【作者】 张烜；

【导师】 张晓东； 叶丽虹；

【作者基本信息】 南开大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 乙型肝炎病毒(hepatitis B virus, HBV)的持续感染是肝细胞癌(hepatocellular carcinoma, HCC)发生的最主要因素。HBV X (HBx)基因在肝细胞基因组中的整合和X蛋白的反式激活作用对HCC的发生和发展具有重要意义,但其致癌的分子机制目前尚不十分清楚。对HBx的研究大多着眼于X蛋白的反式激活功能,而忽略了HBx基因在肝细胞基因组整合的作用。研究表明,在HBV导致肝癌发生过程中,病毒DNA在宿主基因组中有高频率的整合,整合部位随机分布于多条染色体上,这种整合多发生在HBV X基因区域。前期研究发现,稳定整合HBx基因的人正常肝胞系(L-02-X)发生恶性转化,推测HBx基因的整合可能与肝癌发生有直接关系。此外,HBx蛋白对HCC的发生和转移具有重要作用。近年来研究发现,骨桥蛋白(osteopontin, OPN)及钙蛋白酶小亚基1(calpain small subunit 1,Capn4)与肿瘤细胞的生长、增殖、浸润及转移密切相关。因此,HBx蛋白可能通过OPN及Capn4促进肝癌细胞迁移。为了进一步阐明HBx基因/蛋白与肝癌发生发展的关系,本研究应用细胞模型和病人肝癌组织标本,探讨了HBx基因整合在肝癌发生中的直接作用和HBx蛋白促进肝癌细胞迁移作用的分子机制,主要研究内容如下：第一部分：HBx基因整合与肝癌发生关系的研究一、HBx基因突变体稳定整合细胞模型的建立前期研究发现,稳定整合HBx基因的人正常肝胞系(L-02-X)发生了恶性转化,因此推测HBx基因的整合可能与肝癌发生有直接关系。但是,其中包含了HBx蛋白的反式激活作用。为了探讨HBx基因整合与肝细胞转化的直接关系,首先建立了无反式激活功能的HBx基因突变体稳定整合的细胞模型。本研究中,HBx基因被随机分成五个片段,并分别克隆到真核表达载体pCMVTag-2B,获得的质粒分别命名为pCMV-X1、pCMV-X2、pCMV-X3、pCMV-X4和CMV-X5。将此五个质粒及克隆有全长HBx基因的质粒分别瞬时转染人肝细胞L-02使其蛋白过表达,同时检测对核因子-κB(NF-κB)和人端粒酶反转录酶(hTERT)启动子活性的影响,发现与野生型相比,五个突变的HBx基因片段失去了对NF-κB和hTERT启动子活性的上调作用。通过基因转染技术将克隆有这五个片段的质粒分别导入人正常肝细胞系L-O2和人肝癌细胞系H7402中,经G418筛选,获得了稳定转染细胞系,分别命名为L-02-X1、L-O2-X2、L-O2-X3、L-O2-X4和L-02-X5(或H7402-X1、H7402-X2、H7402-X3、H7402-X4和H7402-X5)。二、HBx基因片段整合导致肝细胞恶性转化的作用为了阐明L-02-X1、L-O2-X2、L-O2-X3、L-O2-X4和L-02-X5细胞系的恶性表型,进行了免疫印迹实验。结果显示,与对照组相比,在L-02-X3和L-02-X5细胞中有甲胎蛋白(AFP)表达,而L-02-X1、L-02-X2和L-02-X4则检测不到AFP。软琼脂克隆形成实验显示,与对照组相比,L-02-X3和L-02-X5细胞克隆形成能力显著增强,而L-02-X1、L-02-X2和L-02-X4细胞克隆形成能力与对照组相比无明显差异。进一步研究发现,将L-02-X1、L-02-X2、L-02-X3、L-02-X4和L-02-X5分别皮下接种裸鼠后,L-02-X3和L-02-X5细胞接种的裸鼠在接种部位形成原位瘤,而L-02-X1、L-02-X2和L-02-X4细胞接种的裸鼠则未能成瘤。本结果说明,整合有HBx基因片段的L-02-X3和L-02-X5细胞发生了恶性转化,HBx基因整合与肝细胞的转化直接相关。三、HBx基因整合导致肝细胞转化分子机制的研究1、HBx基因整合模式的鉴定。提取L-02-X细胞基因组DNA,应用HBx-AluPCR方法检测HBx基因整合模式,测序结果显示,HBx基因整合到Alu核心序列和亚端粒DNA(subtelomeric DNA)序列上游,整合后HBx基因发生了3’末端(381-465 bp)缺失,产生HBx基因/Alu核心序列/亚端粒DNA重组体。然后,设计针对该重组体中HBx基因和亚端粒的引物进行PCR,在其他HBx基因稳定整合的细胞系(如H7402-X、3T3-X、HepG2-X和HepG2.2.15)中检测上述重组体是否存在。结果显示,仅在H7402-X细胞系中检测到该重组体。2、HBx基因/Alu核心序列/亚端粒DNA重组体与肝细胞转化的关系。为了阐明该重组体是否参与肝细胞的转化,应用上述特异于HBx基因和亚端粒的引物,在稳定整合HBx基因突变体的细胞系(L-02-X1、L-02-X2、L-02-X3、L-02-X4和L-02-X5；H7402-X1、H7402-X2、H7402-X3、H7402-X4和H7402-X5)中进行验证。结果显示,在上述发现的已发生恶性转化的L-02-X3和L-02-X5(或H7402-X3和H7402-X5)细胞中检测到HBx基因/Alu核心序列/亚端粒DNA重组体,而未发生转化的细胞无该重组体。为了进一步提供HBx基因/Alu核心序列/亚端粒DNA重组导致肝细胞发生转化的证据,应用报告基因及免疫印迹的方法对L-02-X1、L-02-X2、L-02-X3、L-02-X4、L-02-X5和L-02-X(或H7402-X1、H7402-X2、H7402-X3、H7402-X4、H7402-X5和H7402-X)细胞系转录因子启动子活性及癌蛋白表达进行检测,结果显示NF-κB、AP-1和hTERT启动子活性在L-02-X3、L-02-X5和L-02-X、(或H7402-X3、H7402-X5和H7402-X)细胞中显著增强,而在L-02-X1、L-02-X2和L-02-X4则与对照组无明显差异。c-Myc、PCNA和Bcl-2蛋白表达在L-02-X3、L-02-X5和L-02-X、(或H7402-X3、H7402-X5和H7402-X)细胞中显著上调,而在L-02-X1、L-02-X2和L-02-X4细胞中则与对照组无明显差异。提示,HBx基因整合导致的HBx基因/Alu核心序列/亚端粒DNA重组是肝细胞转化的重要原因之一。3、在肝癌和癌旁组织中检测HBx基因/Alu核心序列/亚端粒DNA重组体。应用PCR方法对60例肝癌组织和癌旁组织中HBx基因进行检测,发现HBx在60例肝癌组织及其癌旁组织中有44例(73.3%)。然后,应用PCR方法在44例HBx阳性的肝癌组织及其癌旁组织中检测上述重组体,结果发现其中有5例肝癌组织中存在与细胞模型中相同的HBx基因/Alu核心序列/亚端粒DNA重组体,而在其对应的癌旁组织无上述重组体,临床标本检测结果进一步证实HBx基因/Alu核心序列/亚端粒DNA重组与肝癌发生有密切的关系。本研究发现HBx基因整合与肝细胞转化具有直接的关系,HBx基因/Alu核心序列/亚端粒DNA重组导致基因组不稳定是肝细胞转化的重要原因之一。