circEts-1抑制神经母细胞瘤Ets-1表达及侵袭转移的机制研究

The main death cause for neuroblastoma is tumor invasion and metastasis. Ets-1 is closely related to tumor invasion and lymph node metastasis，however its regulation mechanisms remain unclear. Our previous investigation has established that circEts-1 downregulates Ets-1 protein levels in neuroblastoma cell line, The results of our preliminary experiments showed that circEts-1 bond with VCP, enhanced VCP interaction with COP1, and resulted in Ets-1 ubiquitination and degradation, thus reducing the invasiveness and metastasis of neuroblastoma. Therefore, we proposed a scientific hypothesis: circEts-1 targets Ets-1 ubiquitination and degaradation via enhancing protein interaction of VCP with COP1,thus inhibits neuroblastoma invasion and metastasis. To validate this hypothesis and further explore its underlying mechanisms, we plan to apply various kinds of techiniques including gene trasfection and knockdown, two dimentional electrophoresis and mass-spectrometry, RNA immunoprecipitation, RNA EMSA, co-immunoprecipitation, and protein FRET, along with cell biology assay and animal xenografted tumor model. The results of the study would deepen the knowledge for neuroblastoma occurrence and provide new choices for clinical treatment and prevention.

神经母细胞瘤患儿死亡的主要原因是肿瘤的侵袭转移，Ets-1是调控神经母细胞瘤恶性进展的关键靶标，但调控机制不明。前期研究发现在神经母细胞瘤中COP1可通过泛素化降解下调Ets-1表达；而circEts-1可通过结合VCP蛋白，增强VCP与COP1的相互作用，促进COP1对Ets1的泛素化降解，从而抑制肿瘤侵袭。因此，我们提出“circEts-1通过结合VCP蛋白，增强VCP与COP1的相互作用，促进COP1对Ets-1的泛素化降解，从而抑制神经母细胞瘤侵袭转移”的假说。拟利用基因转染、二维电泳质谱检测、RNA免疫沉淀、RNA-EMSA及蛋白免疫共沉淀、FRET等分子生物学技术，结合细胞学动物学实验方法，进一步研究circEts-1对Ets-1的调节作用，并对其机制进行深入研究，为临床诊治神经母细胞瘤提供新的思路。

本项目拟在前期试验的基础上，检测神经母细胞瘤组织和细胞株中circEts-1表达水平及其与Ets-1表达、患者临床预后的相关性；构建circEts-1过表达及敲低载体，运用免疫共沉淀、Western blot、蛋白酶体抑制剂处理等方法，检测circEts-1对神经母细胞瘤细胞中Ets-1泛素化降解的调控作用；采用RNA免疫共沉淀、RNA EMSA、蛋白免疫共沉淀、荧光能量共振转移 (FRET) 等方法，探索神经母细胞瘤中circEts-1与VCP蛋白的相互作用及其对VCP蛋白结合COP1蛋白、Ets-1泛素化降解的调控作用；筛选稳定过表达或敲低circEts-1、检测circEts-1对神经母细胞瘤细胞体内外侵袭转移的影响，阐明circEts-1调控神经母细胞瘤Ets-1表达的作用及分子机制，深入了解环状RNA在肿瘤发生发展过程中的作用及其分子机制，为提高神经母细胞瘤的诊治水平提供新途径及理论依据。. 课题组根据研究计划进行了为期3年的研究，基本按照研究计划进行，包括明确circEts-1在神经母细胞瘤中的表达水平，解析circEts-1对神经母细胞瘤组织和细胞中Ets-1泛素化降解的调控作用，明确了circEts-1对神经母细胞瘤组织和细胞中VCP与COP1蛋白相互作用，并完成了部分动物试验。