乙型肝炎病毒X蛋白（HBx）对Notch1与Snail的调控及其功能的研究

【作者】 徐洁杰；

【导师】 闻玉梅； 顾建新； 江建海； 魏湲颜； 恽小婧；

【作者基本信息】 复旦大学 ， 病原生物学， 2010， 博士

【摘要】 早在1970年,Sherlock等人的研究发现在慢性肝脏疾病和原发性肝细胞癌患者血清中存在肝炎相关澳大利亚抗原,由此提示慢性乙型肝炎病毒(hepatitis B virus,HBV)感染与肝细胞癌(hepatocellular carcinoma, HCC)发生存在关联。到1981年,Beasley等人通过对中国台湾地区22,707例男性的前瞻性人群调查发现乙型肝炎病毒表面抗原(hepatitis B surface antigen, HBsAg)携带者较非HBsAg携带者罹患HCC的风险高出63倍,由此为HBV感染与HCC发生的关联提供强有力的证据。在HBV基因组编码的所有蛋白中,乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)作为一个重要的非结构多功能调节蛋白在病毒感染、复制、致病和致癌过程中发挥重要作用。尽管已有部分研究证实HBx可通过影响细胞本身信号通路来激活癌基因信号在HBV相关肝癌发生(HBV-associated hepatocarcinogenesis)过程中可能发挥重要的促进肿瘤发生的作用,然而对于HBx在HBV相关肝癌(HBV-associated hepatocellular carcinoma, HBV-HCC)发生发展过程中的作用仍有许多不明之处。已有研究发现作为一个进化上高度保守的局部信号传导通路,Notch1信号在肝癌发生过程中可能发挥抑制肿瘤形成的作用,但是在HBV相关肝癌发生过程中HBx表达与Notch1信号之间的相互作用及其机制仍有待阐明。此外在慢性HBV感染的HCC患者中发现有较高的肿瘤复发和转移率,尽管近年来HBx与HBV相关肝癌发生的病理相关和重要性引起诸多关注,但是对于HBx在肿瘤侵袭和转移中的作用及其机制目前不甚明了,而本研究主要围绕HBx在HBV相关肝癌发生发展和侵袭转移中的作用及其机制展开。在本研究的第一部分,发现在正常肝细胞系和肝癌细胞系中表达HBx可下调内源性的Notch1胞内活性片段(Notch1 intracellular domain, ICN1)蛋白水平及其下游靶基因的mRNA水平。进一步研究发现HBx对Notch1信号通路的负调控是通过减少Notch1的剪切而不是抑制Notch1转录或者其配体表达水平来实现的,而且HBx下调Notch1剪切的作用是经由抑制参与Notch1剪切的γ-分泌酶(γ-secretase)的关键组分presenilinl (Psen1)来介导的。通过HBx瞬时转染实验发现,在体外细胞培养中HBx可通过下调ICN1水平来促进肝癌细胞增殖(cell proliferation)、诱导肝癌细胞G1-S期的细胞周期进展(G1-S cell cycle progression)和抑制细胞衰老(cellular senescence)。进一步用裸鼠皮下肿瘤移植实验证实稳转HBx也可经由下调ICN1水平钝化衰老样生长抑制(senescence-like growth arrest)从而促进肝癌细胞生长和肿瘤形成。最后在HBV相关肝癌患者肿瘤组织样本中证实了下调的Psen1-依赖的Notch1信号和钝化的衰老样生长抑制二者之间的相关性。结论：这部分研究阐明了HBx在肝癌发生过程中的一个新功能,即通过抑制Notch1信号活化来钝化衰老样生长抑制从而促进肿瘤发生,该发现提供了一种可能的HBV相关肝癌发生的新机制。在本研究的第二部分,发现在肝癌细胞中表达HBx可使肝癌细胞形态从上皮型转化为间质型、上皮细胞标志分子下调(如E-cadherin)、β-catenin的核转位以及间质细胞标志分子的上调(如N-cadherin、vimentin和fibronectin),由此提示HBx表达可诱导肝癌细胞发生上皮-间质转化(epithelial-mesenchymal transition,EMT)。进一步研究发现HBx诱导发生EMT依赖于EMT调节转录因子Snail蛋白水平的增加,而Snail蛋白水平的增加是通过HBx表达增强Snail蛋白稳定性,而非通过促进Snail转录来实现的,siRNA干扰Snail可以抑制HBx诱导发生EMT。此外还证实HBx表达通过激活PI3K/AKT/GSK-3β信号通路来增强Snail蛋白稳定性,PI3K/AKT信号通路特异性的抑制剂LY294002和Wortmannin处理、以及组成性激活型的GSK-3βS9A突变体共转染均可抑制HBx表达上调Snail蛋白水平。最后通过体外细胞划痕实验、Transwell细胞迁移实验和Collagen细胞侵袭实验分析发现,在体外细胞培养中HBx表达可促进肝癌细胞的细胞迁移(cell migration)和细胞侵袭(cell invasion),而siRNA干扰Snail可以抑制HBx表达增强细胞迁移和细胞侵袭的能力,由此提示HBx表达可能通过诱导肝癌细胞发生EMT从而参与HBV相关肝癌的侵袭和转移过程。结论：这部分研究阐明了HBx在促进HBV相关肝癌发生发展中的一个新功能,即通过激活PI3K/AKT/GSK-3β信号通路来增强肝癌细胞中Snail蛋白稳定性从而诱导发生EMT、增强细胞迁移和侵袭能力,该发现提供了一种可能的HBV相关肝癌患者肿瘤复发和转移的新机制。更多还原

