调控组蛋白H3K4甲基化对毛细胞的保护作用及其机制研究

Given that hair cells are extremely vulnerable to various kinds of ototoxic insults like noise, drugs and infections, effective protection of hair cells and investigation of the mechanism are particularly important for prevention of sensorineural hearing loss. Histone was confirmed playing an important role in cell differentiation, cell survival, and injury recovery process. Our previous results showed that the regulation of histone methylation transferase G9a/GLP could inhibit Caspase-3 and other classical apoptotic pathway which subsequently protected hair cells from apoptosis. This subject aimed to explore the protective effects and its machenism of H3K4 di-methylation during hair cell damage process. Based on our preliminary results, we will observe the protective effects of cochlear hair cells on both drug- and noise-induced deaf mice model in vivo under the intervention of LSD1 inhibitors. Hair cell purification is going to be performed by flow cytometry cell sorting and laser capture microdissection; Chromatin immunoprecipitation combined with high -throughput sequencing (ChIP-Seq) method will be applied for whole genomic H3K4 methylation analysis of hair cells to identify the relative differentially expressed genes modulating histone modifications and their function as well as their communication networks.

噪声、药物、感染等因素均可造成内耳毛细胞损伤和永久性听力障碍，有效的毛细胞保护及其机制研究在感音神经性聋的防治中尤为重要。组蛋白调控在细胞分化、存活和损伤修复过程中发挥重要作用，我们既往研究发现，调控组蛋白甲基化转移酶G9a/GLP能够抑制Caspase-3等经典细胞凋亡通路，实现毛细胞保护。本课题围绕感音神经性聋防治中听觉保护的关键科学问题，探索H3K4me2修饰在毛细胞保护中的作用及其机制。在前期工作基础上，采用LSD1抑制剂上调H3K4me2水平，研究其在小鼠活体耳聋模型（药物致聋模型和噪声致聋模型）中耳蜗毛细胞保护作用；采用流式细胞分选和激光捕获显微切割技术纯化毛细胞，应用染色质免疫共沉淀结合高通量测序技术，进行全基因组H3K4me2分析，筛选及鉴定组蛋白修饰相关的差异基因及其调控网络，并对表达差异基因进行功能鉴定，揭示LSD1抑制剂及H3K4me2修饰对毛细胞保护作用的分子机制。

噪声、药物、感染等因素均可造成内耳毛细胞损伤和永久性听力障碍，有效的毛细胞保护及其机制研究在感音神经性聋的防治中尤为重要。组蛋白调控在细胞分化、存活和损伤修复过程中发挥重要作用，本课题针对有效的毛细胞保护作用、毛细胞损伤和保护机制等方向进行阐述。首先，我们上调 H3K4me2 水平（LSD1抑制剂S2101 或 CBB1007），研究其在小鼠耳聋模型（主要是在体耳聋模型）中耳蜗毛细胞保护和听力保留作用。然后，我们进一步探索毛细胞损伤和保护机制，为阐述了毛细胞保护的具体分子学机制奠定基础。具体结果和关键数据包括以下几点：1）LSD1抑制剂（ S2101 或 CBB1007）通过上调H3K4me2，抑制新霉素所致的毛细胞死亡，实现毛细胞保护。2）上调H3K4me2的毛细胞保护作用是通过抑制经典凋亡通路caspas-3来实现的。3）我们通过表观遗传学机制研究发现，调控miRNA-182是通过抑制FOXO3a基因的转录而阻碍顺铂引起的毛细胞凋亡。4）另外，我们进一步通过线粒体的研究发现，TRMU表达降低显著增加了毛细胞样细胞对新霉素损伤的敏感性，这可能对我们的毛细胞保护具有重要的临床指导意义。通过本课题，我们通过调控组蛋白H3K4me2 水平提高内耳毛细胞的稳定性及对损伤的耐受能力，最终实现对目前常见感音神经性聋保护的目的，并对毛细胞损伤和保护的机制进行分子生物学探索，为临床毛细胞保护奠定实验依据和理论。