Role of miRNA from amelogenin exon4 in enamel formation

项目总结/摘要 釉质形成的遗传学尚未得到很好的研究。在人类中，遗传性牙釉质缺陷（HED）作为一种遗传性牙釉质缺陷， 非综合征性釉质疾病（釉质形成综合征/AI）或影响组织的综合征性疾病，或 除了牙釉质表型外，还有其他器官。已知至少有73个基因参与HED。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种主要mRNA。在这些事件中， 外显子4总是被剪接掉，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 发表了一种新的microRNA（miRNA）来源于剪接外显子4（miR-exon 4）。这个miR-exon 4 调节成釉细胞中Runx 2的表达。实际上，已知Runx 2与HED相关联，因此， 提示miR-exon 4在釉质形成中的作用。miRNAs在牙齿形成中起重要作用。 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-exon 4如何参与正常和病理性釉质形成尚不清楚。搪瓷是一种 牙齿不可或缺的组织层，因为有缺陷的牙釉质严重影响HED患者的生活质量 导致美观性差、牙齿过敏、机械性能降低和咬合。因此， 迫切需要增加我们对这个新发现的与釉质相关的miR-exon 4的了解 阵我们的长期目标是彻底了解分子信号如何指导釉质形成， 特别是涉及源自釉原蛋白基因的分子。我们这项建议的总体目标 是发现miR-exon 4的形成是如何启动的，以及它如何促进釉质形成。在我们出版的 初步研究发现，miR-exon 4调控包括Runx 2在内的HED致病基因。我们的计算机模拟 分析表明大多数导致X连锁AI的釉原蛋白突变破坏了可变剪接， 这将影响miR-外显子4的产生。综上所述，我们的中心假设是miR-外显子4的产生 通过选择性剪接启动，miR-exon 4调节HED相关分子。这一假设将是 1）确定外显子4的选择性剪接是如何被调节以启动miR-1表达的。 外显子4形成，2）揭示了miR-外显子4在成釉过程中调节Runx 2表达的机制。 3）了解miR-exon 4如何在体内釉质形成中发挥重要作用。这些 知识将促进我们对调控釉质形成的分子机制的理解，特别是， 与X连锁AI病因相关的机制。我们的发现将提供有用的知识 基金会开发精准医学策略，为X连锁AI定制治疗。 此外，随着miR-exon 4的作用将被进一步阐明和确定，miR-exon 4将有可能被用于 作为一种治疗性分子，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.