组蛋白甲基化转移酶G9a调控神经母细胞瘤增殖与自噬的分子机制研究

Neuroblastoma is a common solid tumour in children. Clinically, neuroblastoma is a heterogeneous group of tumors,with the characteristics of high malignancy and high risk of metastasis and recurrence. Therefore, the investigation of the mechanism of neuroblastoma development and the exploration of new targets for therapy is a effective approach to improve neuroblastoma patients survival rate. The result of our recent study showed that, the high expression of the histone methyltransferase G9a was associated with poor prognosis in neuroblastoma patients. Moreover, G9a played an important role in neuroblastoma cell growth and proliferation, and inhibition of G9a induced autophagy in neuroblastoma cells. However, the molecular mechanism of G9a regulation of neuroblastoma autophagy was still unclear. In this study, we investigated the signaling pathway in G9a regulation of neuroblastoma autophagy, and identified the target gene of G9a in autophagy. The main contents of the study are as follows: 1. screening the signaling pathway of G9a regulation of neuroblastoma autophagy by Microarray and transcriptome sequencing. 2. Identification of G9a target gene in G9a-mediated autophagy process by using ChIP-seq or ChIP-qPCR technology. 3. Investigation of the interaction of G9a and target gene by immunoblotting, GST pull-down, and Co-IP experiments. 4. Verification of the function of G9a target gene by inhibition or overexpression of the target gene, combined with amino acids, glucose, and lipids additional experiments. 5. Verification of the signaling pathway in G9a-mediated autophagy process by Flux and Metabolite analysis. 6. Investigation of the mechanism of G9a regulation of target gene in the process of autophagy by luciferase reporter experiment, promoter truncation/deletion, and EMSA experiments. Collectively, the aim of the study is to clarify the link between H3K9 methylation and tumor cell autophagy, and explain the molecular mechanism of G9a regulation of autophagy, then provide G9a as a potential therapeutic target for neuroblastoma treatment.

神经母细胞瘤是儿童最常见的颅外实体肿瘤，具有异质性、恶性程度高、易转移和易复发等特点，研究该肿瘤的发生机理，探索新的治疗方法、寻找新的治疗靶点是提高神经母细胞瘤病人生存率的一个有效途径。申请人近期研究发现，抑制组蛋白甲基化转移酶G9a能够抑制神经母细胞瘤的生长增殖并诱导其发生自噬，但G9a调控神经母细胞瘤自噬的作用机制尚不明确。因此，本项目拟通过Microarray及转录组测序等方法筛选G9a调控肿瘤细胞自噬的信号通路，采用ChIP-seq或ChIP-qPCR鉴定G9a介导自噬过程中调控的靶标基因；通过Co-IP、荧光素酶报告实验、启动子截短/缺失实验、EMSA等实验分析G9a调控自噬过程中作用于相关靶基因的分子机制并验证G9a靶基因的功能。本项目拟阐明H3K9甲基化与肿瘤细胞自噬调控之间的联系，从分子水平上阐释G9a调控自噬的作用机制，为神经母细胞瘤的临床治疗新策略提供理论依据及新思路。

神经母细胞瘤是一种在儿童中最常见的颅外实体肿瘤，具有高度异质性、恶性程度高、易转移和易复发等临床表现。神经母细胞瘤的发病率大约占儿童恶性肿瘤发生的7%-10%，仅低于白血病和中枢神经系统肿瘤。神经母细胞瘤的治疗比较困难，对化疗易产生耐药性，患儿发病后，除早期可以完全切除以外，其余病例即使经过大剂量化疗、手术切除、局部发射治疗、造血干细胞移植及生物治疗等，其长期生存率仍然小于40%，患者预后不良。约15%的患儿天然耐药，对诱导治疗无反应。神经母细胞瘤的治疗效果与诊断时患儿的年龄及临床分期有关， 患儿年龄越大分期越后， 肿瘤越容易扩散， 则预后越差， 即使获得完全缓解后， 50%以上的患儿也最终复发。因此，研究该肿瘤的发生机理，探索新的治疗方法和策略，对提高病人生存率及神经母细胞瘤的防治工作具有重要意义。本项目针对近年来表观遗传修饰在肿瘤发病机制中的调控作用等研究热点，以组蛋白甲基转移酶G9a调控神经母细胞瘤细胞自噬的作用机制为研究对象，通过G9a的小分子抑制剂药理性抑制G9a，以及通过shRNA介导的基因抑制G9a蛋白表达均能在神经母细胞瘤细胞中成功诱发强烈自噬，并且神经母细胞瘤细胞自噬后发生死亡。利用RNA-Seq检测分析，结果显示，G9a调控自噬过程中，前10个显著下调的信号通路为赖氨酸生物合成、DNA复制、细胞周期、p53信号通路、孕酮介导的卵母细胞成熟、碱基切除修复、卵母细胞的减数分裂、核苷酸切除修复、肿瘤中的MicroRNA、及基底细胞癌信号通路。经过qRT-PCR验证，赖氨酸生物合成途径中的关键限速酶基因在抑制G9a后显著下调，细胞周期信号通路中G1期的关键基因显著下调。同时，我们发现G9a与神经母细胞瘤原癌基因N-MYC关系密切，并且调控N-MYC基因启动子的表达。本项目的研究结果提示以G9a为靶点设计靶向药物具有一定的应用前景，为G9a作为肿瘤治疗靶标奠定了理论基础，并为探索神经母细胞瘤治疗新策略提供了思路，同时为开发小分子化合物一类的肿瘤分子靶向药物提供理论支持，具有重要的科学意义和应用转化潜能。