The essential role of miR-27a in craniofacial and body skeletons

miR-27a 在颅面和身体骨骼中的重要作用

• 批准号: 10433673
• 负责人: Takamitsu Maruyama
• 金额: $ 4.05万
• 依托单位: ADA FORSYTH INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-17 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10433673
• 关键词: essential; role; miR; 27a; craniofacial

Abstract MicroRNAs are ~22 nucleotides noncoding RNAs which control gene expression at post-transcriptional levels. Owing to its ability to simultaneously modulate a vast amount of genes, microRNA has been postulated to workas a master regulator for cell type-specific development and function. Although gain-of-function analysis with microRNA overexpression can affect these processes in transgenic mice, loss-of-function study often fails to detect phenotypic alterations and is unable to faithfully determine their role in development and disease. Moreover, results obtained from in vitro analysis sometimes are not supported by functional analyses in vivo. Studies of Dicer, an enzyme essential for biosynthesis of microRNAs, have implicated their importance in boneremodeling. However, the exact miRNA(s) involved has not been identified. The cluster of miR- 23a~27a~24-2 consists of three miRNAs generated from a single transcript. Dysregulation of miR-23a and miR-27a has beenshown in osteoporosis patients but their regulatory role remains elusive. In cell culture study, high levels of miR-23a or miR-27a inhibits OB differentiation, suggesting that they are negative regulators for bone formation.In mice, OB-specific expression of miR-23a~27a~24-2 cluster reveals its effects on osteocyte but not OB differentiation. The animal study does not support the cell culture analysis. There is an urgent need to develop loss-of-function models to definitively assess the function of these microRNAs in vivo. To fill our knowledge gaps, we created a mouse model deficient for miR-27a using CRSPR-Cas9 gene editing. The loss of this single miRNA causes severe osteoporosis in the craniofacial and body skeletons, suggesting the function of miR-27a cannot be substituted. The bone loss phenotype also becomes more prominent with age. New genetic evidence, indicating miR-27a as a positive regulator in bone remodeling, strongly argues against the previous cell culture study. In this application, we will characterize craniofacial and skeletal defects in miR-27adeficient mice. We will examine osteoblast and osteoclast abnormalities associated with the mutation to causean imbalance in skeletal remodeling. To further elucidate the mechanism by which miR-27a regulates bone formation and resorption, we will identify its direct targets using unbiased screening. The completion of this proposal has outstanding potential to advance our knowledge base of epigenetic regulation in bone metabolism, leading to novel strategies for prevention and treatment of craniofacial and skeletal disorders.

摘要 MicroRNAs是一种~22个核苷酸的非编码RNA，在转录后水平控制基因的表达。 由于它能够同时调节大量的基因，所以microRNA被认为是 作为细胞类型特定发展和功能的主要调节者。虽然函数增益分析 由于microRNA的过度表达可以影响转基因小鼠的这些过程，功能丧失的研究经常 未能检测到表型变化，无法忠实地确定它们在发育和发育中的作用 疾病。此外，从体外分析获得的结果有时得不到功能分析的支持 在活体内。DICER是一种对microRNAs的生物合成至关重要的酶，其研究已经暗示了它们的重要性 在骨感重塑方面。然而，涉及的确切miRNA(S)尚未确定。MiR-星团 23A~27A~24-2由单个转录本产生的3个miRNAs组成。MiR-23a和miR-23a的调节失调 已经在骨质疏松症患者中发现了MIR-27A，但它们的调节作用仍然难以捉摸。在细胞培养中 研究表明，高水平的miR-23a或miR-27a抑制OB分化，表明它们是阴性的 骨形成调节剂。在小鼠中，miR-23a~27A~24-2簇的OB特异性表达揭示了其作用 对成骨细胞有影响，但对OB分化无影响。动物实验不支持细胞培养分析。的确有 迫切需要开发功能丧失模型来明确评估这些microRNAs在 活着。为了填补我们的知识空白，我们使用CRSPR-Cas9基因创建了miR-27A缺陷的小鼠模型 正在编辑。这种单个miRNA的丢失会导致头面部和身体骨骼严重的骨质疏松， 提示miR-27A的作用不可替代。骨丢失表型也变得更多 随着年龄的增长而突出。新的遗传学证据表明miR-27A在骨重建中是一个积极的调节因子， 强烈反对之前的细胞培养研究。在本应用程序中，我们将描述颅面部和 MiR-27a缺陷小鼠的骨缺陷。我们将检查与成骨细胞和破骨细胞相关的异常 突变导致骨骼重塑失衡。以进一步阐明其作用机制 MIR-27A调控骨形成和吸收，我们将使用无偏筛选来确定其直接靶点。 这项建议的完成具有极大的潜力来推进我们的表观遗传学知识基础 对骨代谢的调节，导致了预防和治疗颅面和骨关节紊乱的新策略 骨骼疾病。