Role of microRNAs in skeletal development

描述(申请人提供)：小的非编码microRNAs(MiRNAs)主要在转录后水平调节基因表达。MiRNAs在各种生物体中发挥着重要的作用。MiRNA依赖的基因调控缺陷已被认为与癌症和精神障碍等人类疾病有关。然而，关于miRNAs在哺乳动物骨骼系统中的功能的了解是有限的。本研究的目的是确定miRNAs在小鼠骨骼发育过程中的作用。MiRNAs是通过多个处理步骤产生的；miRNAs的初级转录本首先由包含DROSHA和DgCr8作为关键成分的微处理器处理，然后由DICER处理。然后，成熟的miRNAs被装载到RNA诱导的沉默复合体(RISC)上，该复合体包含ArgAerte(AGO)作为核心成分，并通过部分碱基配对与其目标转录本结合。我们在Dester缺陷的小鼠生长板上的发现导致了我们的中心假设，即软骨细胞miRNAs调控细胞的增殖和分化。然而，由于DICER被认为可以处理除miRNAs之外的其他RNA物种，目前还不清楚miRNAs的丢失在多大程度上对DICER缺陷软骨细胞的表型有贡献。在目标I中，我们将通过建立和分析缺失Dgcr8和DROSHA的条件性基因敲除小鼠来消除独立于DICER的miRNAs，以确认miRNAs在调控软骨细胞增殖和分化中的作用。在AIM II中，为了了解miRNAs调控软骨细胞增殖和分化的机制，我们首先将通过对Ago2相关RNA的分析，在实验上鉴定软骨细胞中miRNA调控的转录本。其次，通过流式细胞术对miRNA缺陷软骨细胞的增殖缺陷进行表征。在目标III中，我们将研究软骨细胞特异性miRNAs，miR-140和miR-140*，在骨骼形成中的生理作用。编码这些miRNAs的Mir140基因在脊椎动物中进化上保守，表明它在骨骼系统中发挥着重要作用。缺失Mir140基因的小鼠将被培育出来并进行鉴定。此外，Mir140 miRNAs的目标转录本将通过标记的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。Mir140 miRNA结合的转录本将使用抗地高辛抗体进行免疫纯化。明确miRNAs在软骨细胞中的作用并确定其靶点可能有助于确定骨骼疾病干预的新治疗靶点。

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.