Role of microRNAs in skeletal development

描述（由申请人提供）：小的非编码microRNA（miRNAs）主要在转录后水平调节基因表达。miRNAs在各种类型的生物体中发挥着重要的作用。miRNA依赖性基因调控的缺陷与人类疾病如癌症和精神障碍有关。然而，关于miRNAs在哺乳动物骨骼系统中的功能的知识是有限的。本研究的目的是确定miRNAs在小鼠骨骼发育过程中的作用。miRNA通过多个处理步骤产生; miRNA的初级转录物首先由含有Drosha和DGCR 8作为关键组分的微处理器处理，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，该复合物含有Argonaute（Ago）作为核心组分，并通过部分碱基配对与其靶转录物结合。我们在Dicer缺陷小鼠生长板中的发现导致了我们的核心假设，即软骨细胞miRNA调节细胞增殖和分化。然而，由于已知Dicer加工miRNA以外的RNA种类，因此不清楚miRNA的损失在多大程度上有助于Dicer缺陷型软骨细胞的表型。在目的I中，我们将通过产生和分析缺失DGCR 8和Drosha的条件性敲除小鼠以独立于Dicer消除miRNA来确认miRNA在调节软骨细胞增殖和分化中的作用。在目的II中，为了理解miRNA调节软骨细胞增殖和分化的机制，首先我们将通过分析Ago 2相关RNA来实验性地鉴定软骨细胞中miRNA调节的转录物。其次，将通过流式细胞术表征miRNA缺陷软骨细胞的增殖缺陷。在目的III中，我们将研究软骨细胞特异性miRNAs，miR-140和miR-140* 在骨骼发生中的生理作用。编码这些miRNAs的Mir 140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。将产生缺失Mir 140基因的小鼠并进行表征。此外，Mir 140 miRNA的靶转录物将通过标记的miRNA下拉测定（LAMP）进行实验鉴定。洋地黄毒苷标记的前-Mir 140 miRNA将与软骨细胞混合以在体外重建RISC。Mir 140 miRNA结合转录物将使用抗地高辛抗体进行免疫纯化。 定义miRNAs的作用并确定其在软骨细胞中的靶点可能会导致确定骨骼疾病干预的新治疗靶点。 公共卫生相关性：新发现的通过microRNA的基因调控机制已被证明在包括人类在内的各种生物体中发挥重要的生理和病理生理作用。然而，关于它们在骨骼系统中的作用的知识是非常基本的。该提案旨在确定microRNA在骨骼系统中的作用，并为骨骼疾病的潜在治疗干预提供基础。

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. PUBLIC HEALTH RELEVANCE: The newly discovered gene regulatory mechanism through microRNAs has been shown to play important physiological and pathophysiological roles in a wide variety of organisms including humans. However knowledge about their roles in the skeletal system is very rudimentary. This proposal is aimed to establish the role of microRNAs in the skeletal system and to provide a basis for potential therapeutic interventions for skeletal diseases.