RND3在人脑胶质母细胞瘤细胞增殖调控中的作用及机制研究

Glioblastoma multiforme（GBM）is the most common and deadliest malignant primary brain tumor in humans. There is a huge lack of understanding the mechanisms of tumorigenesis and uncontrollable glioblastoma cell proliferation. In our preliminary study, we observed a significant downregulation of RND3 (a small Rho GTPase) gene expression in human GBM tissues. The RND3 expression levels were reversely correlated with tumor size and patient survival rate. RND3 stable expression in GBM cells significantly reduced the cell growth rate in vitro. Forcing RND3 expression in GBM cells remarkably attenuated GBM growth in vivo in a tumorigenesis experiment in nude mice, suggesting that RND3 is a new anti-GBM factor. Two aims are proposed: 1)To investigate the relationship between RND3 gene expression levels and human GBM growth. A Large amount of human GBM tissues will be collected from our GBM Research and Treatment Center, which will provide a rich tissue resource for the project. Using the same approaches as we conducted in our preliminary study, RND3 expression levels will be assessed along with tumor size, patient survival time, and treatment efficiency. The outcome of the proposal will be to establish direct links between RND3 expression levels and GBM cell proliferation/tumor growth and patient prognoses. 2)To explore the molecular mechanism of RND3-mediated GBM cell proliferation inhibition, eventually leading to tumor repression. Our recent study found that RND3 is a new regulator of Notch signaling. RND3 deficiency resulted in decreased Notch intracellular domain (NICD) protein degradation through repressing NICD protein ubiquitination. This eventually enhanced Notch signaling (PNAS in press). It is well known that upregulation of Notch signaling facilitates GBM tumorigenesis and growth. In this study, we propose that RND3 functions as a new factor that regulates Notch complex in GBM cell proliferation. By using loss- and gain-of-function approaches, we propose to demonstrate that RND3 manipulates GBM cell proliferation and tumor growth partially through Notch signaling pathway. We have established human GBM cell lines with both stable RND3 expression and RND3 deficiency. Meanwhile, we generated RND3 knockout mice, which provide a unique mouse model for our mechanistic study. The innovation of proposal includes:1)elucidation of RND3 downregulation in human GBM patients;2)establishment of the reverse relationship of RND3 expression levels with GBM tumor size and patient prognosis;3) demonstration of the anti-GBM role of RND3;4)elucidation of the RND3→NICD→Notch signaling→GBM cell proliferation inhibition→GBM repression mechanism;5)implication of manipulating RND3 expression levels as a potential therapeutic target. Since RND3 downregulation was observed in GBM patients, understanding its pathogenic effect and the associated mechanism has clear basic and clinical significance. The results will provide new information in GBM growth and treatment.

本课题研究小G蛋白RND3基因在人脑胶质母细胞瘤（GBM）细胞增殖中的作用和机制。RND3是Rho激酶抑制因子，在人脑GBM中的作用尚未研究。我们前期工作发现，RND3表达水平在人脑GBM中明显低于正常脑组织。过表达RND3可显著地抑制GBM细胞在体外的增殖和小鼠脑内的生长，提示RND3参与GBM细胞的增殖调控。机制实验揭示RND3介导的肿瘤抑制效应可能与Notch1信号通路有关。前期实验证实，RND3可以与Notch1活性形式NICD结合进而降低细胞核NICD蛋白水平。因此我们提出假说：RND3抑制GBM细胞增殖， 其机制是通过降低细胞核内NICD蛋白水平，进而抑制Notch1信号通路。具体实验包括：1）检测RND3在人脑GBM中的表达量并分析其与患者预后的关系。2）体内外实验探讨RND3在GBM细胞增殖及Notch1信号通路中作用与机制，并进一步研究RND3结合降低NICD的分子机制。

胶质母细胞瘤是成人中枢神经系统最常见最致命的原发性恶性肿瘤。RND3是Rho激酶抑制因子，在人脑GBM中的作用尚未研究。本项目研究RND3在人GBM细胞增殖中的作用及机制。实验发现：RND3 在人脑 GBM 组织内异常低表达，其表达量与患者的生存时间、肿瘤大小及 HistoneH3 磷酸化水平均呈负相关；RND3 可以抑制 GBM 细胞的增殖； RND3 通过 Notch1 信号通路调节 GBM 细胞的增殖；RND3 通过调节 NICD-CSL-Maml1 集团调节 Notch1 信号通路，而这一过程中起关键作用的是 RND3 对 NICD 的调节。综上我们得出结论： RND3 是一个抑癌基因，其可以通过调节 Notch1 信号通路调控 GBM 细胞的增殖，分子机制是降低 NICD-CSL-Maml1 集团的表达量，而这一过程中起关键作用的是 RND3 对 NICD 的调节。本项目的研究成果有助于促进RND3治疗胶质瘤的临床研发，并有助于研究联合应用分子靶向治疗和化疗治疗胶质瘤的新方法，为胶质瘤的治疗开辟新的途径。