【作者】 陈实；

【导师】 彭承宏；

【作者基本信息】 上海交通大学 ， 外科学（肝胆胰外科）， 2016， 博士

【摘要】 背景:Polycomb成员Ring1B和EZH2,作为两种重要的表观遗传修饰标志物,分别能催化组蛋白H2A在K119发生单泛素化(H2AK119Ub1)和组蛋白H3在K27发生三甲基化(H3K27Me3),其基因沉默作用在个体胚胎发育和肿瘤发生过程中有重要意义。转录因子Twist是上皮-间质转化的重要调控因子,其抑制基因转录和诱导EMT过程的表观机制尚不清楚。本课题将探讨Ring1B和EZH2及其介导的H2AK119Ub1和H3K27Me3作用与Twist抑制基因转录的调控机制在胰腺恶性肿瘤中的作用。方法:我们首先通过免疫组化检测Ring1B、H2AK119Ub1、EZH2、H3K27Me3四种蛋白在80例胰腺导管腺癌组织芯片中的表达分布,分析四种蛋白表达水平与临床预后的相关性。在细胞中通过沉默Ring1B和EZH2来检测两种组蛋白修饰的水平,同时观察细胞在体外以及小鼠体内的生长。其次通过免疫组化检测Twist在90例胰体尾癌中的表达,接着通过免疫共沉淀等实验证实Ring1B、EZH2与Twist之间的相互作用。然后在缺氧培养下的胰腺癌细胞中分别敲除Twist、Ring1B和EZH2,通过实时定量PCR和免疫印迹检测Twist靶基因E-cadherin和P16的表达水平变化,并通过Transwell和小鼠模型验证Ring1B、EZH2对Twist介导细胞增殖和迁移能力的影响。最后通过染色质免疫沉淀检测Ring1B、H2AK119Ub1和EZH2、H3K27Me3等在Twist靶基因E-cadherin和P16启动子区的富集水平。结果:Ring1B和EZH2在胰腺导管腺癌肿瘤组织中表达升高,H2AK119Ub1和H3K27Me3在胰腺癌中的协同作用与临床预后相关联。在胰腺癌细胞中敲除Ring1B/EZH2后,H2AK119Ub1和H3K27Me3水平明显下降,HOX基因家族的HOXC10的m RNA水平出现上调。在脾动脉浸润的胰体尾癌中TWIST呈高表达且与预后差相关,TWIST表达升高与肿瘤缺氧环境形成相关。TWIST与Ring1B、EZH2存在特异性结合作用。在缺氧诱导的胰腺癌细胞中敲除Ring1B和EZH2,能削弱缺氧诱导的Twist对E-cadherin和P16的转录抑制作用;同样,Twist介导细胞迁移和增殖的能力亦受到削弱。最后通过染色质免疫共沉淀发现,Twist可以先后招募EZH2、Ring1B至靶基因E-cadherin和P16启动子区介导H3K27Me3和H2AK119ub1。结论:这些结果阐述了Ring1B和EZH2介导的组蛋白修饰H2AK119Ub1和H3K27Me3可以作为生物学标志物应用于胰腺癌的预后分期。Ring1B/EZH2对缺氧诱导下高表达的TWIST介导的靶基因转录抑制具有表观调控作用。更多还原

【Abstract】 Background: Polycomb group(Pc G)proteins Ring1 B and EZH2,which have been characterized as catalyzing the two epigenetic modifications H2AK119 monoubiquitination(H2AK119Ub1)and H3K27 trimethylation(H3K27Me3),are well-known epigenetic silencers implicated in embryonic development and tumorigenesis.Transcriptional repressor Twist is a master regulator of epithelialmesenchymal transition,yet the epigenetic mechanism governing Twist to induce EMT is poorly understood.Here we studied the epigenetic mechanism that Ring1B/EZH2-mediated H2AK119Ub1 and H3K27me3 involves in the Twist-induced transcriptional repression and tumor metastasis.Methods: First immunohistochemistry was performed on tissue microarrays of human resected pancreatic duct adenocarcinoma to detect Ring1 B,H2AK119Ub1,EZH2,and H3K27Me3 expressions in tumor tissues,and the correlations between these proteins and clinical prognosis were analyzed.Then we silence Ring1 B and EZH2 via sh RNA to detect the level of H2AK119Ub1 and H3K27Me3 in the pancreatic cancer cells,and observe the tumor cell growth in vitro and in tumor xenograft models.Next,we performed immunohistochemistry on tissue microarrays of human distal pancreatic cancer specimens to assess the expression levels of Twist,then performed co-immunoprecipitation to verify the interaction between Ring1B/EZH2 and Twist.Twist,Ring1 B and EZH2 were simultaneously knocked down via sh RNA in hypoxic pancreatic cancer cells,and the Twist target gene E-cadherin and P16 was checked by real-time PCR and Western blotting,and cell growth and migration ability were tested thereafter by transwell assay and mice model.Finally,Chromatin-immunoprecipitation was employed in the above cells to detect Ring1 B,H2AK119Ub1,EZH2,and H3K27Me3 enriching at the promoter region of Twist target locus.Results: Here we demonstrated that both Ring1 B and EZH2 were elevated in pancreatic ductal adenocarcinoma(PDAC),and H2AK119Ub1 and H3K27Me3 cooperate in tumors and are associated with the clinical prognosis in combinatorial patterns.Simultaneous silencing of Ring1 B and EZH2 via sh RNA depleted H2AK119Ub1 and H3K27Me3 in the pancreatic cancer cells,enhanced HOX gene derepression.TWIST was overexpressed in distal pancreatic cancer invading splenic artery and was associated with the worst prognosis,which might be activated by hypoxic microenvironment.We identified Ring1 B and EZH2 as Twist interacting proteins.Ring1B/EZH2 knocking down simultaneously in pancreatic cancer cells resulted in derepression of Twist target gene E-cadherin and P16,as well as impairement of Twist-induced cell growth and migration ability.Finally,Ch IP on the above stable cells demonstrated that Twist recruited Ring1B/EZH2 to E-cadherin and P16 gene promoter region to catalyze H2AK119Ub1 and H3K27Me3.Conclusion: This study shows that Ring1B/EZH2-mediated H2AK119Ub1 and H3K27me3 may serve as discriminatory biomarkers for molecular staging of pancreatic cancer,and Ring1B/EZH2 involving in hypoxic Twist-mediated transcriptional repression.更多还原