组蛋白甲基化转移酶SETDB1调控神经母细胞瘤增殖分化及分子机制研究

Neuroblastoma is a common childhood malignant tumor of neural crest origin, accounting for approximately 6%-10% of pediatric cancers and 15% of cancer-related deaths in children. Neuroblastoma is also a common malignant extracranial solid tumor of children, its annual incidence is about 1/10 million, just below the leukemia and central nervous system tumors. Malignant degree of neuroblastoma is high and its clinical manifestation includes high heterogeneity, easy metastasis, easy recurrence and poor prognosis, thus the treatment of neuroblastoma is difficult. Therefore, it is important to study the pathogenesis of neuroblastoma, explore the key targets for tumor cell proliferation and differentiation, and illustrate the mechanism of cell proliferation and differentiation, which can provide a new strategy for clinical treatment of neuroblastoma..Epigenetic regulation of tumor cell growth is one of the current research focuses, some reports show that histone methylation plays a key role in the growth of the tumor. SETDB1 is a histone methyltransferase, mainly involved in H3K9me3 methylation, the reports show that SETDB1 participate in mouse embryonic development, especially in nervous system development and the differentiation. In zebrafish experiments SETDB1 has been shown to promote the growth of melanoma, but the effects of cell proliferation and differentiation of SETDB1 in neuroblastoma has not been well studied. .In this study, we will manipulate the SETDB1 expression by up-regulation, down-regulation and rescue in neuroblastoma, and determinate the cell proliferation, self-renewal ability and differentiation capacity. Then we will investigate the molecular mechanisms of SETDB1 regulation cell proliferation and differentiation by microarray, Co-IP, GST pull-down, ChIP-qPCR, etc. in neuroblastoma. The main contents of the study are as follows: 1) Clarificate the effects of SETDB1 on cell proliferation of neuroblastoma. 2) Verify the effects of SETDB1 on cell differentiation of neuroblastoma. 3) Investigate of the mechanism of SETDB1 regulation cell proliferation and differentiation of neuroblastoma. 4) study on the effects of SETDB1 on cell proliferation and differentiation of neuroblastoma in vivo. Collectively, the aim of the study is to clarify the link and the molecular mechanism between SETDB1and neuroblastoma cell proliferation and differentiation, then provide SETDB1 as a potential therapeutic target for neuroblastoma treatment.

抑制神经母细胞瘤（NB）增殖及诱导分化成为治疗NB的新方向。组蛋白H3K9me3甲基化转移酶SETDB1对细胞增殖分化有重要作用，研究SETDB1对NB细胞增殖分化的影响，揭示其分子机制具有重要意义。本项目通过在NB细胞中对SETDB1的表达进行干预，研究NB细胞的增殖、自我更新能力及分化能力，揭示SETDB1对NB细胞增殖分化的影响；进而通过表达谱芯片、Co-IP、GST pull-down、ChIP-qPCR等手段，解析SETDB1参与NB细胞增殖分化的分子机制。主要包括以下研究内容：1) SETDB1对NB细胞增殖的影响；2) SETDB1对NB细胞分化的影响；3) SETDB1影响NB细胞增殖分化的分子机制研究；4) SETDB1影响NB细胞在免疫缺陷鼠体内成瘤实验研究。本项目从分子、细胞、动物模型三个层次解析SETDB1对NB细胞增殖分化的作用及机制，为NB的临床治疗提供新理论。

神经母细胞瘤是一种儿童最常见的颅外实体肿瘤，临床表现具有恶性度高、多样性和易转移等特点。该病多发于10岁以下的儿童，大约占儿童肿瘤发生的8-10%，年发病率大约为1/10万，仅低于白血病和中枢神经系统肿瘤，虽少数可自行缓解，但是大多数预后较差，神经母细胞瘤具有明显的异质性，对化疗易产生耐药性，即使经过大剂量化疗、手术切除、局部放射治疗、造血干细胞移植及生物治疗等，其长期生存率仍然小于40%。大约15%的患儿天然耐药，对诱导治疗无反应。获得完全缓解后50%以上的患儿最终复发，因此探索新的治疗方法是提高NB病人生存率的一个有效途径。.组蛋白H3K9me3甲基化转移酶SETDB1对细胞增殖有重要作用，研究SETDB1对神经母细胞瘤细胞增殖的影响，揭示其分子机制具有重要意义。本项目通过在神经母细胞瘤细胞中对SETDB1的表达进行干预，研究神经母细胞瘤的增殖、自我更新能力以及小鼠成瘤能力，揭示SETDB1对神经母细胞瘤增殖能力的影响；进而通过表达谱芯片，解析SETDB1参与神经母细胞瘤增殖的分子机制。主要得到以下研究结果：1）明确了SETDB1对神经母细胞瘤增殖的影响，2）通过表达谱芯片检测下调SETDB1引起神经母细胞瘤增殖抑制的信号通路，筛选出SETDB1显著影响神经母细胞瘤的固醇类生物合成、DNA复制、细胞周期、嘌呤代谢、嘧啶代谢等信号途径，其中对固醇类生物合成途径影响是最为明显的通路之一。3）固醇类合成途径的来源主要是胆固醇，进一步研究了SETDB1对神经母细胞瘤胆固醇代谢的影响及分子机制。4）通过对临床数据库及表达谱芯片结果分析显示，SETDB1与MYCN有密切的关系，为了明确SETDB1与MYCN的功能的影响，对MYCN影响神经母细胞瘤胆固醇代谢影响进行了研究。5）研究SETDB1调控固醇调控元件结合蛋白SREBF2，从而影响神经母细胞瘤胆固醇代谢的分子机制。