【作者】 陈键；

【导师】 郑树森；

【作者基本信息】 浙江大学 ， 外科学（普通外科）， 2021， 博士

【摘要】 背景:肝细胞癌是最常见的恶性肿瘤之一,严重危害人民生命健康。以外科手术为主的多学科综合治疗是目前肝癌的主要治疗方式,但肝癌总体疗效欠佳。因此,寻找肝癌治疗新靶点是亟待解决的关键问题。持续不断的细胞分裂是肿瘤重要的生物学特征。胞质分裂是细胞分裂的最后阶段,是形成两个子细胞的过程。胞质分裂保证了肿瘤细胞的快速增殖,胞质分裂障碍则会形成多核多倍体细胞而影响细胞增殖。寻找胞质分裂相关靶点,特异性靶向肿瘤细胞的胞质分裂过程有望进一步丰富肝癌综合治疗体系,为肝癌治疗提供新策略。目的:本课题旨在研究胞质分裂相关基因ANLN(Anillin actin binding protein,ANLN)在肝癌发生及发展中的作用;阐明ANLN调控胞质分裂促进肝癌增殖的分子机制;探究ANLN在肝癌组织中的表达水平与肿瘤临床病理特征及患者预后的相关性;明确肝癌进展中ANLN调控的胞质分裂与细胞多倍体化的关系;探索ANLN与肝癌索拉菲尼治疗应答之间的相关性,以期为靶向ANLN治疗肝癌提供理论依据。方法:1、基于TCGA数据库和肝癌临床标本筛选与肝癌进展相关的基因,在组织水平验证基因表达及其与肿瘤临床病理特征和患者预后的相关性。2、构建肝脏ANLN特异性敲除小鼠,结合小鼠尾静脉高压注射诱导肝癌模型研究ANLN在肝癌发生中的作用。3、通过细胞增殖、细胞周期、细胞多倍体、细胞凋亡和DNA损伤检测等实验技术体外水平研究ANLN对肝癌细胞增殖的作用。4、利用裸鼠皮下移植瘤模型、裸鼠肝脏原位移植瘤模型体内水平研究ANLN对肝癌肿瘤生长的作用。5、使用免疫荧光、免疫共沉淀、Pull-down、磷酸化位点突变等实验研究ANLN调控肝癌细胞胞质分裂的分子机制。6、结合转录组测序、肝癌PDX模型等方法研究肝癌中ANLN表达与其对索拉菲尼治疗应答的关系。结果:1、ANLN在肝癌组织中较癌旁高表达;ANLN在肝癌组织中表达量与肿瘤组织学分级、大小、TNM分期和患者预后相关;肝癌组织中ANLN表达量与双核多倍体比例呈负相关。2、小鼠肝脏ANLN特异性敲除抑制肝癌形成;敲低ANLN可抑制肝癌细胞体内成瘤能力。3、敲低ANLN在体外水平抑制肝癌细胞增殖,导致细胞周期G2/M期阻滞,介导胞质分裂障碍并形成多核细胞和多倍体,诱导细胞发生DNA损伤、有丝分裂灾难与凋亡。4、ANLN与RACGAP1结合并促进RACGAP1磷酸化;ANLN可作为支架蛋白促进PLK1激酶与RACGAP1结合;ANLN通过促进PLK1激酶磷酸化RACGAP1进而激活下游Rho A,最终介导胞质分裂。5、敲低ANLN可抑制ERK通路激活从而增强肝癌细胞对索拉菲尼的敏感性;肝癌患者肿瘤中ANLN的表达量与肿瘤对索拉菲尼治疗反应性相关。结论:1、ANLN在肝癌组织中高表达且与肿瘤临床病理特征、患者预后及肿瘤中双核多倍体比例相关。2、小鼠肝脏ANLN敲除抑制肝癌形成,敲低ANLN可在体内外水平抑制肝癌增殖。3、敲低肝癌细胞ANLN导致胞质分裂障碍、促进多核细胞形成并诱导细胞发生DNA损伤与凋亡。4、ANLN作为支架蛋白促进PLK1磷酸化RACGAP1进而激活Rho A,促进肝癌细胞胞质分裂。5、敲低ANLN可增加肝癌细胞对索拉菲尼的敏感性,ANLN可作为生物标志物预测肝癌患者对索拉菲尼治疗反应性。更多还原

