Prdx1-Spy1通过增强CDK2介导的p27降解在肝细胞肝癌发生中的意义

p27kip1 is an important tumor suppressor that is critically involved in the development of Hepatocellular carcinoma (HCC). The degradation of p27 was mainly attributed to CDK2-mediated phosphorylation on Thr187 residue.We and other research groups have recently identified Spy1 as a novel CDK2 activator. Spy1 binds and promotes the activation of CDK2, thereby triggering the phosphorylation and consequent degradation of p27 as well as the progression of HCC. In the present study, we show that Peroxiredoxin 1 (Prdx1) is a novel binding partner of Spy1, and the interaction of the two protein promotes the degradation of p27. Based on these findings, we aim to first clarify the correlation among the expression of Prdx1, Spy1 and p27 in HCC clinical specmens. Furthermore, we will explore the impact of Prdx1-Spy1 interaction on the kinase activity of CDK2, the phosphorylation and degradation of p27. Moreover, the influence of the molecular mechanism underlying Prdx1-Spy1 interaction on the proliferation and migration will be analyzed. Finally, we will investigate the role of Prdx1-Spy1 interaction on the progression of HCC via the degradation of p27 using nude mouse model. Our study may help gain a better insight into the mechanism underlying HCC progression.

p27kip1是肝细胞肝癌（Hepatocellular carcinoma，HCC）发病过程中的一个重要抑癌基因。p27的降解主要受CDK2介导的磷酸化调节。我们和其他课题组先前的研究表明，Spy1蛋白作为一个CDK2激活分子，可结合并活化CDK2，促进p27的磷酸化降解和HCC的进程。本课题中，我们鉴定发现过氧化物酶1（peroxiredoxin 1，Prdx1）是Spy1的一个新的相互作用蛋白，并且Prdx1具有促进p27降解的能力。基于此，我们拟首先在HCC临床组织中阐明Prdx1、Spy1和p27的表达关系，进而分析Prdx1和Spy1的结合对CDK2的激酶活性以及p27磷酸化和降解的影响，并研究此作用分子机制对HCC细胞增殖和迁移的影响；最后，我们拟在裸鼠动物模型中分析Prdx1和Spy1通过促进p27的降解对肝细胞肝癌发生发展的影响。

氧化应激对肿瘤形成和进程的促进作用一直被研究者所关注，但其机理一直未明。近年来发现Prdx家族分子可作为氧化应激的感应分子，触发氧化应激下游信号传导。本研究通过免疫沉淀-质谱分析（IP-MS）在肝癌组织中鉴定出与Spy1存在相互作用的蛋白质Prdx1，通过免疫沉淀和免疫荧光在肝癌细胞中验证两者的相互作用及相互作用结构域，并且通过GST-pull down实验证实两者存在直接相互作用。为了阐明Prdx1与Spy1相互作用的分子机制，本研究中成功构建了Prdx1的抗氧化位点突变体，证实野生型和突变型Prdx1与Spy1均存在相互作用，且两者的相互作用可以促进p27的泛素化降解。在细胞水平，CCK-8、Edu和克隆形成实验证明Prdx1/Spy1的干扰表达可以抑制肝癌细胞的增殖和集落形成。在动物水平，裸鼠皮下成瘤实验证实Prdx1通过与Spy1相互作用促进肝癌的发生发展。在临床水平，选取8对新鲜的肝癌及配对的癌旁组织，Western blot结果显示p27kip在肝癌组织中表达下降，而Prdx1、Spy1在肝癌组织中表达上调。同时选取100例具有完整临床病理学资料的肝癌患者组织，免疫组化显示p27kip与Prdx1、Spy1的表达水平呈负相关，且三者表达水平与肝癌的组织学分级、TNM分期密切相关，与年龄、性别、肿瘤大小等参数无相关性。P27kip的低表达、Prdx1高表达、Spy1高表达与肝癌患者的不良预后显著相关。我们的研究结果表明，Prdx1/Spy1介导的p27泛素化可能成为肝癌发生的新型机制，为肝癌的临床治疗提供新的实验依据。