Unraveling the biological roles of specific miRNAs, from experimental target identification through functional characterization

从实验目标识别到功能表征，揭示特定 miRNA 的生物学作用

• 批准号: 10566442
• 负责人: Jennifer F. Kugel
• 金额: $ 31.67万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-10 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10566442
• 关键词: Binding; Biological; Biological Assay; Biological Models; Biological Process; Cell Differentiation process; Cell Fate Control; Cell Line; Cell Proliferation; Cell physiology; Cells; Complex; Data; Disease; Down-Regulation; Embryonic Development; Gene Expression; Genes; Goals; Individual; Injury; Knock-out; Knowledge; Mediating; Messenger RNA; Methods; MicroRNAs; Modeling; Molecular; Mus; Muscle Fibers; Myoblasts; Pathologic; Pathway interactions; Phenotype; Play; Process; Proliferating; RNA; Regulation; Regulator Genes; Research; Role; Techniques; Testing; Undifferentiated; Validation; Work; biological systems; experimental study; in vivo; in vivo Model; mRNA Expression; mouse model; muscle regeneration; myogenesis; premature; prevent; skeletal muscle differentiation; success

PROJECT SUMMARY/ABSTRACT Precisely controlling the expression levels of genes is essential to any biological process. microRNAs (miRNAs) play important roles in controlling gene expression by binding to specific target mRNAs and downregulating their expression. To understand how a miRNA functions it is critical to both identify the set of mRNA targets to which it binds in a specific biological context, and to determine how this pairing interaction regulates gene expression to ultimately impact cellular processes and phenotypes. Some miRNAs can be controlled by competing endogenous RNAs (ceRNAs), which bind miRNAs and prevent them from downregulating expression of their mRNA targets, creating a complex network of regulation that can be difficult to experimentally unravel. The proposed research will develop a comprehensive experimental approach to uncover the biological functions of a miRNA. As a model system the work focuses on two miRNAs (miR-1 and miR-206) that control myogenesis, the differentiation of skeletal muscle cells. The experiments use cultured mouse C2C12 cells, a widely used model for myogenesis, as well as in vivo experiments with mice. In Specific Aim 1 experiments will identify the direct RNA targets of miR-1 and miR-206 in undifferentiated C2C12 cells and during differentiation. Supporting data demonstrate the success of a target identification technique, which will be used with miR-1 and miR-206 knockout cells to identify mRNAs uniquely targeted by each miRNA. In Specific Aim 2, newly identified targets of miR-1 and miR-206 during myogenesis will be screened for their contributions to differentiation phenotypes. Experiments will also test the function of miRNA/target pairing in controlling the expression levels of targets during myogenesis. In addition, the regulatory relationships of specific miRNA/ target pairs will be studied using mouse models. In Specific Aim 3 experiments will test the hypothesis that RNA targets of miR-1 and miR-206 in undifferentiated cells function as ceRNAs to maintain proliferation and prevent premature differentiation. The absolute levels of miRNAs and their targets in undifferentiated cells will be quantified, targets will be screened to identify those important for maintaining cellular proliferation, and regulatory mechanisms of ceRNAs will be investigated. Together the proposed studies develop an experimental framework to understand the functional roles of two important miRNAs. This work will not only advance understanding of the roles of miR-1 and miR-206 in myogenesis, but will also provide a comprehensive strategy that could be applied to other miRNAs in a variety of biological systems.

项目摘要/摘要 精确控制基因表达水平对于任何生物学过程都是必不可少的。 microRNA （miRNA）通过与特定靶标mRNA和 下调他们的表达。要了解miRNA的功能是如何识别的 它在特定的生物学环境中结合的mRNA靶标，并确定这种配对相互作用如何 调节基因表达以最终影响细胞过程和表型。一些mirnas可以是 由竞争的内源性RNA（CERNAS）控制，该RNA结合miRNA并防止它们 下调其mRNA靶标的表达，建立一个复杂的调节网络，可能很难 实验拆卸。 拟议的研究将开发一种全面的实验方法来揭示生物学 miRNA的功能。作为模型系统，工作着重于控制两个miRNA（miR-1和miR-206） 肌发生，骨骼肌细胞的分化。实验使用培养的小鼠C2C12细胞A 广泛用于肌发​​生的模型以及小鼠的体内实验。在特定目标中1实验将 在未分化的C2C12细胞中以及分化过程中确定miR-1和miR-206的直接RNA靶标。 支持数据证明了目标识别技术的成功，该技术将与mir-1一起使用 和miR-206敲除细胞，以鉴定每个miRNA唯一靶向的mRNA。在特定目标2中，新的 在肌发生过程中，将鉴定出miR-1和miR-206的靶标，将筛选其对它们的贡献 分化表型。实验还将测试miRNA/目标配对的功能 肌发生过程中靶标的表达水平。此外，特定miRNA/ 目标对将使用鼠标模型进行研究。在特定目标中，3实验将检验以下假设。 未分化的细胞中miR-1和miR-206的RNA靶标充当维持增殖和 防止过早差异。 miRNA的绝对水平及其在未分化细胞中的靶标将 可以量化目标，将筛选目标以识别那些对于维持细胞增殖的重要的目标，并且 将研究CERNAS的调节机制。 拟议的研究共同开发了一个实验框架，以了解 两个重要的miRNA。这项工作不仅会进一步了解mir-1和miR-206在 肌发生，但还将提供一种全面的策略，可以应用于其他miRNA 生物系统。