Role of miRNA from amelogenin exon4 in enamel formation

釉原蛋白外显子 4 的 miRNA 在牙釉质形成中的作用

• 批准号: 10192498
• 负责人: Yukiko Nakano
• 金额: $ 38.36万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10192498
• 关键词: Affect; Alternative Splicing; Ameloblasts; Amelogenesis Imperfecta; Animal Model; Binding; Biological Assay; CXCL11 gene; CYP27B1 gene; Cells; Data; Defect; Dental Enamel; Development; Disease; Electrophoretic Mobility Shift Assay; Enamel Formation; Enamel Organ; Enhancers; Esthetics; Etiology; Event; Exons; Foundations; Future; Genes; Genetic; Goals; Growth; Human; Hypersensitivity; In Vitro; Infection; Inherited; Knockout Mice; Knowledge; LAMC2 gene; Link; Messenger RNA; MicroRNAs; Molecular; Mus; Mutate; Mutation; NFIA gene; Organ; Outcome Study; Pathologic; Patients; Phenotype; Play; Preparation; Process; Production; Proteins; Publishing; Quality of life; RNA; RNA Splicing; Reporter; Role; SRSF2 gene; Signal Transduction; Syndrome; TSC1 gene; Testing; Therapeutic; Tissues; Tooth structure; Validation; Variant; amelogenin; gain of function; improved; in silico; in vivo; individualized medicine; leucine-rich amelogenin peptide; loss of function; mechanical properties; novel; precision medicine; skeletal

Project Summary/Abstract Genetics of enamel formation is not well studied yet. In humans, hereditary enamel defects (HED) occurs as a non-syndromic enamel conditions (amelogenesis imperfecta/AI) or syndromic conditions affecting tissues or organs in addition to the enamel phenotype. At least 73 genes are known to involve in HED. Amelogenin gene is the most essential one of those genes, and causes X-linked AI when mutated. Amelogenin gene is processed via alternative splicing to produce the two major mRNAs for proteins with distinct functions. During these events, exon4 is always spliced out, and the importance of the spliced-out exon4 has not been well studied. We recently published that a novel microRNA (miRNA) is derived from the spliced-out exon4 (miR-exon4). This miR-exon4 regulates expression of Runx2 in ameloblasts. In tooth, Runx2 is known associate with HED, thus the critical role of miR-exon4 for enamel formation is suggested. miRNAs play important roles in tooth formation. Nevertheless, clear links between the miRNAs, tooth development and diseases have yet to be established. Particularly, how miR-exon4 is involved in normal and pathologic enamel formation is not clear. Enamel is an indispensable tissue layer of the tooth, since defective enamel severely affects the quality of life for HED patients by causing poor aesthetics, hypersensitive tooth, reduced mechanical property and occlusion. Hence, there is an urgent necessity to increase our knowledge of this newly discovered miR-exon4 associated with enamel formation. Our long-term goal is to thoroughly understand how molecular signaling directs enamel formation, particularly the involvement of molecules derived from amelogenin gene. Our overall objective for this proposal is to discover how miR-exon4 formation is initiated and how it contributes to enamel formation. In our published and preliminary studies, we found that miR-exon4 regulates HED-causative genes including Runx2. Our in silico analysis demonstrated that most of the amelogenin mutations causing X-linked AI disrupt alternative splicing, which will affect production of miR-exon4. Taken together, our central hypothesis is that production of miR-exon4 is initiated via alternative splicing, and miR-exon4 regulates molecules involved in HED. This hypothesis will be tested with the following specific aims; 1) Determine how alternative splicing of exon4 is regulated to initiate miR- exon4 formation, 2) Reveal the mechanism by which miR-exon4 regulates Runx2 expression during enamel formation, and 3) Understand how miR-exon4 plays important roles in enamel formation in vivo. These knowledge will advance our understanding of molecular mechanisms regulating enamel formation, in particular, the mechanisms associated with the etiology of X-linked AI. Our findings will provide useful knowledge foundation to develop the strategies in the precision medicine to customize the treatment for X-linked AI. Moreover, as the role of miR-exon4 will be further clarified and established, miR-exon4 will potentially be used as a therapeutic molecule to improve the quality of the enamel in the future.

项目概要/摘要 牙釉质形成的遗传学尚未得到充分研究。在人类中，遗传性牙釉质缺陷 (HED) 作为一种 非综合征性牙釉质病症（釉质生成不全/AI）或影响组织或的综合征性病症 除了牙釉质表型之外的器官。已知至少有 73 个基因与 HED 相关。釉原蛋白基因 是这些基因中最重要的一个，突变时会导致 X 连锁 AI。釉原蛋白基因被加工 通过选择性剪接产生具有不同功能的蛋白质的两个主要 mRNA。在这些活动期间， exon4总是被剪接的，并且剪接的exon4的重要性尚未得到很好的研究。我们最近 发表了一种新的 microRNA (miRNA) 来自剪接的外显子 4 (miR-exon4)。这个miR-外显子4 调节成釉细胞中 Runx2 的表达。实际上，Runx2 已知与 HED 相关，因此关键 提示 miR-exon4 在牙釉质形成中的作用。 miRNA 在牙齿形成中发挥着重要作用。 然而，miRNA、牙齿发育和疾病之间的明确联系尚未确定。 特别是，miR-exon4 如何参与正常和病理性牙釉质形成尚不清楚。搪瓷是一种 牙齿不可或缺的组织层，因为牙釉质缺陷严重影响 HED 患者的生活质量 造成美观性差、牙齿过敏、机械性能下降和咬合。因此，有 迫切需要增加我们对这种新发现的与牙釉质相关的 miR-exon4 的了解 形成。我们的长期目标是彻底了解分子信号如何指导牙釉质形成， 特别是源自牙釉蛋白基因的分子的参与。我们此提案的总体目标 目的是发现 miR-exon4 的形成是如何启动的以及它如何促进牙釉质的形成。在我们发表的 初步研究发现，miR-exon4 调节 HED 致病基因，包括 Runx2。我们的计算机模拟 分析表明，大多数导致 X 连锁 AI 的牙釉蛋白突变会破坏选择性剪接， 这将影响 miR-exon4 的产生。综上所述，我们的中心假设是 miR-exon4 的产生 通过选择性剪接启动，miR-exon4 调节参与 HED 的分子。这个假设将是 测试的具体目标如下； 1) 确定如何调节外显子4的选择性剪接来启动miR- exon4形成，2）揭示miR-exon4在牙釉质过程中调节Runx2表达的机制 形成，3) 了解 miR-exon4 如何在体内牙釉质形成中发挥重要作用。这些 知识将增进我们对调节牙釉质形成的分子机制的理解，特别是 与 X 连锁 AI 病因学相关的机制。我们的研究结果将提供有用的知识 基金会开发精准医学策略，定制 X 连锁人工智能的治疗方法。 此外，随着miR-exon4的作用将进一步阐明和确立，miR-exon4将有可能被使用 作为未来改善牙釉质质量的治疗分子。