【Abstract】 Background Hepatocellular carcinoma(HCC)is a common malignancy,which seriously endangers life and health.Surgery-based multi-discipline therapy is the main therapeutic option for HCC,but the curative effect is not ideal.Therefore,novel therapeutic strategies are urgently needed in the treatment of HCC.Continuous cell division is a characteristic feature of cancer.Cytokinesis is the final stage of cell division that generates two daughter cells.Accurate cytokinesis ensures the rapid proliferation of cancer cells,while cytokinesis failure leads to multinucleated cells and polyploidy,which affect cell proliferation.Thus,targeting cytokinesis-related elements is a promising strategy for HCC treatment.Objectives1.To investigate the role of Anillin actin binding protein(ANLN)in the tumorigenesis and progression of HCC.2.To elucidate the molecular mechanism of ANLN-modulated cytokinesis in promoting HCC proliferation.3.To explore the correlation between the expression of ANLN and the clinicopathological characteristics and prognosis of HCC patients;4.To investigate the relationship between ANLN-modulated cytokinesis and cell polyploidy in the progression of HCC.5.To study the role of ANLN in the efficacy of sorafenib treatment in HCC.6.To provide the theoretical basis on targeting cytokinesis for HCC therapy.Methods1.We screened the HCC-related genes based on TCGA database and clinical samples.The correlation between gene expression and HCC patients’ clinicopathological characteristics was studied.2.Liver conditional ANLN knockout mice were constructed to investigate the role of ANLN in the tumorigenesis of HCC by utilizing HCC hydrodynamic venous injection model.3.The effect of ANLN on cell proliferation was studied in vitro by cell proliferation,cell cycle,cell polyploidy,cell apoptosis and DNA damage detection experiments.4.The effect of ANLN on tumor growth was studied in vivo using HCC subcutaneous xenograft model and HCC orthotopic xenograft model.5.Immunofluorescence,co-immunoprecipitation assay,pull-down assay,and phosphorylation site mutation assay were used to study the molecular mechanism of ANLN in regulation of cytokinesis.6.RNA sequencing and HCC PDX models were performed to investigate the correlation between ANLN expression and sorafenib treatment efficacy in HCC.Results1.ANLN was highly expressed in HCC tissues compared with paracancerous tissues.The expression of ANLN in HCC tissues was correlated with tumor histological grade,tumor size,TNM stage and patients’ prognosis.The expression of ANLN in HCC tissues was negatively correlated with the proportion of binucleated polyploidy.2.Liver conditional ANLN knockout inhibits the growth of tumor during HCC tumorigenesis in mice.3.ANLN knockdown inhibits the proliferation of HCC cells in vitro.ANLN inhibition promotes G2/M phase arrest and leads to the formation of multinucleated cells and polyploidy due to cytokinesis failure,ultimately inducing DNA damage and apoptosis.4.ANLN interacts with RACGAP1 to promote its phosphorylation.ANLN can act as a scaffold to promote the interaction between PLK1 and RACGAP1.ANLN activates Rho A by promoting PLK1-mediated RACGAP1 phosphorylation in regulation of cytokinesis.5.ANLN knockdown inhibits ERK activation and enhances the sensitivity of HCC cells to sorafenib treatment.The level of ANLN in the tumors of HCC patients correlates with tumor response to sorafenib treatment.Conclusion1.The expression of ANLN is correlated with the clinicopathological characteristics of tumor,patients’ prognosis and the proportion of binucleated polyploidy in HCC.2.Liver conditional ANLN knockout can inhibit HCC tumorigenesis,and knockdown of ANLN inhibits the growth of HCC in vivo and in vitro.3.ANLN inhibition leads to cytokinesis failurer,multinucleated cell formation,DNA damage and apoptosis in HCC cells.4.ANLN can act as a scaffold to promote PLK1-mediated RACGAP1 phosphorylation,which then activates Rho A to ensure the fidelity of cytokinesis in HCC cell.5.ANLN knockdown increases the sensitivity of HCC cells to sorafenib,and ANLN can be used as a biomarker to predict tumor response to sorafenib in HCC treatment.更多还原