这一发现首次为揭示HBV DNA在肝细胞基因组中整合的致癌作用提供了直接证据,对于肝癌的预防具有重要的指导作用。第二部分：HBx蛋白促进肝癌细胞迁移分子机制的研究近年研究发现OPN和Capn4与肿瘤细胞的生长、增殖、浸润及转移密切相关。因此,HBx蛋白可能通过OPN及Capn4促进肝癌细胞迁移,为此进行了以下研究：一、HBx蛋白促进肝癌细胞迁移信号传导途径的研究本研究对HBx蛋白促进肝癌细胞迁移的信号传导途径进行了探讨。前期研究发现HBx蛋白通过NF-κB上调肝癌细胞HepG2和H7402中Capn4的表达；在此基础上,进一步研究显示HBx蛋白在启动子转录活性、mRNA及蛋白等水平通过5-脂氧化酶(5-LOX)上调肝癌细胞HepG2和H7402中OPN的表达；进而,OPN可以通过NF-κB上调肝癌细胞HepG2和H7402中Capn4的表达；而Capn4则以正反馈的形式对肝癌细胞HepG2和H7402中OPN的表达起调节作用。体外划痕实验显示,HBx蛋白可显著增强肝癌细胞HepG2的迁移能力,而对HepG2-X细胞中OPN或Capn4进行RNA干扰后,其迁移能力受到明显抑制。表明,HBx蛋白通过正反馈环HBx/5-LOX/OPN/NF-κB/Capn4/OPN促进肝癌细胞迁移。二、HBx蛋白突变体HBxΔ127促进肝癌细胞迁移分子机制的研究实验室前期研究发现了一个新的HBx蛋白突变体,将其命名为HBx△127。研究表明,HBx△127具有明显促进肝癌细胞增殖的作用。在此基础上,本研究探讨了HBx△127对肝癌细胞迁移的影响,结果发现HBx△127具有促进肝癌细胞迁移的作用。HBx△127可通过5-LOX上调肝癌细胞中OPN的表达,进而促进肝癌细胞迁移。与野生型HBx蛋白相比,HBx△127促进肝癌细胞迁移的作用更强。上述实验结果进一步丰富了HBx蛋白及其突变体促进肝癌细胞迁移的分子机制,为肝癌的治疗提供了理论基础。更多还原

【Abstract】 Infection of hepatitis B virus (HBV) is closely related to the development of hepatocellular carcinoma (HCC). The integration of HBV DNA into the host genome and the trans-activation of HBx protein play crucial roles in the development of HCC. However, the molecular mechanisms underlying HBV-induced HCC remain to be elucidated. Most researches related to HBx focused on its trans-activation role in the cells, but the role of HBV DNA integration in hepatocarcinogenesis was ignored. It has been reported that HBV DNA, especially X gene, can randomly integrate into the host genome with a very high integration rate of more than 80% in HBV-related HCC. Previously, we reported that human immortalized liver L-O2 cells could be transformed by HBx in a model of stably HBx gene-transfection cells. Therefore, we supposed that the integration of HBx gene may be directly involved in hepatocarcinogenesis. Meanwhile, HBx protein also plays an important role in the metastasis of HCC. Accordingly, osteopontin (OPN) and calpain small subunit 1 (Capn4) are related to tumor cell adhesion, migration and invasion. Therefore, we supposed that OPN and Capn4 may be involved in the promotion of hepatoma cell migration mediated by HBx. In the present study, we took advantage of cell models and clinical HCC specimens to investigate the direct role of the HBx gene-integration in hepatocarcinogenesis and the mechanism of hepatoma cell migration promoted by HBx protein. This study includes two parts as follows:Part I:Investigation of relationship between HBx gene integration and hepatocarcinogenesisEstablishment of cell models stably transfected with HBx mutantWe previously reported that the L-O2 cells stably transfected with full-length HBx gene (L-O2-X cells) could be transformed, suggesting that HBx integration may be involved in hepatocarcinogenesis. However, there were the possibilities of trans-activation by HBx proteins in the transformation mediated by full-length HBx-integration. To investigate the direct role of HBx integration in the transformation, we tried to establish cell models involving HBx-mutant gene integration, in which the mutant HBx protein lost the trans-activation function. Firstly, we randomly divided the full-length HBx