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Defining the Oncogenic Functions of Hepatitis B Virus-Human Fusion Transcripts in Hepatocellular Carcinoma.

机译：定义乙型肝炎病毒-人类融合转录本在肝细胞癌中的致癌作用。

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摘要

Chronic hepatitis B (HBV) infection IS a major etiologic factor in the development of human hepatocellular carcinoma (HCC). It is long recognized that viral integration into the host HCC genome can be detected in >85% cases, yet the inertional mutagenesis remains largely unclear. In an attempt to define viral integrants, our lab has previously defined recurring HBx/8p 11 integration site using the technique of restriction site-polymerase chain reaction (RS-PCR). This study was elaborated by cloning the full-length HBx/8p 11 chimeric transcript by 5' and 3' Rapid Amplification of cDNA Ends (RACE). Investigations conducted in this thesis revealed that this novel viral-human HBx/8pll fusion transcript functions as long non-coding RNA (ncRNA). It regulates the epithelial-to-mesenchymal transition (EMT) and triggers Wnt signalling pathway, all of which are considered important processes in the liver oncogenesis.;The full-length HBx/8p11 chimeric transcript of 674bp contains a microhomology sequence of GTG shared at the flanking juncture. Expression of HBx/8p11 fusion transcript can be detected in 23.3% of HBV-positive HCC tumors (21 out of 90 cases), where a significant correlation with shorter patient survival ( P = 0.015) and cox prognostic indicative value (P = 0.014) could be drawn. Functional knockdown of HBx/8pll resulted in marked inhibition on cell migration (P = 0.04) and invasion (P = 0.04), with upregulation of epithelial markers E-cadherin and γ-catenin expressions, and downregulation of mesenchymal marker fibronectin, suggesting its likely role in EMT phenotype. A corresponding diminution of β-catenin nuclear localization and reduced β-catenin trans activating activity (P = 0.0076) were also revealed, which enforced a role for HBx/8p11 in the membrane-bound ECadherin/β-catenin complex. Conversely, ectopic expression of full-length HBx/8p11 in immortalized hepatocyte L02 promoted cell invasiveness (P = 0.005) and migration ( P = 0.028) via promoting EMT. This effect was not observed in L02 transfected with empty vector, HBx component or flanking 8p11 sequence. GFP-tagged constructs further suggested protein encoded from HBx/8p11 was similar to the flanking HBx component, suggesting the functional phenotype observed on HBx/8p11 was likely attributed to a non-protein coding RNA transcript (ncRNA). Ectopic expression of full-length HBx/8p11 in L02 also induced colony growth. We further demonstrated in liver-specific transgenic model that mice with HBx/8p11 trans gene were more susceptible to DEN-induced tumor formation than non-transgenic littermate. These data highlights the importance for the HBx/8p11 ncRNA transcript in conferring oncogenic advantages, and may represent an elemental predisposing factor in the initiation and progression of HCC.;During the course of RACE cloning for HBx/8p11 full-length sesquence, another viral-human transcript HBx/22q11 (477bp) was also identified. Similar to the HBx/8p11 characterizations of HBx/22q11 were also carried out. HBx/22q11 expressed in 24.9% HBV-positive HCC cases (12 out of 49 cases). However, HBx/22q11 expressions do not correlate with patients' survival and clinicopathological parameters of these patients. Functional studies also revealed insignificant effect on cell migration, invasion and cell growth.;In summary, novel recurrent viral integration sites leading to the expression fusion transcripts were identified. In particular, HBx/8p11 functions as long ncRNA to promote cell motility, cell proliferation and tumor growth. Taken together with its high prevalence and significant effect in overall survival, HBx/8p11 fusion transcript is a possible molecular target for the development of successful therapeutic interventions in HCC.
机译：慢性乙型肝炎（HBV）感染是人类肝细胞癌（HCC）发生的主要病因。长期以来，人们认识到在> 85％的情况下可以检测到病毒整合到宿主HCC基因组中，但是惯性诱变作用仍然不清楚。为了定义病毒整合体，我们的实验室先前使用限制性酶切位点聚合酶链反应（RS-PCR）技术定义了复发性HBx / 8p 11整合位点。这项研究是通过cDNA末端的5'和3'快速扩增（RACE）克隆全长HBx / 8p 11嵌合转录本来完成的。本论文进行的研究表明，这种新型的病毒-人HBx / 8pll融合转录本具有较长的非编码RNA（ncRNA）的功能。它调节上皮到间质转化（EMT）并触发Wnt信号通路，所有这些通路均被认为是肝癌发生中的重要过程。全长674xbp的HBx / 8p11嵌合转录本包含GTG的微同源序列侧面的关头。在23.3％的HBV阳性HCC肿瘤中（90例中有21例）可以检测到HBx / 8p11融合转录物的表达，这与患者生存期较短（P = 0.015）和Cox预后指示值（P = 0.014）有显着相关性可以画。 HBx / 8pll的功能性抑制可显着抑制细胞迁移（P = 0.04）和侵袭（P = 0.04），上皮标记物E-cadherin和γ-catenin表达上调，间质标记物纤连蛋白下调，表明其可能在EMT表型中的作用。还揭示了β-连环蛋白核定位的相应减少和β-连环蛋白反式激活活性的降低（P = 0.0076），这在膜结合的ECadherin /β-连环蛋白复合物中增强了HBx / 8p11的作用。相反，在永生化肝细胞L02中全长HBx / 8p11的异位表达通过促进EMT促进了细胞侵袭性（P = 0.005）和迁移（P = 0.028）。在用空载体，HBx组分或侧翼8p11序列转染的L02中未观察到这种效果。带有GFP标签的构建体进一步表明，从HBx / 8p11编码的蛋白质与侧翼HBx组分相似，这表明在HBx / 8p11上观察到的功能表型可能归因于非蛋白质编码RNA转录物（ncRNA）。 L02中全长HBx / 8p11的异位表达也诱导菌落生长。我们进一步在肝特异性转基因模型中证明，与非转基因同窝仔相比，具有HBx / 8p11转基因的小鼠更容易受到DEN诱导的肿瘤形成。这些数据凸显了HBx / 8p11 ncRNA转录本在赋予致癌性方面的重要性，并且可能代表了HCC起始和进展的基本诱因。在RACE克隆过程中，HBx / 8p11全长后继是另一种病毒还鉴定了人转录本HBx / 22q11（477bp）。与HBx / 8p11类似，还对HBx / 22q11进行了表征。 HBx / 22q11在24.9％HBV阳性HCC病例中表达（49例中有12例）。但是，HBx / 22q11表达与这些患者的生存率和临床病理参数无关。功能研究也显示出对细胞迁移，侵袭和细胞生长的微不足道的影响。总之，鉴定了导致表达融合转录本的新的复发性病毒整合位点。尤其是HBx / 8p11可以作为长ncRNA来促进细胞运动，细胞增殖和肿瘤生长。 HBx / 8p11融合转录本及其高患病率和对整体生存的显着影响，可能是成功开发HCC治疗干预措施的可能的分子靶标。

著录项

  • 作者

    Lau, Chi Chiu.;

  • 作者单位

    The Chinese University of Hong Kong (Hong Kong).;

  • 授予单位 The Chinese University of Hong Kong (Hong Kong).;
  • 学科 Biology Molecular.;Health Sciences Pathology.;Biology Virology.;Health Sciences Oncology.
  • 学位 Ph.D.
  • 年度 2011
  • 页码 159 p.
  • 总页数 159
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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