Cullin3/TNFAIP1泛素连接酶复合体调控抑癌蛋白RhoB降解的机制与功能研究

During the onset and progression of cancer, the degradation of tumor suppressor proteins regulated by ubiquitin-proteasome system is the most important mechanism for promoting the tumorigenesis of cancer. Our previous study found that Cul3/TNFAIP1 ubiquitin ligase is over-expressed in liver cancer, which resulting in the degradation of RhoB. Moreover, the inhibition of Cul3/TNFAIP1 pathway could cause RhoB marked accumulation and prolong the half-life of RhoB. These findings suggested that: Cul3/TNFAIP1 ubiquitin ligase may regulate the degradation of RhoB and then promote the progression of the liver cancer. The inhibition of Cul3/TNFAIP1 pathway could cause RhoB accumulation, leanding to a significant anticancer effect. To test this hypothesis, we plan to carry out intensive studies in this project to elucidate the interaction between Cul3/TNFAIP1 and RhoB and the degradation mechanism of RhoB regulated by Cul3/TNFAIP1 pathway. We will further evaluate the feasibility of anti-hepatocarcinoma therapy through the accumulation of tumor suppressor RhoB induced by the inhibition of Cul3/TNFAIP1 ubiquitin ligase. The success of this project will validate the role of Cul3/TNFAIP1 ubiquitin ligase for the degradation of RhoB and identify the Cul3/TNFAIP1 ubiquitin ligase as the novel anti-hepatocarcinoma target.

在肿瘤发生发展过程中，泛素-蛋白酶体系统介导的抑癌蛋白底物降解是一种重要的促癌机制。研究发现，Cullin3/TNFAIP1多亚基泛素连接酶在肝癌组织内显著高表达，与抑癌蛋白RhoB的表达呈显著负相关；而且，灭活Cullin3/TNFAIP1导致RhoB显著积聚，蛋白半衰期明显延长。据此，我们提出以下假说：Cullin3/TNFAIP1泛素连接酶可通过调控RhoB的降解而促进肝癌发生发展，而靶向灭活Cullin3/TNFAIP1可通过诱导RhoB的积聚产生抗肝癌效果。为检验该假说，我们拟研究Cul3/TNFAIP1与RhoB的蛋白相互作用及其对RhoB泛素化修饰的调控作用，进而评价通过靶向灭活Cul3/TNFAIP1诱导RhoB积聚杀伤肝癌细胞的治疗策略。本项目的成功完成有助于进一步阐明肝癌发病机理并进而鉴定Cul3/TNFAIP1为新的抗肝癌靶点。

在肿瘤发生发展过程中，泛素-蛋白酶体系统介导的抑癌蛋白底物降解是一种重要的促癌机制。我们前期研究发现，Cullin3/TNFAIP1多亚基泛素连接酶在肝癌组织内显著高表达，与抑癌蛋白RhoB的表达呈显著负相关；而且，灭活Cullin3/TNFAIP1导致RhoB显著积聚，蛋白半衰期明显延长。本项目旨在探讨Cullin3/TNFAIP1与RhoB的蛋白的相互作用及其对RhoB泛素化修饰的调控作用，进而评价通过靶向灭活Cullin3/TNFAIP1诱导RhoB积聚杀伤肝癌细胞的治疗策略。. 我们经免疫共沉淀证明TNFAIP1与RhoB蛋白间存在相互作用，并且发现Cullin3及TNFAIP1均能明显促进肝癌细胞中RhoB的泛素化降解。ATPlite及克隆形成分析结果显示，下调Cullin3可显著抑制肝癌细胞HepG2的增殖与克隆形成。此外，回复实验证实下调Cullin3可诱导肝癌细胞中RhoB蛋白的显著积聚，而敲低RhoB不仅能部分恢复Cullin3下调导致的RhoB蛋白积累，也能部分恢复Cullin3下调对肝癌细胞增殖和克隆形成的抑制作用。. 本项目成果证实了Cullin3/TNFAIP1通过诱导RhoB蛋白的泛素化降解进而刺激肝癌细胞增殖，从而促进肝癌的发生发展。本项目为靶向灭活Cullin3/TNFAIP1泛素连接酶诱导抑癌蛋白RhoB积聚杀伤肝癌细胞的治疗策略提供了科学依据。