gene into five fragments, which were subcloned into the pCMVTag-2B vector. The constructs were termed as pCMV-X1, pCMV-X2, pCMV-X3, pCMV-X4 and pCMV-X5, respectively. We observed that the overexpression of the 5 fragments of HBx gene failed to affect the promoter activities of NF-κB and human telomerase reverse transcriptase (hTERT) relative to wild type HBx protein in the transiently HBx mutant-transfected L-O2 cells, suggesting that the mutant HBx proteins lost the trans-activation roles. The constructed plasmids were transfected into L-O2 (or H7402) cells and selected by G418 for two weeks. The engineered cells were termed as L-02-X1,L-O2-X2, L-O2-X3, L-O2-X4 and L-O2-X5 (or H7402-X1, H7402-X2, H7402-X3, H7402-X4 and H7402-X5), respectively.Effect of HBx gene integration on the transformation of L-O2 cellsWe observed the malignant phenotype of above HBx-fragments engineered L-O2 cells. The results showed that alpha-fetoprotein (AFP) was detectable in the L-O2-X3 and L-O2-X5 cells, suggesting that L-O2-X3 and L-O2-X5 cells become HCC cells. Moreover, the two cell lines generated more clones in soft agar. Animal transplantation showed that nude mice injected with L-O2-X3 and L-O2-X5 cells gave rise to tumors after transplantation, while the others failed to form any visible tumors. This finding suggests that L-O2-X3 and L-O2-X5 cells are transformed and that HBx gene-integration is directly involved in the transformation of L-O2 cells.The mechanism of HBx gene integration-meditated transformationWe found that HBx-integration was involved in the transformation of L-O2 cells, and then we tried to identify the HBx-integration manner by HBx-Alu PCR approach in the L-02-X cells. We randomly picked up one band from the positive PCR products. The sequencing data revealed that HBx gene inserted into the upstream of a 20 bp Alu core sequence with a secondary deletion of 382-465 bp at the 3’end of HBx gene, followed by subtelomeric DNA, suggesting that HBx gene-integration results in a recombination of HBx/Alu core sequence/subtelomeric DNA in the host genome. Accordingly, we designed the PCR primers to amplify the sequence from the integrated HBx to subtelomeric DNA, to confirm the recombination in H7402-X (human hepatoma H7402 cells stably transfected HBx gene),3T3-X (NIH3T3 cell line stably transfected HBx gene), HepG2.2.15 and HepG2-X (human hepatoma HepG2 cells stably transfected HBx gene). The same recombination could be only detected in H7402-X cells.To investigate whether the recombination involved the transformation of L-O2 cells, we examined the recombination in above ten engineered cell lines stably transfected with five HBx fragments by PCR using the primers from each of the five fragments to subtelomeric DNA. Interestingly, we found that the recombination was found in the transformed L-O2-X3 and L-O2-X5 (or H7402-X3 and H7402-X5) cells but not in the non-transformed cells. The corresponding recombination just matched the L-O2-X3 and L-O2-X5 cell lines which were transformed, suggesting that the recombination of HBx