恶性胶质瘤诱导分化中核孤儿受体Nor1的转录调控功能研究

Glioma is the first commonly malignant tumor in central nervous system and is noted for its poor prognosis. So far there is no effective treatment, partly because of its poor differentiation phenotype. We first demonstrated that Cholera toxin can induce glioma differentiation. We used cDNA microarray analysis and had found that the orphan nuclear receptors NR4As family number Nor1 was predominantly up-regulated within the C6 glioma cell differentiation. Then we confirmed the indispensable effect of NR4As for the differentiation. To explore the specific mechanism of NR4As facilitating the differentiation, we constructed fusion vector bait gene of Nor1 and found SNF5. Meantime, we found that Nor1 was located in the nuclear during differentiation. Based on these, we focus our research on the transcriptional regulation of Nor1. We plan to use confocal, CoIP to confirm the protein interaction between Nor1 and SNF5, then use FL and truncated form of Nor1 to narrow down the binding domain with SNF5. After that, we use EMSA to test the transcription activity of Nor1, and use Dominant negative expression to check downstream gene expression. With these multiple confirmations, we can demonstrate that the transcriptional regulation of Nor1 plays a critical role in the differentiation. The research will bring the light on the transcriptional regulation mechanism of Nor1 as an essential activator of glioma differentiation, and provides new theoretical basis for the differentiation therapy of malignant glioma.

恶性胶质瘤是中枢神经系统第一高发肿瘤，其分化幼稚，恶性度高，目前仍缺乏有效治疗手段。本组首次证明霍乱毒素对恶性胶质瘤细胞具诱导分化作用。后用基因芯片检测发现核孤儿受体Nor1等在分化中持续过表达，并证实了其在恶性胶质瘤C6细胞分化中的关键作用。为深入探讨其作用机制，我们设计了Nor1序列诱饵基因进行共作用因子筛选，得到转录调节因子SNF5，同时发现了分化中Nor1的细胞核定位线索。基于以上工作基础，本研究以Nor1转录活性调控为中心，拟采用激光共聚焦、免疫共沉淀确认Nor1与SNF5的蛋白质相互作用；进而找寻其与SNF5结合的蛋白功能结构域；利用EMSA检测Nor1的转录活性；通过负显性表达检测下游基因表达和分化表型，以证实其转录调控活性在C6分化中的重要性。本研究的完成有望阐明胶质瘤诱导分化中Nor1转录调控的分子生物学机制，并为建立恶性胶质瘤的诱导分化疗法提供新的理论依据和精确的靶点。

恶性胶质瘤是中枢神经系统最常见的肿瘤，其分化幼稚，恶性度高，目前仍缺乏有效的治疗手段。本研究在前期基因芯片测试的基础上，筛选出在生物毒素-霍乱毒素诱导大鼠胶质瘤细胞C6分化中持续高表达的核孤儿受体家族成员Nor1、Nur77为研究对象，利用SiRNA和过表达技术正负调控Nur77的胞内水平，双向证明了其在恶性胶质瘤分化中的关键作用；在此基础上，针对预实验中筛选得到的转录调节因子SNF5，我们构建Nor1系列截短体载体以探究2者的结合序列，实验结果证实SNF5确与Nor1有蛋白质相互结合左右，且结合位点是Nor1的染色质结合区（DBD）；最后将基因芯片得到的结果与其他肿瘤的研究结合，为探讨胶质瘤及其他类型肿瘤细胞治疗中可能的关键性分子作用提供线索，为恶性胶质瘤的诱导分化疗法提供新的理论依据和精确的靶点。