MKP-4调节ERK信号通路在肝细胞癌发生发展中的意义

Mitogen-activated protein kinase phosphatases-4 (MKP-4) was a potential tumor suppressor gene. As a new member of dual-specificity phosphatases, MKP-4 enables extracellular signal-regulated kinase (ERK) inactivation by dephosphorylation and changes its biological effects. In our preliminary work, we have revealed the direct relationships between the expression of MKP-4 and the clinicopathological features and prognosis of hepatocellular carcinoma (HCC). In this research, we aim to investigate the role of MKP-4 during the development of HCC and the regulation of ERK signaling pathway. Further study will be performed to validate the relationship between the expression of MKP-4 and relevant proteins of ERK signal pathway in HCC tissues and adjacent non-cancerous liver tissues and the prognosis of HCC. We will construct the MKP-4 expression plasmids and the MKP-4 interference plasmid and explore the biological roles of MKP-4 over-expression or down-regulation in vitro, observe cell growth, proliferation, apoptosis, and migration and the expression of downstream nuclear transcription factor of ERK signaling pathway; investigate the interaction between MKP-4 and ERK and the dephosphorylation sites of ERK; verified by the intervention of inhibitor PD98059 and the transfection of phosphorylation mutant. Finally, tumorigenicity assay of MKP-4 and ERK signaling pathway in nude mice will be performed via inoculating the stable cell lines. This study will elucidate the mechanism of tumor suppressor gene of MKP-4, the regulation of ERK signaling pathway in HCC, and may provide a potential new target for the development of anti-cancer therapy.

丝裂原活化蛋白激酶磷酸酶-4（MKP-4）作为新的双位点特异性磷酸酶，能够使细胞外信号调节激酶（ERK）去磷酸化失活，改变其生物学效应。课题前期研究显示MKP-4 mRNA和蛋白的表达水平与肝癌的临床特征及预后密切相关，是潜在的抑癌基因。本课题将进一步探讨MKP-4在肝癌发病中的作用及对ERK信号通路的调控机制。拟分析肝癌组织中MKP-4及ERK信号通路蛋白表达的相关性及对预后的影响；构建MKP-4表达及干扰质粒，研究其过度表达和表达抑制后对肝癌细胞生长、增殖、凋亡、迁移及ERK通路下游核转录因子表达的影响；探讨MKP-4与ERK的相互作用及去磷酸化位点，并通过PD98059干预及磷酸化突变体转入加以验证；通过裸鼠皮下成瘤实验在体内水平验证MKP-4对ERK信号通路的调控作用。本研究将阐明潜在抑癌基因MKP-4的功能及对ERK信号通路的调控机制，为肝癌的治疗寻找新的调控靶点。

肝癌的发生发展是一个多因素、多阶段、连续发展极其复杂的过程；由多个基因、环境因素及其之间的关系决定。课题组前期研究报道MKP-4在肝癌发生发展中有重要的预后价值。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MKP-4存在相互作用的蛋白质ERK1/2，并且通过免疫沉淀和免疫荧光在肝癌细胞中进一步验证两者的相互作用。在细胞水平，CCK-8、Edu、克隆形成和干细胞成球实验证明MKP-4的干扰表达可以促进肝癌细胞的增殖、集落形成和干细胞球的形成，同时MKP-4过表达或者MKP-4干扰表达加ERK1/2抑制剂PD98059预孵育可以明显抑制肝癌细胞的增殖、集落形成和干细胞球的形成。Western blot结果显示MKP-4过表达或者MKP-4干扰表达加PD98059预孵育可以显著降低ERK1/2的磷酸化水平以及ERK1/2信号通路的下游基因CyclinD1和c-Myc的表达。为了阐明MKP-4与ERK1/2相互作用的分子机制，本研究中成功构建了ERK1/2的磷酸化位点突变体，且通过Western blot实验明确MKP-4主要通过对ERK1/2的去磷酸化抑制其入核发生相互作用。在动物水平，裸鼠皮下成瘤、Western blot、免疫组化、组织荧光实验证实MKP-4通过对ERK1/2去磷酸化抑制肝癌的进展。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MKP-4在肝癌组织中表达下调，而ERK1/2、p-ERK1/2表达上调。同时选取160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MKP-4与ERK1/2、p-ERK1/2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。MKP-4的低表达、ERK1/2高表达、p-ERK1/2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，MKP-4介导的ERK1/2去磷酸化可能成为肝癌发生的新型肿瘤抑制机制，为肝癌的临床治疗提供新的实验依据。