gene/Alu core sequence/subtelomeric DNA was involved in the transformation. Furthermore, we provided more evidence of the transformation mediated by HBx-integraiton at the molecular level. The promoter activities of NF-κB, activator protein-1 (AP-1) and hTERT were increased in the engineered cells with the HBx integration-meditated recombination (such as, L-O2-X, L-O2-X3, L-O2-X5, H7402-X, H7402-X3 and H7402-X5). The proteins involved in proliferation and transformation, such as c-Myc, proliferating cell nuclear antigen (PCNA) and Bcl-2 were remarkably upregulated in the corresponding cells as well. However, the cell lines without the HBx meditated-recombination had no the phenotype. These results indicate that HBx integration-meditated recombination is one of crucial causes of transformation of hepatocytes.To examine the recombination in human HBV-related HCC tissues, we first detected the HBx gene in 60 cases of HCC tissues and their non-tumor ones by PCR. The positive rates of HBx gene were 73.3%(44/60) in HCC tissues and 73.3%(44/60) in their non-tumor tissues. Then, we examined the recombination of HBx/Alu core sequence/subtelomeric DNA in the above HBx-positive tissues by PCR using the primers from HBx to subtelomeric DNA. The recombination was detectable in 5 out of 44 cases HCC tissues, while in their non-tumor tissues the recombination was negative. In summary, our finding first provides evidence that the HBx integration is directly related to the hepatocarcinogenesis involving a recombination of HBx/Alu core sequence/subtelomeric DNA. It suggests that the prevention of HCC is very important.Part II:The mechanism of hepatoma cell migration promoted by HBx proteinInvestigation of the signal pathways involved in hepatoma cell migration promoted by HBx proteinRecently, we reported that HBx protein could upregulate the expression of Capn4 in the hepatoma cells. In this study, we revealed that HBx could upregulate OPN through 5-lipoxygenase (5-LOX) in hepatoma HepG2 (or H7402) cells. Moreover, OPN could upregulate the expression of Capn4 through NF-κB in the cells. Interestingly, Capn4 could regulate the expression of OPN in a positive feedback manner. In function, wound healing assay showed that OPN and Capn4 were involved in the promotion of hepatoma cell migration mediated by HBx. Thus, we conclude that HBx promotes hepatoma cell migration via a positive feedback loop involving HBx/5-LOX/OPN/NF-KB/Capn4/OPN.The mechanism of hepatoma cell migration promoted by HBx protein mutant (HBxΔ127)We previously identified a natural mutant of the HBx gene, termed HBxΔ127, which was able to promote cell proliferation. In the present study, we investigated the role of HBxΔ127 in hepatoma cell migration. The results display that HBxΔ127 strongly promotes hepatoma cell migration via activating OPN involving 5-LOX relative to wild type HBx protein.Taken together, our findings provide new insight into the mechanism of hepatoma cell migration mediated by HBx protein. Potentially, OPN and Capn4 may serve as key therapeutic targets.更多还原