利用条件性fgf8基因打靶与维甲酸诱导舌发育异常动物模型研究miRNA调控舌肌分化的分子机制

Tongue muscle differentiation is the basis of the tongue development and function, which is the key initiating factor of maxillofacial regions. The mechanism of tongue differentiation is still unclear, which through interactions between tongue myocytes and neural crest-derived mesenchymal cells, especially the function of targeting regulation of miRNA is still in lack of research. In the retinoic acid-induced tongue malformation models of our group, we screened and selected differentially expressed miRNA, and also verified that the miR-27b can target the epigenetic event. Furthermore, we also found that in Osr2-Cre; Rosa26R-fgf8 mouse, up- regulation of fgf8 gene in tongue mesenchymal cells spatially and temporally can induce similar phenotype of tongue which is caused by retinoic acid. This project will first study the molecular mechanisms of tongue muscle differentiation induced by interactions between tongue muscle and mesenchyme using these animal models. Moreover, positively or negatively regulated miRNA and these target molecular networks will be determined by microarray and bioinformatics and verification in vitro. Additionally, the molecular mechanism will be explored that miRNA can coordinate with epigenetic events to regulate the development of the tongue by detecting which miRNA targeting regulate HDAC4/5 and DNMT and whether miRNA appear methylation sites. Our study will provide a new target for clinical diagnosis and treatment of maxillofacial congenital developmental disorders.

舌肌分化是舌作为颌面部发育始动因素而行使功能的基础，但舌肌分化的分子调控机制尚缺乏研究，尤其是miRNA对其靶向调控以及舌肌与神经嵴来源间充质相互作用的机制不清。在本课题组制作的维甲酸诱导舌异常模型中我们筛选出相关差异表达miRNA，并验证了miR-27b等可靶向作用于表观遗传事件；同时我们还发现在Osr2-Cre; Rosa26R-fgf8小鼠舌间充质细胞中时空特异性敲入fgf8基因可出现与维甲酸诱导相似的舌异常表型。本项目将利用上述动物模型，首先研究舌肌与间充质相互作用对舌肌分化的分子调控机制；进而通过芯片筛选与生物信息学预测和体外细胞学验证，确定正向与负向调控舌肌分化的miRNA及其靶向分子网络；并通过检测miRNA靶向调控HDAC4/5和DNMT以及miRNA基因DNA甲基化状况，探究miRNA与表观遗传事件协同调控舌发育的分子机制，为颌面部先天性发育畸形的临床诊治提供新靶点。

舌肌分化是舌作为颌面部发育始动因素而行使功能的基础，舌肌分化的分子调控机制尚缺乏研究，尤其是miRNA对其靶向调控以及舌肌与神经嵴来源间充质相互作用的机制尚不清楚。在本课题组制作的维甲酸诱导舌异常模型中我们筛选出相关差异表达miRNA，并验证了miR-27b等可靶向作用于表观遗传事件；同时我们还发现在Osr2-Cre; Rosa26R-fgf8小鼠的舌间充质细胞Fgf8基因被时空特异性敲入时可出现与维甲酸诱导相似的舌异常表型。本项目首先对维甲酸诱导舌异常模型中miRNA与表观遗传改变的相互作用对舌发育的调控机制进行了研究，明确了miR-31-5p通过调控HDAC4/5参与调控维甲酸诱导舌异常发育的分子机制。接着，尝试揭示维甲酸对小鼠舌肌发育的表观遗传调控机制，结果提示过量维甲酸可能通过不同方式调控HDAC4/5进而抑制舌肌发育。最后，利用Osr2-cre;Rosa26R-Fgf8条件性转基因动物模型，首次分析研究了颌面部发育过程中间充质过表达Fgf8所出现的舌及下颌表型，研究表明Osr2-cre;Rosa26R-Fgf8小鼠表现出与Pierre Robin序列征患者极为相似的颅面部特征，如小颌畸形、舌畸形及腭裂，并明确了舌外周间充质持续性表达Fgf8，抑制了舌结缔组织分化，改变其张力和应力，引起大舌畸形。而该模型中的小颌畸形很可能不是由于下颌骨本身缺陷引起的，而是继发于咬肌肌腱缺失导致的。上述研究结果为颌面部先天性发育畸形的病因学机制和治疗靶点的研究提供了新的思路。