Role of miRNA from amelogenin exon4 in enamel formation

项目摘要/摘要 釉质形成的遗传学还没有得到很好的研究。在人类中，遗传性釉质缺陷(HED)以一种 非综合征性牙釉质状况(釉质发育不全/AI)或影响组织或组织的综合征状况 除了釉质表型外，还有其他器官。已知至少有73个基因与HED有关。釉原蛋白基因 是这些基因中最重要的一个，当突变时会导致X连锁人工智能。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mRNAs。在这些活动中， 外显子4总是被剪接出来，而剪接掉的外显子4的重要性还没有得到很好的研究。我们最近 一种新的microRNA(MiRNA)是从剪接的外显子4(miR-exon4)中衍生出来的。这个miR-外显子4 调控Runx2在成釉细胞中的表达。在牙齿中，已知Runx2与HED有关，因此关键 提示miR-外显子4在釉质形成中的作用。MiRNAs在牙齿形成过程中发挥着重要作用。 然而，miRNAs、牙齿发育和疾病之间的明确联系尚未建立。 特别是，miR-外显子4如何参与正常和病理性釉质的形成尚不清楚。釉质是一种 牙齿不可缺少的组织层，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，机械性能降低，咬合不良。因此，有 迫切需要增加我们对新发现的与釉质有关的miR外显子4的了解 队形。我们的长期目标是彻底了解分子信号如何引导牙釉质的形成， 尤其是釉原蛋白基因衍生分子的参与。我们对这项建议的总体目标 是为了发现miR-外显子4是如何形成的，以及它是如何促进釉质形成的。在我们出版的 初步研究发现，miR-exon4调控包括Runx2在内的Hed致病基因。我们的硅胶 分析表明，大多数导致X连锁AI的釉原蛋白突变破坏了选择性剪接， 这将影响miR-外显子4的生产。总而言之，我们的中心假设是miR-外显子4的产生 是通过选择性剪接启动的，而miR-外显子4调节参与HED的分子。这一假设将是 测试具有以下特定目的：1)确定外显子4的选择性剪接如何调节以启动miR- 外显子4的形成，2)揭示了miR-外显子4调控Runx2在釉质中表达的机制 3)了解miR-外显子4如何在体内牙釉质形成中发挥重要作用。这些 知识将促进我们对调控牙釉质形成的分子机制的理解，特别是， X连锁AI的病因学相关机制。我们的发现将提供有用的知识 基金会将制定精准医学中的战略，以定制X连锁AI的治疗方法。 此外，随着miR-外显子4作用的进一步阐明和确立，miR-外显子4将有可能被使用。 作为一种治疗分子，在未来改善牙釉质的质量。

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.