Role of let-7 microRNAs in skeletal development

描述(申请人提供)：小调控RNAs，microRNAs(MiRNAs)，在各种类型的生物体中发挥重要的生物学作用。在人类中，miRNA依赖的基因调控缺陷与几种疾病有关。尽管有这些事实，但miRNAs在哺乳动物骨骼系统中的功能几乎没有被研究过。Let-7家族的miRNAs构成了软骨细胞miRNAs中最大的部分。Let-7miRNAs调控多种生物学功能，包括调节发育时间、细胞增殖和分化、肿瘤转移和干细胞自我更新。然而，let-7 miRNAs在骨骼发育中的生理作用尚不清楚。本研究的目的是确定let-7miRNAs在骨骼形成过程中的作用。我们将通过抑制和过度表达let-7 miRNAs来研究let-7 miRNAs在小鼠骨骼系统中的作用。在小鼠中，let-7miRNAs在8个基因组座位上编码。这种广泛的遗传多样性使得使用传统的基因靶向策略进行功能丧失的研究变得困难。为了以组织特异性的方式抑制多个let-7 miRNAs，我们将使用依赖Cre的二进制系统在软骨中错误表达RNA结合蛋白Lin28。LIN28特异性地抑制let-7 miRNAs初级转录本的处理，从而抑制let-7的表达。我们将分析LIN28错误表达对小鼠的影响。在AIM II中，将通过功能增益研究来研究let-7 miRNAs的功能。使用相同的二元基因表达系统，let-7 miRNAs将在软骨中过度表达。LET-7也将与LIN28共表达，以确定LIN28的具体功能。该项目应该为let-7miRNAs在骨骼形成中的生理作用提供深入的见解。此外，LIN28和LET-7依赖于Cre的表达系统将成为研究这些基因在其他组织中功能的有用工具。了解单个miRNAs对于开发潜在的基于miRNA的骨骼疾病治疗是必要的。与公共健康相关：小的调控RNA，microRNA主要在转录后水平调节基因表达。Let-7microRNA是骨骼组织中含量最丰富的miRNA物种。然而，let-7miRNAs在骨骼组织中的生理作用尚不清楚。本项目旨在利用新的基因工具阐明let-7在小鼠骨骼系统中的功能。了解单个miRNAs的作用将为骨骼疾病的潜在治疗干预提供基础。

DESCRIPTION (provided by applicant): The small regulatory RNAs, microRNAs (miRNAs), play important biological roles in various types of living organisms. In humans, defects in miRNA-dependent gene regulation are implicated in several diseases. Despite these facts, functions of miRNAs in the mammalian skeletal system have barely been studied. The let-7 family miRNAs constitute the largest potion of chondrocytic miRNAs. let-7 miRNAs regulate diverse biological functions that include regulation of developmental timing, cell proliferation and differentiation, cancer metastasis, and stem cell self-renewal. However, the physiological role of let-7 miRNAs in skeletal development is unknown. The goal of this research is to establish the role of let-7 miRNAs during skeletogenesis. We will investigate the role of let-7 miRNAs in the mouse skeletal system by suppressing and overexpressing let-7 miRNAs. In mice, let-7 miRNAs are encoded at 8 genomic loci. This extensive genetic multiplicity makes it difficult to perform a loss-of-function study using the conventional gene targeting strategy. To suppress multiple let-7 miRNAs in a tissue-specific fashion, we will misexpress the RNA-binding protein, Lin28, in the cartilage using a Cre-dependent binary system. Lin28 specifically inhibits processing of primary transcripts of let-7 miRNAs and thereby suppresses let-7 expression. The consequences of Lin28 misexpression in mice will be analyzed. In Aim II, functions of let-7 miRNAs will be investigated by a gain-of-function study. let-7 miRNAs will be overexpressed in the cartilage using the same binary gene expression system. let-7 will also be co-expressed with Lin28 to determine the specific function of Lin28. This project should provide insights into the physiological role of let-7 miRNAs in skeletogenesis. In addition, the Cre-dependent expression systems for Lin28 and let-7 will be useful tools for studying the function of these genes in other tissues. Understanding of individual miRNAs is necessary for the development of potential miRNA-based treatment of skeletal diseases. PUBLIC HEALTH RELEVANCE: The small regulatory RNAs, microRNAs regulate gene expression mainly at the post transcriptional level. let-7 microRNAs are the most abundant miRNA species in skeletal tissues. However, the physiological roles of let-7 miRNAs in skeletal tissues are unknown. This project aims to elucidate let-7 function in the mouse skeletal system using novel genetic tools. Understanding roles of individual miRNAs will provide a basis for potential therapeutic interventions for skeletal diseases.