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.

项目概要/摘要 精确控制基因的表达水平对于任何 microRNA 都是至关重要的。 (miRNA) 通过与特定靶 mRNA 结合在控制基因表达中发挥重要作用 为了了解 miRNA 的功能，识别这组 miRNA 至关重要。 mRNA 在特定的生物环境中与其结合的目标，并确定这种配对如何相互作用 调节基因表达，最终影响细胞过程和表型。 受竞争性内源性 RNA (ceRNA) 控制，该内源性 RNA 与 miRNA 结合并阻止它们 下调其 mRNA 靶标的表达，创建一个复杂的调控网络，这可能很困难 来通过实验来揭开。 拟议的研究将开发一种全面的实验方法来揭示生物 作为一个模型系统，该工作重点关注控制两个 miRNA（miR-1 和 miR-206）。 肌生成，骨骼肌细胞的分化 该实验使用培养的小鼠 C2C12 细胞。 广泛使用的肌肉生成模型以及小鼠体内实验 In Specific Aim 1 实验将进行。 鉴定未分化 C2C12 细胞和分化过程中 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，以识别每个 miRNA 独特靶向的 mRNA。 将筛选在肌生成过程中确定的 miR-1 和 miR-206 靶标，了解它们对肌生成的贡献 分化表型实验还将测试 miRNA/靶标配对在控制 此外，特定 miRNA/ 的调控关系。 在特定目标 3 中，将使用小鼠模型来研究目标对，实验将检验以下假设： 未分化细胞中 miR-1 和 miR-206 的 RNA 靶标充当 ceRNA 以维持增殖和 防止未分化细胞中 miRNA 及其靶标的绝对水平。 进行量化，将筛选目标以确定那些对于维持细胞增殖重要的目标，以及 ceRNA 的调控机制将得到研究。 拟议的研究共同开发了一个实验框架来了解 这项工作不仅将促进对 miR-1 和 miR-206 在细胞中的作用的理解。 肌发生，但也将提供一个全面的策略，可以应用于多种领域的其他 miRNA 的生物系统。