锌指蛋白ZER6/p21信号通路在细胞周期调控中的作用机制研究

Zinc finger protein ZER6 is a member of Cys2His2 like (C2H2) zinc finger protein family. ZER6 displays aberrantly low expression level in tumor lesions; however, its biological function is poorly characterized. Cell cycle is a tightly controlled fundamental process in living cells, and is closely related to other biological functions, including cell proliferation and apoptosis. Cell cycle progresses in a directional manner following well-ordered events, and defects in cell cycle regulation are closely linked to various abnormalities, such as unlimited cell proliferation that leads to cancer. Despite of its critical function, cell cycle control mechanism remains as a basic scientific problem that has not been totally elucidated. In our previous experiment, we have performed a screening for determining factors that could regulate the transcriptional activity of p21, a master regulator of cell cycle, by using a short hairpin RNA (shRNA) expression vector library. Through this screening, we found that knocking down ZER6 significantly increased p21 transcriptional activity; however, the detail mechanism of ZER6 regulation on cell cycle progression remains to be elucidated. Accordingly, in this study, we will unravel the novel role of ZER6 in cell cycle regulation. To this end, we will perform investigations as below: (1) we will elucidate the role and molecular mechanism of ZER6 in regulating p21 and cell cycle; (2) we will unravel the function of tumor suppressor gene p53, which is an upstream transcriptional regulator of p21, in regulating ZER6/p21 pathway; (3) we will elucidate the roles of ZER6/p21 pathways in tumor cells proliferation and apoptosis, and subsequently, in tumorigenesis. This study will not only uncover novel biological roles of ZER6, especially in cell cycle regulation, but also will provide new insights regarding the molecular mechanisms of cell cycle. Subsequently, our study will also give new perspective regarding the potential of using ZER6 as a marker for diseases related to cell cycle defect, as well as its potential as novel molecular target for treating them.

锌指蛋白ZER6属于Cys2His2 like（C2H2）型锌指蛋白，在肿瘤细胞中呈异常表达，然而有关它在生物功能中的作用机制报道极少。细胞周期调控机制紊乱是导致肿瘤细胞无限增殖的根本原因。课题组前期应用shRNA表达质粒文库对影响细胞周期抑制因子p21转录活性的因子进行高通量筛选，发现敲减锌指蛋白ZER6显著促进p21转录活性，然而，ZER6调控细胞周期的详细分子机制尚待深入探讨。本课题拟从以下三方面展开研究：（1）揭示锌指蛋白ZER6对p21的作用机制及其对细胞周期调控的影响；（2）阐明肿瘤抑制因子p53在ZER6/p21信号通路中的作用机制；（3）解析ZER6/p21信号通路在细胞周期及其相关的细胞增殖及凋亡中的作用。通过本项目研究将揭示锌指蛋白ZER6/p21信号通路在细胞周期调控中的分子机制，为细胞周期调控提供新的理论基础，也为肿瘤等细胞周期紊乱相关疾病的诊治提供生物学新靶标。

锌指蛋白在细胞周期进程中发挥重要作用，与疾病的发生发展密切相关。锌指蛋白ZER6属于C2H2型锌指蛋白，于2002年被发现。然而有关它在生理病理的功能及作用机制尚未有研究报道。课题组前期通过应用shRNA敲减质粒文库对细胞周期抑制因子p21转录活性的调控因子进行高通量筛选，研究发现ZER6在临床结肠癌组织中异常高表达，ZER6敲减后能显著促进p21转录活性。通过本项目的开展，课题组已取得了如下研究成果：（1）本项目首次发现锌指蛋白ZER6亚型p52-ZER6能通过增强抑癌基因p53与MDM2的结合促进p53蛋白泛素化诱发p53蛋白质降解，抑制p53及其下游周期因子p21的表达，促进肿瘤细胞周期进程。（2）项目研究揭示了锌指蛋白ZER6两种亚型p71-ZER6和p52-ZER6的氨基酸序列和结构差异是导致它们生物活性功能不同的关键因素。研究结果显示p71-ZER6具有p52-ZER6所含有的氨基酸序列，而p52-ZER6氮末端缺乏完整KRAB结构域和HUB1结构域，由于它们蛋白结构的差异导致了它们表现出不同的生物学功能。研究结果表明p52-ZER6能促进p53蛋白质降解从而抑制p21的转录水平，然而，p71-ZER6则不能促进p53蛋白降解，研究结果揭示了位于p71-ZER6亚型的氮末端HUB1结构域阻碍了这一重要生物功能，从而使其无法促进p53蛋白降解。（3）本项目研究同时也探讨了p52-ZER6作为判断肿瘤患者是否适用MDM2-p53结合抑制剂类抗癌药物，以及基于p52-ZER6抑制剂与MDM2-p53结合抑制剂联合使用治疗的抗肿瘤治疗新策略。研究结果显示p52-ZER6能显著抑制肿瘤细胞对MDM2-p53结合抑制剂的敏感性，因此，p52-ZER6高表达的患者不适合使用基于MDM2-p53结合抑制剂的抗肿瘤治疗。通过动物荷瘤实验，我们证明了p52-ZER6抑制剂能增强MDM2-p53结合抑制剂的治疗敏感性，p52-ZER6抑制剂和MDM2-p53结合抑制剂表现出显著的抗肿瘤协同作用。这些研究结果表明p52-ZER6抑制剂与MDM2-p53结合抑制剂的联合疗法有望成为有效的抗肿瘤新策略。综上所述，本项目揭示了p52-ZER6是一个调控MDM2/p53通路的新致癌基因，并阐明了其调控MDM2/p53通路的分子机制，同时探讨了该新机制在抗肿瘤精准治疗方面的应用。