NEK-1和NEK-8参与介导pVHL维持肾癌细胞原纤毛结构稳定的机制研究

The primary cilia is a microtubule-based antenna-like structure that emanates from the surface of cell. It is a sensory organelle that receives both mechanical and chemical signals from the environment, and serves as hub for the transduction of various signaling pathways including Hedgehog, PDGF and Wnt. Loss and dysfunction of primary cilia are related to occurrence and development of cancer. It is reported that NEK-1 and NEK-8 are involved in ciliary disassembly. However, the precise mechanism remains unelucidated. Moreover, the mechanisms underlying NEK-1 and NEK-8 regulation are poorly understood. In the present project, we found that the Von Hippel-Lindau tumor suppressor protein, also known as pVHL (the protein encoded by the VHL gene), deregulated the expression of NEK-1 and NEK-8, and we would like to investigate the inherent mechanism in the next step. Based on an overall consideration of pVHL deregulating the expression of NEK-1 and NEK-8, and cells deficient in pVHL can not form primary cilia, we supposed that pVHL maintained primary cilia stability by regulating the NEK-1 and NEK-8 in renal cell carcinoma cells. Our current study will help to shed a new light on understanding NEK-1 and NEK-8 regulation, and improve our understanding the role of pVHL in primary cilia control. The present project will promote exploring the possibilities of discovering new anti-cancer drugs by targeting NEK kinase family or primary cilia.

原纤毛是以微管为结构基础并突出于细胞表面的"天线状"细胞器，其结构和功能异常与肿瘤发生发展密切相关。文献报道，NEK蛋白激酶家族成员NEK-1和NEK-8参与调控原纤毛结构，但相关调控机制及NEK-1、NEK-8自身表达和活性调控机制均有待于进一步研究。本项目中，我们首次发现抑癌基因VHL缺失的肾癌细胞NEK-1和NEK-8表达水平较高，且我们已证实pVHL参与NEK-1和NEK-8的表达调控，并将探讨相关机制。鉴于VHL缺失细胞不能形成原纤毛结构，我们推测NEK-1、NEK-8参与介导pVHL维持原纤毛的结构稳定，并拟加以试验考证、阐明相关机制。本项目结果将有助于阐明NEK-1和NEK-8表达及活性调控的机制，完善pVHL维持肾癌细胞原纤毛结构稳定的机制研究，拓展NEK-1和NEK-8在肿瘤学领域的功能性研究。课题成果将有助于推进靶向NEK家族蛋白和调控原纤毛结构的抗肿瘤新药的研发。

原纤毛（primary cilia）亦称不动纤毛，是以微管为结构基础并突出于细胞表面的“天线状”细胞器，其在细胞增殖、分化、神经生成、组织修复等过程中发挥重要功能，其结构和功能异常与多囊肾病、肾结核、色素性视网膜炎、Bardet-Biedl综合征及癌症等多种疾病的发生发展密切相关。现已证实NEK蛋白激酶家族成员NEK-1和NEK-8参与调控原纤毛结构，但NEK-1、NEK-8自身表达和活性调控机制、以及其调控原纤毛结构的机制均有待于进一步研究。. 本项目中，我们首次发现发现抑癌基因VHL缺失的肾癌细胞NEK-1和NEK-8表达水平较高，且证实pVHL参与NEK-1和NEK-8的表达调控。相关机制探讨结果显示，缺氧可诱导肿瘤细胞nek8基因表达上调。进一步分析nek8基因序列，发现其非编码区含有多个缺氧反应元件（HRE，5’-RCGTG-3’，R=A/G），Ch-IP实验证实nek8基因序列启动子区-643~-639片段为HIF识别元件。在siRNA介导HIF-1/2α表达下调的细胞中，缺氧仅可微弱上调NEK8表达；体内缺氧动物模型实验中，HIF-1及NEK8表达也呈不同程度上调。上述实验结果提示，nek8可能为缺氧诱导因子（HIFs）新靶基因。. NEK1蛋白调控试验中，除发现缺氧可上调NEK1表达外，VHL尚可促进NEK1的蛋白泛素化降解。提示，VHL可从HIF依赖性和HIF非依赖性两个途径下调NEK1/8蛋白水平，进而维持原纤毛结果稳定。. 免疫组织化学实验中，我们发现临床肾癌样本中VHL表达与NEK1/8表达呈负相关，且NEK1/8在癌组织中表达水平明显高于癌旁；siRNA介导的NEK1及NEK8表达下调可明显抑制肾细胞癌细胞系的体外增殖和迁移，推测NEK1/8可能参与肿瘤的发生/发展进程。. 本项目结果初步阐明NEK-1和NEK-8参与介导pVHL维持原纤毛结构稳定的生物学进程；并提示NEK-1和NEK-8在临床样品癌组织中的表达高于癌旁、下调其蛋白水平可明显抑制肿瘤细胞的增殖和迁移；课题延续工作将围绕“靶向NEK家族蛋白和原纤毛结构的抗肿瘤新药的研究开发”领域展开。