【作者】 张飞；

【导师】 刘颖斌；

【作者基本信息】 上海交通大学 ， 外科学（普外科）， 2017， 博士

【摘要】 目的胆囊癌是胆道系统中最常见的恶性肿瘤,致死率高,预后差,五年生存率小于5%。eIF3d作为e IF3亚基中的一员能选择性的调节特定蛋白翻译,而这些蛋白参与细胞的增殖调控,从而促进了肿瘤的发生发展。通过研究eIF3d在胆囊癌中的功能及其机制,为寻找新的胆囊癌靶向治疗药物提供理论依据。方法利用免疫组化技术检测胆囊组织中的eIF3d表达情况,进一步统计分析eIF3d表达与胆囊癌患者临床病理特征和预后的关系。接着通过慢病毒介导的基因敲除技术和质粒过表达方法构建敲减和过表达eIF3d的胆囊细胞株,利用CCK-8,克隆形成,划痕实验,transwell侵袭实验检测eIF3d对胆囊癌细胞增殖和转移的影响,通过流式细胞仪检测eIF3d对胆囊癌细胞周期进程和凋亡情况的影响。通过裸鼠皮下成瘤实验和小鼠转移模型检测eIF3d在体内对胆囊细胞增殖和转移的影响。蛋白免疫印迹技术检测细胞周期和凋亡相关蛋白变化。接着利用酵母双杂交技术筛选eIF3d下游相互作用蛋白,并通过免疫共沉淀来验证。放线菌酮脉冲实验检测蛋白降解速度,泛素蛋白抗体检测eIF3d对GRK2泛素化的影响。利用天然产物样品库筛选GRK2激酶抑制剂,通过GRK2激酶活性检测试剂盒检测人参皂苷Rg-3对GRK2激酶活性的抑制作用。并通过CCK-8,克隆形成,细胞周期,细胞凋亡实验检测人参皂苷Rg-3对胆囊癌细胞生物学功能的影响,裸鼠皮下成瘤实验检测人参皂苷Rg-3对胆囊癌细胞体内生长的影响。结果eIF3d在胆囊癌组织中异常高表达,并与胆囊癌患者的临床分期,转移以及预后相关,细胞生物功能学研究结果显示,eIF3d通过促进细胞周期进程和抑制细胞凋亡促进胆囊癌细胞的增殖,同时eIF3d通过调节胆囊癌细胞的EMT促进胆囊癌细胞侵袭转移能力。通过酵母双杂交系统和免疫共沉淀发现eIF3d能直接结合GRK2,并通过抑制GRK2泛素-蛋白酶体途径的降解增加GRK2蛋白的稳定性,上调的GRK2激活PI3K/AKT信号通路促进胆囊癌细胞的增殖和转移。最后通过天然产物样品库筛选出GRK2激酶抑制剂人参皂苷Rg-3,人参皂苷Rg-3能显著的抑制胆囊癌细胞在体内体外的增殖。结论胆囊癌中高表达的eIF3d通过GRK2介导的PI3K/ATK信号通路激活促进胆囊癌的发生发展,人参皂苷Rg-3抑制GRK2激酶活性阻断eIF3d-GRK2作用轴抑制胆囊癌的进展,为胆囊癌的治疗提供新的方法。更多还原

【Abstract】 Object Gallbladder cancer is most common malignancy in the biliary tract system,high mortality,poor prognosis and five-year survival rate less than 5%.EIF3d as a member of the e IF3 subunit can selectively regulate the translation of specific protein which involved in cell proliferation and thus promote the progression and development of tumor.Investigating the function and mechanism of eIF3d in gallbladder cancer provides us theoretical basis for gallbladder cancer targeted therapyMethod Immunohistochemistry analysis was performed to detect eIF3d protein expression in gallbladder cancer tissue.The association between the expression of eIF3d and the clinicopathological characteristics and prognosis of gallbladder cancer were analyzed statistically.EIF3d was knocked down by lentivirus-mediated gene knockdown method or over-expressed by plasmid overexpression means in gallbladder cancer cells,respectively.CCK-8 assay,clone formation assay,wound heal assay and transwell invasion assay were used to evaluate the proliferation and migration of gallbladder cancer cells.The cell cycle progression and cell apoptosis of gallbladder cancer cells were analyzed by flow cytometry.Nude mouse xenograft model and mouse metastasis model were used to assess the tumorigenic and metastatic ability of the stable cells in vivo.Immunoblotting analysis was performed to analyze cell cycle and apoptosis related proteins expression.Using the yeast two-hybrid to screen the eIF3d interacting protein.Co-immunoprecipitation assay was carried out to validate the specific interaction.Cycloheximide pulse-chase experiments detect the protein degradation speeds.The GRK2 kinase inhibitor was obtained by screening the natural product library.The inhibitory effect of ginsenoside Rg-3 on GRK2 kinase was detected by GRK2 kinase activity assay kit.CCK-8,colony formation,cell cycle and apoptosis assay were used to evaluate the effect of ginsenoside Rg-3 on gallbladder cancer cells.Nude mouse xenograft assay were used to assess the effect of ginsenoside Rg-3 on gallbladder cancer cell tumorigenic ability in vivoResult EIF3d is highly expressed in gallbladder cancer patients,which is related to the clinical stages,metastasis and prognosis of gallbladder cancer patients.The cell biological function study shows that eIF3d promotes the proliferation of gallbladder cancer cells via promoting cell cycle progression and inhibiting cell apoptosis.Moreover,EIF3d promotes the invasion and metastasis of gallbladder cancer cells by regulating the EMT procession.The yeast two-hybrid system and immunoprecipitation results showed that eIF3d could directly interact with GRK2.EIF3d enhances GRK2 protein stability by inhibiting the ubiquitin-proteasome degradation of GRK2.Upregulation of GRK2 activated PI3K/AKT signaling pathway to promote proliferation and metastasis of gallbladder cancer cells.Finally,ginsenoside Rg-3 as a GRK2 kinase inhibitor was obtained through screening the natural product sample library.Ginsenoside Rg-3 could significantly suppress the proliferation of gallbladder cancer cells both in vitro and in vivo.Conclusion Overexpression of eIF3d promotes the gallbladder cancer progression through the GRK2-mediated activation of PI3K/AKT signaling pathway.Ginsenoside Rg-3 inhibits GRK2 kinase activity and blocks e IF3d-GRK2 axis in gallbladder cancer to suppress gallbladder cancer progression,which provides us a new treatment for gallbladder cancer.更多还原