Role of microRNAs in skeletal development

描述（由申请人提供）：小的非编码microRNAs （miRNAs）主要在转录后水平调控基因表达。mirna在各种类型的生物体中起着至关重要的作用。mirna依赖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mirna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mirna在小鼠骨骼发育过程中的作用。mirna是通过多个加工步骤生成的；mirna的初级转录本首先由含有Drosha和DGCR8作为关键成分的微处理器处理，然后由Dicer处理。然后将成熟的mirna装载到含有Argonaute （Ago）为核心成分的rna诱导沉默复合体（RISC）上，并通过部分碱基配对与目标转录物结合。我们在Dicer缺陷小鼠生长板中的发现导致了我们的中心假设，即软骨细胞mirna调节细胞增殖和分化。然而，由于已知Dicer可以加工mirna以外的RNA物种，因此尚不清楚mirna的缺失在多大程度上导致Dicer缺陷软骨细胞的表型。在Aim I中，我们将通过生成和分析缺少DGCR8和Drosha的条件敲除小鼠来独立于Dicer消除miRNAs，从而证实miRNAs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rna来实验鉴定软骨细胞中mirna调节的转录物。其次，利用流式细胞术表征mirna缺失软骨细胞的增殖缺陷。在Aim III中，我们将研究软骨细胞特异性mirna miR-140和miR-140*在骨骼形成中的生理作用。编码这些mirna的Mir140基因在脊椎动物中是进化保守的，这表明它在骨骼系统中起着重要作用。缺失Mir140基因的小鼠将被生成并鉴定。此外，Mir140 miRNA的靶转录本将通过标记miRNA下拉试验（LAMP）进行实验鉴定。地高辛标记的pre- Mir140 miRNA将与软骨细胞混合，在体外重建RISCs。使用抗地高辛抗体对Mir140 mirna结合转录物进行免疫纯化。确定mirna在软骨细胞中的作用并确定其靶点可能会导致骨骼疾病干预的新治疗靶点的鉴定。

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.