一个新的Rb泛素连接酶在肝癌发生中的分子机制研究和临床意义探讨

We previously identified a novel Rb E3 ubiquitin ligase (NRBE3), which binds Rb and promotes Rb degradation. We found recently that NRBE3 is regulated by E2F, suggesting that NRBE3 might form a regulation loop with Rb/E2F1. Western blotting shows that NRBE3 protein band is about 40kD larger than the size deduced from its sequence. Bioinformatics analysis suggests that two phosphorylation sites and two SUMOylation sites exist in NRBE3, which may cause slower migration of NRBE3 on Western blot. It is of important that expression of NRBE3 is up-regulated in hepatocellular carcinoma (HCC) tumor tissues and NRBE3 band migrates slower in HCC tissues than that in the non-tumorous counterpart liver tissues. These results raise following questions: How is NRBE3 up-regulated in HCC? Is the posttranslational modification of NRBE3 changed during tumorigenesis of HCC and how does the changed posttranslational modification of NRBE3 function in the tumorigenesis of HCC? In addition, it was suggested that NRBE3 might be regulated by a microRNA. In this proposal we will investigate the transcriptional and post-translational regulation of NRBE3 aiming to elucidate the mechanism by which NRBE3 is up-regulated in HCC. We will evaluate the effect of the posttranslational modifications of NRBE3 on its biological function and study the posttranslational modification change of NRBE3 in HCC aiming to uncover a novel mechanism by which NRBE3 functions in tumorigenesis of HCC. Furthermore, expression profile of NRBE3 in HCC will be evaluated in a large sample. The relationship between NRBE3 and the development of HCC, the clinical characteristics and the survival of HCC patients will be analyzed to explore the potential of NRBE3 as a indicator for diagnosis and/or prognosis of HCC. We will establish a mouse model for NRBE3 knockout aiming to elucidate the function of NRBE3.

我们发现一个新的Rb 泛素连接酶NRBE3 (A Novel Rb E3 ligase)，结合Rb促进Rb泛素化和降解。NRBE3受到E2F1的调节，与Rb/E2F1存在反馈调节；NRBE3蛋白比预测大40kD，信息学分析NRBE3存在磷酸化和SUMO化位点，提示NRBE3存在翻译后修饰；NRBE3在肝癌组织中表达升高、肝癌组织中的蛋白条带比肝硬变中移动慢，提示NRBE3在肝癌发生过程中可能发生了突变或翻译后修饰改变。信息学发现一个NRBE3相关的microRNA。本课题将研究NRBE3的转录调节、microRNA对NRBE3的转录后调节、翻译后修饰对其生物学功能的影响、NRBE3在肝癌发生进展中表达上调的机理以及存在的突变或修饰改变在癌变过程中的作用、探讨NRBE3与肝癌临床分期和预后的相关性。建立NRBE3 knockout小鼠，观察实验动物表型和对肿瘤的罹患性，阐明NRBE3的功能。

RB是一个重要的抑癌基因产物，一些肿瘤相关蛋白通过结合并降解RB在肿瘤的发生发展中发挥作用，然而，只有MDM2被发现是促进RB蛋白泛素化后在蛋白酶体降解的E3泛素连接酶。本研究发现了一个新的促进RB降解的E3泛素连接酶NRBE3(A Novel RB E3 ligase)，通过结合并降解RB促进肿瘤细胞增殖，并发现NRBE3通过U-box发挥E3泛素连接酶的作用；进一步发现，NRBE3在乳腺癌转移中通过调节E-cadherin的表达促进乳腺癌转移；而且，NRBE3在肝癌中的表达升高，并与患者不良预后相关。对NRBE3的翻译后修饰进行研究发现了两个SUMO化位点，这两个位点的SUMO化修饰是NRBE3降解RB所必需，并提示NRBE3的SUMO化可能与其它翻译后修饰相互调节，目前正在进行深入研究。同时，在研究NRBE3是RB蛋白的泛素连接酶时，发现NRBE3的一个结合蛋白hUTP14a也具有降解RB蛋白的功能，对hUTP14a降解RB的分子机制进行深入研究发现：hUTP14a通过一个新的E3 ligase motif 发挥作用，这个motif富集了亮氨酸(Leucine Rich Region, LRR)，这个研究结果为鉴定新的泛素连接酶提供了科学依据。本研究已经得到了Nrbe3 基因敲除小鼠，目前正在对基因敲除小鼠的表型进行研究。