miR-18a通过Kdm5b调节H3K4甲基化促进毛细胞分化及再生的研究

The mechanism of inner ear sensory epithelia and hair cells malformation induced by miR-18a knocking-down is not clear. H3K4me3 can promote the differentiation of embryonic stem cell to neural progenitor, and contribute to the neural development and differentiation. However, its function in the development of inner ear is still unclear. Based on the previous researches and our studies, we hypothesize that miR-18a-Kdm5b-H3K4me3 pathway participate in the math1 transcriptional activation to promote hair cell differentiation and regeneration. We aimed to: explore the precise function and mechanism of miR-18a-Kdm5b-H3K4me3 pathway on the inner ear development and hair cell differentiation; study the relationship between histone methylation and the inner ear development and hair cell differentiation; discuss the possibility of hair cell regeneration promoted by H3K4me3 up-regulation mediated by Kdm5b and miR-18a with Immunofluorescence, Immunobloting, In Situ Hybridization, Quantitive-PCR, embryonic injection, Electromicroscopy, gene transfection and knocking-down techniques on mouse and zebrafish. This study further investigate the mechanism of inner ear development, hair cell differentiation and regeneration from a novel scope, and provide an new insight on the research of hair cell regeneration, to help clinical prevention and therapy for hearing loss with great significance.

miR-18a敲除导致内耳感觉上皮及毛细胞发育障碍，其机理不清。H3K4me3促进胚胎干细胞向神经系前体细胞分化，与神经元发育及分化有关，其在内耳的作用不清。本项目结合文献及前期工作基础，提出miR-18a-Kdm5b-H3K4me3途径参与math1转录激活调控，促进毛细胞分化及再生这一假设。并以小鼠及斑马鱼为研究对象，采用免疫荧光、免疫印迹、原位杂交、定量PCR、胚胎注射、电镜、基因转染及敲除等技术，研究miR-18a-Kdm5b-H3K4me3对内耳发育及毛细胞分化作用及机制；分析组蛋白甲基化与内耳发育、毛细胞分化的关系；探讨通过调节miR-18a及Kdm5b上调H3K4me3水平促进毛细胞再生的可能性；本项目从一个全新的角度探讨内耳发育、毛细胞分化及再生机理，深入研究有望为毛细胞再生研究提供新的思路，为耳聋防治研究提供新的线索及理论依据，具有重要的理论意义。

内耳的发育及毛细胞的分化是一个多阶段、多步骤、多条信号通路、多种机制参与的复杂过程。本课题通过系统研究胚胎鼠发育过程中内耳miRNA-18a、Kdm5b、H3K4me1、H3K4me2、H3K4me3、H3K9me3、H3K27me3的表达变化，并进一步阐述了miR-18a-Kdm5b-H3K4me3 途径参与Math1转录激活调控过程。我们发现：1）miRNA-18a、Kdm5b、H3K4me1、H3K4me2、H3K4me3、H3K9me3、H3K27me3在内耳发育的全程呈现动态表达过程；2）通过双荧光素酶报告系统确认了miR-18a对Kdm5b基因的靶向调控，并使用Q-PCR及Western Blot对其进行了验证；3）通过染色质免疫共沉淀—PCR技术发现Math1基因的启动子区域分布有H3K4me3，表明Math1基因转录水平受到了启动子区域的甲基化状态的调控；4）利用基因干扰技术干扰Kdm5b基因的表达将提高细胞H3K4me3，即提升组蛋白甲基化水平，并进而显著提升毛细胞分化指标Math1、Myosin6等基因的表达，同时能降低干细胞指标如Nestin、Sox2等的表达。这项研究的完成，一方面探索了miR-18a及组蛋白去甲基化在内耳发育及毛细胞分化中的作用，有望开发一个具有潜在临床价值的干预靶点，具有较重要的科学价值；并为进一步研究毛细胞的再生并探索可能的干预提供一个新的思路。