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 MicroRNA是一种约22个核苷酸的非编码RNA，在转录后水平控制基因表达。 由于其能够同时调节大量基因，因此microRNA被假定为 作为细胞类型特异性发育和功能的主调节剂。虽然功能增益分析 microRNA过表达可以影响转基因小鼠的这些过程，功能丧失研究通常 不能检测到表型改变，不能忠实地确定它们在发育中的作用， 疾病此外，从体外分析获得的结果有时不支持功能分析 in vivo. Dicer是microRNA生物合成所必需的一种酶， in bone骨remodeling重塑.然而，涉及的确切miRNA尚未确定。miR-1基因簇 图23 a ~ 27 a ~24-2由三个miRNAs组成，它们由一个转录本产生。miR-23 a的失调和 miR-27 a在骨质疏松症患者中已被证实，但其调控作用仍不明确.在细胞培养物中 在一项研究中，高水平的miR-23 a或miR-27 a抑制OB分化，表明它们是负性的。 在小鼠中，OB特异性表达的miR-23 a ~ 27 a ~24-2簇揭示了其对骨形成的影响。 对成骨细胞的分化无明显影响。动物研究不支持细胞培养分析。有 迫切需要开发功能丧失模型，以明确评估这些microRNA的功能， vivo.为了填补我们的知识空白，我们使用CRSPR-Cas9基因创建了miR-27 a缺陷的小鼠模型， 编辑.这种单一miRNA的缺失会导致颅面和身体骨骼的严重骨质疏松， 提示miR-27 a的功能不可替代。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-27 a是骨重建的正调节因子， 强烈反对先前的细胞培养研究。在本申请中，我们将描述颅面和 miR-27 a缺陷小鼠的骨骼缺陷。我们将研究成骨细胞和破骨细胞异常与 突变导致骨骼重塑失衡。为了进一步阐明 miR-27 a调节骨形成和骨吸收，我们将使用无偏筛选来确定其直接靶点。 这项提案的完成具有突出的潜力，以推进我们的知识基础表观遗传 调节骨代谢，导致新的策略，预防和治疗颅面和 骨骼疾病