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发挥着根本性的重要作用。 miRNA 依赖性基因调控的缺陷与癌症和精神障碍等人类疾病有关。然而，关于哺乳动物骨骼系统中 miRNA 功能的知识有限。本研究的目的是确定 miRNA 在小鼠骨骼发育过程中的作用。 miRNA是通过多个处理步骤产生的； miRNA 的初级转录物首先由含有 Drosha 和 DGCR8 作为关键组件的微处理器处理，然后由 Dicer 处理。然后，成熟的 miRNA 被加载到以 Argonaute (Ago) 作为核心成分的 RNA 诱导沉默复合物 (RISC) 上，并通过部分碱基配对与其目标转录物结合。我们在 Dicer 缺陷小鼠生长板中的发现得出了我们的中心假设：软骨细胞 miRNA 调节细胞增殖和分化。然而，由于已知 Dicer 可以加工 miRNA 以外的 RNA 种类，因此尚不清楚 miRNA 的丢失在多大程度上对 Dicer 缺陷软骨细胞的表型有影响。在目标 I 中，我们将通过生成和分析缺失 DGCR8 和 Drosha 的条件敲除小鼠来独立于 Dicer 消除 miRNA，从而确认 miRNA 在调节软骨细胞增殖和分化中的作用。在目标 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 RNA 来实验性地鉴定软骨细胞中 miRNA 调节的转录本。其次，缺乏 miRNA 的软骨细胞的增殖缺陷将通过流式细胞术进行表征。在目标 III 中，我们将研究软骨细胞特异性 miRNA、miR-140 和 miR-140* 在骨骼形成中的生理作用。编码这些 miRNA 的 Mir140 基因在脊椎动物中在进化上是保守的，这表明它在骨骼系统中发挥着重要作用。将生成缺失 Mir140 基因的小鼠并对其进行表征。此外，Mir140 miRNA 的靶转录物将通过标记 miRNA Pull-down 测定 (LAMP) 进行实验鉴定。地高辛标记的 pre-Mir140 miRNA 将与软骨细胞混合以在体外重建 RISC。 Mir140 miRNA 结合转录物将使用抗地高辛抗体进行免疫纯化。 定义 miRNA 的作用并确定其在软骨细胞中的靶点可能会导致骨骼疾病干预的新治疗靶点的确定。