【作者】 陈军；

【导师】 郑树森；

【作者基本信息】 浙江大学 ， 临床医学（专业学位）， 2019， 博士

【摘要】 背景:肝细胞肝癌(Hepatocellular carcinoma,HCC)是一种常见的恶性肿瘤,在全球范围内发病率位居第五,肿瘤相关死亡率居第三。临床上仅有10%-20%的肝癌能通过外科手术完全切除,术后5年复发率超过70%。目前,缺乏有效的靶点仍是肝癌治疗难点。EAG1(Kv10.1,KCNH1,Ether-a-go-go-1)是一种电压门控钾通道,已证实在宫颈癌、肺癌、乳腺癌、结肠癌、前列腺癌和肝癌中高表达,EAG1表达与多种肿瘤的临床预后密切相关。深入研究EAG1在肝癌中的表达与肝癌预后的关系,阐明EAG1调控肝癌增殖和迁移侵袭的相关分子机制,有望为肝癌的防治提供新的理论依据,具有重要的科学意义和临床价值。目的:本研究拟在肝癌患者肝癌组织及其配对的癌旁组织中比较EAG1的表达差异,了解EAG1表达差异是否与肝癌的不良预后相关。通过进一步的体内和体外研究阐明EAG1对肝癌增殖和迁移侵袭能力的调节作用,并深入研究其分子机制。探索EAG1抑制剂在肝癌治疗中是否发挥作用。为肝癌的防治提供新的理论依据和治疗方法。方法:1.收集233对肝癌患者肿瘤及配对癌旁组织,分别使用RT-qPCR、Western Blot、免疫组化技术研究EAG1在肝癌组织与癌旁组织的表达差异,并分析EAG1的表达强度与肝癌患者临床指标、临床预后是否相关。2.使用LM3和Huh7细胞系,构建EAG1稳定沉默或过表达的细胞,通过体内及体外实验研究EAG1对肝癌增殖能力的影响。通过周期分析、转录组组织芯片筛选、信号通路蛋白验证,研究EAG1可以通过调控细胞周期影响肝癌细胞增殖,并寻找下游关键分子。进一步通过功能补救实验研究EAG1与下游调控分子的相互作用关系及调节方式。3.通过体内外实验研究EAG1对肝癌迁移、侵袭能力的影响,并研究是否是因为细胞结构功能变化导致迁移、侵袭能力的改变。4.联合使用EAG1的抑制剂和肝癌化疗药物,探索EAG1肝癌治疗中的意义。结果:1.在肝癌患者肿瘤组织中EAG1较癌旁组织高表达,并且EAG1的高表达和肝癌患者的不良预后相关。2.体外及体内实验均证明EAG1能促进肝癌细胞增殖。周期分析、转录组基因芯片信号通路富集分析发现EAG1异常表达能影响细胞周期,周期信号通路相关蛋白表达差异验证了这一结果,并发现下游调控分子SKP2。SKP2功能补救及药物抑制证明EAG1通过调控SKP2影响细胞增殖。进一步研究发现EAG1是通过抑制SKP2的泛素化降解促进SKP2蛋白含量升高。3.体外及体内实验证明EAG1可以促进肝癌细胞的迁移及侵袭能力,并且EAG1是通过促进肝癌细胞形成细胞伪足促进肝癌迁移、侵袭。4.使用EAG1的抑制剂(阿司咪唑)可以增强阿霉素对肝癌细胞的杀伤作用。结论:EAG1在肝癌组织中高表达,并且和肝癌患者的不良预后相关;EAG1可以通过抑制SKP2的泛素化降解而促进细胞周期进展,进而促进肝癌的增殖;EAG1可以通过促进细胞伪足形成促进肝癌细胞的迁移侵袭能力;阿司咪唑(一种EAG1的抑制剂)可以促进阿霉素对肝癌的抗肿瘤作用(概述图1-1)。以上研究表明EAG1在肝癌的发生发展中发挥重要作用,并且提供了一种肝癌的潜在治疗方法。更多还原

【Abstract】 Background:Hepatocellular carcinoma(HCC)is a common malignant tumor,which accounting for the fifth incidence cancer and the third cancer-related mortality worldwide.Only 10%to 20%of hepatocellular carcinomas can be cured by surgery.More than 70%patients recurred after operation in 5 years.Therefore,it is important to explore new pathogenesis and therapeutic targets for the diagnose and treatment of HCC.As one of the potassium channels,ether-a-go-go-1(EAG1)is involved in various physiological processes and plays an important role in the tumorigenesis of many kinds of human cancers.However,the role of EAG1 in hepatocellular carcinoma(HCC)remains unclear and needs to be further explored.Objectives:Quantitative real-time PCR,western blot and immunohistochemistry staining were used to detect EAG1 expressions in HCC tissues.The correlations between EAG1 expression and clinicopathologic features were analyzed.The cell proliferation assay and colony formation assay were applied to evaluate proliferation ability,while trans-well invasion assays were used to evaluate migratory and invasive abilities.For in vivo experiments,subcutaneous xenografted tumors and pulmonary colonization assays were performed.The value of astemizole in the treatment of HCC was evaluated by drug combination experiments.Results:1.EAG1 was overexpressed in HCC tissues and was associated with a poor clinical prognosis.2.In vitro and in vivo experiments showed that EAG1 could promote the proliferation of HCC by inhibiting the ubiquitination of S-phase kinase-associated protein 2(SKP2)and promoting cell cycle progression.3.Our research also revealed that EAG1 could promote the migration and invasion of HCC by promoting cell pseudopod formation.4.Furthermore,we found that astemizole,an EAG1 inhibitor,could promote the anti-tumor effects of doxorubicin on HCC.Conclusion:EAG1 could promote cell proliferation through modulating SKP2 protein level and migration through facilitating pseudopod formation.As EAG1 plays an important role in the progression of HCC,the combined use of astemizole and doxorubicin might be a potential treatment method for HCC.更多还原