USP10去泛素化E-cadherin抑制胃癌细胞迁移和侵袭的机制研究

The role of the ubiquitin-specific protease 10 (USP10), a novel deubiquitinating enzyme, in regulating the oncogenesis and tumor progression has become a hot topic of research now. Our previous work have shown that USP10 was down-regulated in tissues of gastric cancer patient as well as gastric cancer cell lines, and its expression was associated with invasion, metastasis and prognosis of gastric cancer. However, the mechanism of USP10 inhibits gastric cancer cell motility and invasiveness has not been elucidated yet. E-cadherin is a cell–cell adhesion protein. In our preliminary experimental, a positively correlation between the expression of USP10 and E-cadherin protein was found in gastric cancer patients. In addition, up-regulation of USP10 could increase the expression of E-cadherin in protein level, but not in mRNA level, which could further inhibit gastric cancer cell migration. Thus, we speculated that USP10 maybe inhibit gastric cancer cell invasion and metastasis by regulating deubiquitination and degradation of E-cadherin. The aim of the present study is to investigate：(1) Could USP10 bind with E-cadherin directly? (2) Was the stability of E-cadherin protein regulated by USP10. (3) If USP10 could reverse Mdm2/Hakai-mediated E-cadherin protein ubiquitination and degradation by deubiquitination. (4) Whether the expression of USP10-E-cadherin was associated with the motility and invasiveness in gastric cancer cells. This research will elucidate the mechanism of USP10 inhibiting the invasion and metastasis of gastric cancer cells, which could provide fresh view for suppression of gastric cancer progression.

泛素特异性蛋白酶10（USP10）是一种新的去泛素化酶，该酶在肿瘤中的作用已成为研究热点。申请人以往研究表明USP10在胃癌中表达下调，与胃癌的浸润、转移及预后密切相关，然而作用机制尚未明确。上皮钙粘蛋白（E-cad）是一种细胞间粘附分子，前期预实验发现胃癌组织中USP10与E-cad表达呈正相关；促进USP10表达后，E-cad mRNA未明显改变，但蛋白增多，胃癌细胞迁移能力减弱。推测USP10可能是通过调控E-cad的去泛素化及蛋白降解，参与胃癌的浸润和转移。本项目在此研究基础上，拟进一步明确①USP10与E-cad蛋白能否结合；②E-cad蛋白稳定性是否受USP10调控；③USP10能否发挥去泛素化作用，逆转Mdm2/Hakai导致的E-cad泛素化降解；④在此过程中胃癌细胞迁移和侵袭能力的改变。该研究将阐明USP10抑制胃癌浸润和转移的作用机制，为阻止胃癌的进展提供新的方向。

背景与目的：申请人以往研究发现泛素特异性蛋白酶10（ubiquitin-specific protease 10，USP10）在胃癌（gastric cancer，GC）组织中表达下调，与浸润深度、淋巴结转移、患者预后及上皮性钙粘蛋白（epithelial cadherin, E-cadherin）的表达密切相关，然而USP10是否能够调控GC细胞的浸润和转移尚有待进一步探讨。本研究拟以E-cadherin为突破口，阐明USP10抑制GC细胞迁移和侵袭的分子作用机制，从而为阻止GC的进展提供新的思路。.材料与方法：构建USP10表达抑制或过表达的GC细胞株；通过体外Transwell、细胞划痕及体内裸鼠转移瘤实验观察GC细胞迁移及侵袭的变化；采用Western blot及RT-PCR检测上皮间充质转化（epithelial-mesenchymal transition, EMT）中的关键分子及其上游转录因子的表达变化；通过免疫共沉淀及放线菌酮实验探讨USP10能否直接结合E-cadherin蛋白并调控其蛋白稳定性；联用泛素化修饰蛋白质组学、定量蛋白质组学及转录组学，在GC细胞中高通量筛选出可被USP10去泛素化调控的下游靶分子。.结果：在GC细胞中过表达USP10后，肿瘤细胞迁移、侵袭能力减弱；反之，采用shRNA抑制USP10蛋白表达后，细胞迁移、侵袭能力增强；在USP10表达下调后，E-cadherin、Occludin及ZO-1也随之下调，而N-cadherin并未见明显改变，提示USP10可通过EMT过程抑制GC细胞迁移和侵袭。进一步的免疫共沉淀及放线菌酮等实验表明USP10并不能直接调控E-cadherin蛋白的稳定性，而是通过转录因子Twist 1作为桥梁的间接调控。多组学联合检测分析发现TNFRSF10B在内的9种蛋白在USP10表达上调后，其泛素化程度下降，总蛋白含量增加，而mRNA未见明显改变，提示这9种蛋白在GC细胞中可被USP10去泛素化，是Twist 1-E-cadherin-EMT轴的上游调控分子。.结论：USP10通过去泛素化作用上调TNFRSF10B在内的9种蛋白表达，进而通过Twist 1-E-cadherin轴调控GC细胞的EMT过程，抑制GC细胞迁移和侵袭。