COP1调控ZBRK1/HMGA2信号通路促进胶质瘤增殖的机制研究

Malignant proliferation of glioma is an important factor to the deterioration and even death of the patients. However, the key biological mechanism of this process is still unclear. COP1, an ubiquitin ligase that discovered in recent years, is related to the development and progression of human cancers. In our preliminary experiments, we found that COP1 was highly expressed in the glioma tissues, which was able to promote the proliferation of glioma cells by increasing the transcription of HMGA2. To further explore the underlying mechanisms of COP1 promoted glioma cell proliferation, the COP1 was subjected for a screen in the human brain cDNA library. The ZBRK1 protein was identified to associate with COP1 in the screen experiment and the Co-IP result showed that ZBRK1 and COP1 could form into a complex. It has been reported that ZBRK1 is a transcriptional inhibitor of HMGA2. ZBRK1 always functions as a tumor suppressor in the development and progression of human cancers. Furthermore, COP1 was found to promote the ubiquitination and degradation of ZBRK1. These findings suggest that ubiquitination of ZBRK1 mediated by COP1 may be an important factor of HMGA2 activation. Thus, by using the glioma cells, clinical human glioma tissues and nude mice, we intended to study and clarify the mechanism that COP1 promoted the proliferation of glioma by inducing the ubiquitination and degradation of ZBRK1 to increase the transcription of HMGA2. Our results will provide a valuable idea and potential targets for molecular targeting therapy of gliomas.

胶质瘤恶性增殖是造成患者病情恶化甚至死亡的关键因素，然而介导这一进程的生物学机制尚不明确。近年来发现的COP1是一个与肿瘤发生发展密切相关的泛素连接酶。我们前期研究发现COP1蛋白在胶质瘤组织中表达上调，并可增强HMGA2转录水平而促进胶质瘤细胞增殖。为了深入研究COP1调控HMGA2转录水平的分子机制，我们通过酵母双杂交筛选获得了COP1的新相互作用蛋白ZBRK1。而ZBRK1作为HMGA2转录的负调控因子发挥抑癌作用。进一步的预实验发现COP1可促进ZBRK1的泛素化降解，提示COP1可能通过调控转录抑制子ZBRK1的泛素化降解而导致HMGA2转录异常激活，最终促进胶质瘤细胞增殖。因此，本项目拟采用胶质瘤细胞、胶质瘤标本和裸鼠原位模型，在分子、细胞与整体水平阐明COP1通过ZBRK1调控HMGA2转录而促进胶质瘤细胞增殖的分子机制，预期结果将为胶质瘤的靶向治疗提供新的思路和潜在靶点。

组成型光形态建成蛋白1 (Constitutively photomorphogenic 1，COP1)是首先在植物中被发现的一个高度保守的泛素连接酶。也是近年发现的一个与肿瘤发生发展密切相关的泛素连接酶。然而，COP1在胶质瘤中的作用和机制尚不清楚。因此，本项目拟采用体外培养的胶质瘤细胞、胶质瘤手术标本和裸鼠原位胶质瘤模型为研究对象，从分子、细胞与整体水平阐明COP1调控胶质瘤恶性增殖的作用和机制。. 在本项目的资助下，我们主要进行了如下研究：1、COP1在胶质瘤患者中的表达及在胶质瘤细胞增殖中的作用。2、COP1相互作用蛋白的筛选和鉴定。3、COP1与ZBRK1蛋白的相互作用验证。4、COP1与ZBRK1的调控关系。5、COP1/ZBRK1与HMGA2的调控关系。6、COP1与ZBRK1在胶质瘤患者中的表达相关性。7、COP1在体内移植瘤生长中的作用。获得如下研究结果：1、COP1在胶质瘤患者中高表达，沉默COP1抑制胶质瘤细胞增殖，相反过表达COP1则促进胶质瘤细胞增殖。2、发现ZBRK1是COP1的一个潜在相互作用蛋白。3、通过 Co-IP、WB 实验证明 COP1 和 ZBRK1存在相互作用。4、COP1不调控ZBRK1本身的蛋白水平。5、Q-PCR实验表明下调ZBRK1或COP1时，显著促进HMGA2的转录，相反过表达ZBRK1或COP1时，HMGA2的转录则被抑制；另外，共同下调ZBRK1和COP1后HMGA2转录的增加程度显著高于分别下调；并且，沉默COP1引起的HMGA2上调可以被过表达ZBRK1部分抑制。6、COP1在胶质瘤患者中表达显著上调，而ZBRK1在胶质瘤患者中表达显著下调，且两者呈现负相关。7、沉默COP1抑制体内移植瘤生长。. 研究结果表明：1、在胶质瘤中，COP1高表达，ZBRK1低表达，且呈现负相关性。2、COP1与ZBRK1相互作用，共同调节HMGA2的转录，ZBRK1的低表达，使其对HMGA2的转录抑制作用减弱，而导致HMGA2高表达，促进胶质瘤细胞增殖。这些研究结果提示：COP1/ZBRK1/HMGA2信号通路在胶质瘤恶性增殖中发挥重要作用，有可能作为胶质瘤分子靶向治疗的一个靶点。.