Cul9泛素化降解YES1在抑制胃癌发展中的机制研究

The therapeutic effect of gastric cancer is poor and its mechanism is unknown. To clarify the molecular mechanism of its occurrence and development is essential to improve the therapeutic effect. YES1 has been reported to play a key role in tumor development. Our previous study also showed that YES1 can promote gastric cancer cell proliferation and invasion, but its regulatory mechanism unknown. Ubiquitin ligase Cul9 has recently been reported to be associated with cancer, but so far there has been no study about it in gastric cancer. Our previous results showed that interaction between Cul9 and YES1 protein, overexpression of Cul9 can reduced YES1 whereas reducing Cul9 can decrease the ubiquitin of YES1, and Cul7 can inhibit the role of Cul9. YES1 is a kind of phosphorylated kinase, so we speculate that Cul9 can inhibit the proliferation and invasion of gastric cancer cells by ubiquitin degradation YES1,and Cul7 competitively inhibits Cul9 ubiquitination while YES1 phosphorylation suppresses Cul9, forming a negative feedback regulation pathway. Based on the previous study, this study will explore the relationship between Cul9, Cul7 and YES1 and tumor differentiation, pathological staging and prognosis by using tissue microarray, and explores the mechanism of Cul9/Cul7/YES1 pathway in the development of gastric cancer and provides a new strategy for the treatment of gastric cancer.

胃癌治疗效果差且机制不明。研究其内在分子机制，对其治疗至关重要。有研究YES1是肿瘤发展中关键癌蛋白，我们报道了YES1上调可促进胃癌细胞增殖侵袭，而其蛋白调控机制仍不明；Cul9被报道与肿瘤相关，但迄今未在胃癌中研究。我们发现：Cul9和YES1蛋白可互作；过表达Cul9，可使YES1蛋白下调，而敲减则相反；Cul7能抑制Cul9的泛素化作用；Cul9在胃癌中低表达，YES1与Cul7则高表达；Cul9敲减促进胃癌细胞增殖侵袭，同时敲减YES1则逆转此效果。因此,我们推测Cul9可通过泛素化降解YES1抑制胃癌增殖侵袭，且Cul7可抑制Cul9的泛素化作用，YES1可磷酸化抑制Cul9，从而形成负反馈通路。本课题拟在前期基础上，利用组织芯片分析Cul9、Cul7和YES1与胃癌分化、病理和预后的相关性，并探究Cul9/Cul7/YES1通路在胃癌发展中的机制，为治疗胃癌提供新的策略。

Cul9与人类肿瘤的发生相关。然而，Cul9的上游调节因子和在胃癌发病机制中的作用仍然未知。甲基化CpG‐DNA免疫沉淀和实时PCR检测Cul9启动子区的甲基化水平；CCK8、Edu测定、异种移植用于研究Cul9在胃癌中的作用；生物信息学分析、质谱和无偏激酶库筛选用于确定Cul9下游的靶效应物；高通量药物筛选用于鉴定胃癌中Cul9的激活剂。研究结果发现胃癌中Cul9基因的启动子是高甲基化的，并与不良预后相关；生物信息学、质谱和无偏激酶筛选确定酪氨酸激酶YES1是Cul9致癌行为的关键调节因子；YES1在Y1505磷酸化Cul9；而Cul9与YES1结合并破坏YES1的稳定性，进而建立反馈调节；DNA去甲基化药物HG78激活CpG甲基化读取器MBD2蛋白，上调Cul9表达进而抑制胃癌细胞增殖；YES1抑制剂CH6953755或MMF会影响Cul9失调后的胃癌进展；在胃癌的临床样本上发现，p-Y1505-Cul9、Cul9、YES1和核苷酸代谢酶之间存在显著的相关性，并影响胃癌的临床预后。本研究揭示了Cul9-YES1环在胃癌中的未知作用，揭示其在胃癌上潜在的治疗靶点。