Role of let-7 microRNAs in skeletal development

描述（由申请人提供）：小调节RNA，microRNA（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依赖性基因调控的缺陷与几种疾病有关。尽管有这些事实，miRNAs在哺乳动物骨骼系统中的功能几乎没有研究。let-7家族miRNAs构成软骨细胞miRNAs的最大部分。let-7 miRNAs调节多种生物学功能，包括调节发育时间、细胞增殖和分化、癌症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-7 miRNAs在骨骼发生中的作用。我们将通过抑制和过表达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-7 miRNAs在8个基因组位点编码。这种广泛的遗传多样性使得难以使用常规基因靶向策略进行功能丧失研究。为了以组织特异性方式抑制多个let-7 miRNA，我们将使用Cre依赖的二元系统在软骨中错误表达RNA结合蛋白Lin 28。Lin 28特异性地抑制let-7 miRNA的初级转录物的加工，从而抑制let-7表达。将分析小鼠中Lin 28错误表达的后果。在目的II中，let-7 miRNA的功能将通过功能获得研究来研究。let-7 miRNA将使用相同的二元基因表达系统在软骨中过表达。let-7也将与Lin 28共表达以确定Lin 28的特异性功能。该项目将为let-7 miRNAs在骨骼发生中的生理作用提供见解。此外，依赖Cre的Lin 28和let-7表达系统将是研究这些基因在其他组织中功能的有用工具。了解单个miRNA对于开发潜在的基于miRNA的骨骼疾病治疗是必要的。公共卫生相关性：小调控RNA、microRNA主要在转录后水平调节基因表达。let-7 microRNA是骨骼组织中最丰富的miRNA种类。然而，let-7 miRNAs在骨骼组织中的生理作用尚不清楚。该项目旨在利用新的遗传工具阐明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.