MPP3调控肝癌细胞迁移和侵袭的分子机制研究

It has been demonstrated that membrane-associated guanylate kinase (MAGUK) family participate in the regulation of development of cancer in recent studies, but their roles in hepatocellular carcinoma (HCC) remain unclear. Our previous study ,supported by the National Key Sci-Tech Special Project of China （2008ZX10002-021）,has shown that membrane palmitoylated protein 3(MPP3), a member of MAGUK family ,is significantly up-regulated in HCC cell lines of high metastatic potential and HCC tissues ,and the overexpression of MPP3 correlated with poor prognosis in patients with HCC. Furthermore, we demonstrated that MPP3 promotes HCC cell migration and invasion. However, its molecular mechanism has not elucidated yet. In our follow-up study, we found MPP3 interacting with cyclin-dependent kinase 5(Cdk5) could be phosphorylated by Cdk5.In according to our precious work as well as literature research, we propose that MPP3 phosphorylation by Cdk5 regulates HCC cell migration and invasion,which will extend the finding of our precious work and unfold the molecular mechanism of MPP3 in regulation of HCC cell migration and invasion. By means of this study, it will provide important theoretical basis for elucidating the mechanism of invasion and metastasis and finding therapeutic targets of anti-metastasis and relapse of HCC.

新近研究表明膜结合鸟苷酸激酶（MAGUK）家族作为细胞内支架蛋白参与调控癌症的发生和发展，但其在肝癌中的作用未知。我们的前期研究在国家科技重大专项基金（2008ZX10002-021）的支持下，首次发现MAGUK家族成员棕榈酸酰化膜蛋白3（MPP3）在高转移潜能肝癌细胞株和肝癌组织中均显著性上调，且其过表达与肝癌患者预后差有关，并进一步证实MPP3促进肝癌细胞的迁移和侵袭，但其分子机制未明。我们的后续研究发现MPP3能与细胞周期素依赖蛋白激酶5（Cdk5）外源互作且在体外能被Cdk5磷酸化。结合文献检索和现有研究基础，我们提出假设：Cdk5磷酸化MPP3调控肝癌细胞的迁移和侵袭。本研究将延续前一课题，进一步揭示MPP3调控肝癌细胞迁移和侵袭的分子机制。通过以上机制的研究，为阐明肝癌侵袭、转移机理，寻找治疗肝癌的转移与复发的分子靶标提供重要理论依据。

课题组前期研究发现MAGUK家族成员棕榈酸酰化膜蛋白3（MPP3）在高转移潜能肝癌细胞株和肝癌组织中均显著性上调，且其过表达与肝癌患者预后差有关，并进一步证实MPP3促进肝癌细胞的迁移和侵袭，但其分子机制未明。基于MPP3作为细胞内支架蛋白的特性，寻找MPP3蛋白的互作蛋白是进一步理解MPP3蛋白功能和阐明其调控肝癌细胞迁移与侵袭的一个重要途径。因此，我们利用酵母双杂交技术大规模筛选MPP3的互作蛋白,并由此捕获互作候选蛋白CDK5。为此，本研究通过免疫共沉淀和细胞共定位实验技术等多种体内外实验手段，首次发现并证实了CDK5是MPP3的互作蛋白，通过构建显性抑制型CDK5和组成激活型CDK5细胞模型和体外激酶实验等体内外实验，首次证实了MPP3 是 CDK5的磷酸化底物，并且第60位丝氨酸是CDK5磷酸化MPP3的氨基酸位点 。体外细胞迁移和侵袭实验结果显示，抑制CDK5激酶活性显著减弱肝癌细胞的迁移和侵袭能力，证实 CDK5 参与调控肝癌细胞的迁移和侵袭，并且首次发现MPP3促进肝癌细胞的迁移和侵袭能力受CDK5调控。而CDK5磷酸化MPP3第60位丝氨酸的突变并不影响肝癌细胞的迁移和侵袭能力，表明CDK5调控肝癌细胞的迁移和侵袭并非通过磷酸化MPP3第60位丝氨酸所介导。通过以上机制研究，为阐明肝癌侵袭、转移机理，寻找治疗肝癌转移与复发的分子靶标提供重要理论依据。