【Abstract】 As early as 1970, Sherlock and his colleagues found chronic liver disease and primary liver-cell cancer with hepatitis-associated (Australia) antigen in serum, which indicated the association between chronic hepatitis B virus (HBV) infection and the development of hepatocellular carcinoma (HCC). In 1981, a powerful substantiation of the association between chronic HBV infection and HCC development was the result of a prospective cohort study in which Beasley and his colleagues followed 22, 707 Chinese men in Taiwan (China) and found that incidence of HCC among carriers of HBV surface antigen (HBsAg) is 63 times higher than among HBsAg non-carriers.Among all proteins encoded by HBV genome, hepatitis B virus X protein (HBx), a key nonstructural multifunctional regulatory protein of the virus, is at the intersection of HBV infection, replication, pathogenesis, and carcinogenesis. Although previous investigations have revealed that HBx might promote HBV-associated hepatocarcinogenesis through activating some oncogenic signal pathways, the role of HBx in the molecular mechanism for the development and progression of HBV-associated hepatocellular carcinoma (HBV-HCC) remains poorly understood.Although it has been reported that Notch1 signaling, as an evolutionary conserved local cell interaction mechanism, could exert a tumor suppressive function in hepatocarcinogenesis, the interaction between HBx expression and Notch1 signaling remains further elucidation. Besides, a high incidence of tumor recurrence and metastasis has been reported in hepatocellular carcinoma (HCC) patients with chronic hepatitis B virus (HBV) infection. Although the pathological relevance and significance of HBx in HBV-associated hepatocarcinogenesis attracted much attention in recent years, the role and molecular mechanism for HBx in tumor invasion and metastasis remain not fully understood.In the part one of this thesis, we report that HBx expression in hepatic and hepatoma cells resulted in decreased endogenous protein level of Notch1 intracellular domain (ICN1) and mRNA levels of its downstream target genes. These effects were shown due to reduction of Notch1 cleavage by HBx via suppressing presenilin1 (Psen1) transcription rather than inhibiting Notch1 transcription or its ligands expression. By transient HBx expression, decreased ICN1 resulted in enhanced cell proliferation, induced G1-S cell cycle progression, and blunted cellular senescence in vitro. Furthermore, the effect of blunted senescence-like growth arrest by stable HBx expression via suppressing ICN1 was shown in a nude mouse xenograft transplantation model. The correlation of inhibited Psenl-dependent Notchl signaling and blunted senescence-like growth arrest was also observed in HBV-associated HCC patient tumor samples. Our results revealed a novel function of HBx in blunting senescence-like growth arrest through decreasing Notchl signaling, which could be a putative molecular mechanism mediating HBV-associated hepatocarcinogenesis.In the part two of this thesis, we found that HBx expression could induce change of cell morphology from epithelial-like phenotype to mesenchymal-like phenotype, downregulation of epithelial marker expression (e.g. E-cadherin), nuclear translocation of P-catenin, upregulation of mesenchymal marker expression (e.g. N-cadherin, vimentin, and fibronectin), which indicates HBx expression could induce epithelial-mesenchymal transition (EMT) in hepatoma cells. This effect was shown due to increased EMT regulatory transcription factor Snail protein level by HBx transfection through enhancing Snail protein stability rather than upregulating Snail transcription, which could be reversed by siRNA Snail cotransfection with HBx. It was further elucidated HBx expression could enhance Snail protein stability via activating PI3K/AKT/GSK-3P signal pathway, which were substantiated by PI3K specific inhibitor LY294002 and Wortmannin treatment or constitutively active GSK-3βS9A mutant plasmid cotransfection with HBx. Moreover, it was also found that HBx expression in hepatoma cells could enhance cell migration ability and cell invasion ability in vitro, which could be reversed by siRNA Snail cotransfection, through wound healing assay, transwell cell migration assay, and collagen cell invasion assay. These results revealed a novel function of HBx in promoting EMT through stabilizing Snail protein via activating PI3K/AKT/GSK-3βsignaling, thus facilitating cell migration and invasion, which could provide a potential molecular mechanism for tumor invasion and metastasis in HBV-associated HCC patients.更多还原