软骨细胞miRNA调节细胞增殖和分化。但是，由于已知DICER会处理除miRNA以外的其他RNA物种，因此尚不清楚miRNA的丧失在多大程度上有助于二氧化碳缺陷型软骨细胞的表型。在目标I中，我们将通过产生和分析缺少DGCR8和DROSHA的条件敲除小鼠以独立于DICER来消除miRNA的条件敲除小鼠来确认miRNA在调节软骨细胞增殖和分化中的作用。在AIM II中，为了了解miRNA调节软骨细胞增殖和分化的机制，首先，我们将通过分析AGO2-相关的RNA在软骨细胞中实验中识别miRNA调节的转录本。其次，缺乏miRNA的软骨细胞的增殖缺陷将以流式细胞仪为特征。在AIM III中，我们将研究软骨细胞特异性miRNA，miR-140和miR-140*在骨骼生成中的生理作用。编码这些miRNA的miR140基因在脊椎动物中是进化保守的，表明其在骨骼系统中的重要作用。缺少miR140基因的小鼠将产生和表征。此外，将通过标记为miRNA下拉测定法（LAMP）实验鉴定miR140 miRNA的目标转录本。二氧基素标记的Pre-MiR140 miRNA将与软骨细胞混合，以在体外重新构建RISC。 miR140 miRNA结合的转录本将使用抗二氧素抗体进行免疫菌株。 定义miRNA的作用并确定其在软骨细胞中的靶标可能会导致鉴定出用于骨骼疾病干预的新型治疗靶标。