基于宿主基因组HBV整合位点分析寻找新的肝癌相关基因

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. In china, more than 80% of HCC cases were related to Hepatitis B Virus (HBV) infection. HBV integration is considered to be one of the major cause factors of carcinogenic HBV infection. However the underlying mechanism has not been fully understood. Recently, using traditional cloning based sequencing and next generation sequencing techniques, a large number of host-viral junction sequences have been collected from both tumor and their matched adjacent non-cancerous tissues. Sequencing analysis of the viral sequences of this large ensemble of integrated HBV fragment failed to reveal differences of viral mutation or pattern of viral end-break points between tumor and non-tumor tissues. On the other hand, bioinformatic analysis was carried out to compare the potentially integration-affected host genes derived from tumor and non-tumor tissues. We revealed significantly differences of enrichment in biological processes and signal pathways, between tumor and adjacent non-cancerous tissues. Based on the above observations, we postulate that, when targeted to/near host gene which possessing cancer related functions, the risk of HBV integration induced hepatocyte transformation and HCC development will be great. In support of our hypothesis, it has been shown that almost all of the host genes known to be frequently integration targeted, such as hTERT, MLL4 and CCNE1were carcinogenic related. Therefore, we further postulate that the integration repeatedly targeted host gene(s) (RTGs) in tumor tissues are most likely hepatocarcinogenic related, and therefore could be considered as the candidates of HCC related genes. In this research, the tumor related functions of TUFT1 and UBXN8 gene will be profoundly investigated, to prove their relation to hepatocarcinogenesis; we will also investigate their expressed and functional aberrations in preliminary HCC tissues, to evaluate their clinical significance.

病毒整合被认为乙肝病毒（HBV）感染致癌密切相关，但确切机制尚未完全阐明。我们最近建立了较大规模的HBV整合信息数据集。通过对该数据集中整合病毒片段的序列在断点分布、突变及所携带的病毒调控元件等方面的分析，未见癌和癌旁组织存在差异；但对该数据集宿主序列的生物信息学分析发现，肝癌组织中潜在受HBV整合影响的宿主基因所参与的生物学过程和信号通路与癌旁组织存在显著差异，且肝癌组织中较为常见的整合位点所涉及的宿主基因如hTERT、MLL4、CCNE1等基因的功能均与肿瘤发生密切相关。我们推测受整合影响的宿主基因的功能和生物学特性在HBV整合致癌中具有重要作用，那些在不同肝癌组织重复发生的HBV整合位点附近的宿主基因可能具有一定的肿瘤相关性。本研究将对前期工作中获得的重复整合基因TUFT1和UBXN8进行深入的功能研究，并通过分析其在原发性肝癌样本中的表达和功能异常，确定其与肝癌发生的相关性及临床意义。

乙型肝炎病毒（HBV）的慢性感染是肝细胞癌发生的重要诱导因素之一，而HBV基因组DNA片段在人类肝细胞基因组中的整合被认为对肝癌的发生发展起重要的促进作用。近年来的研究提示，病毒整合对附近宿主肿瘤相关基因表达和功能的影响，可能是HBV整合的重要致癌机制。据此，我们认为肝癌组织中HBV整合周围的宿主基因可能与肝癌的发生、发展相关，这些基因将是筛选HBV感染相关肝癌相关基因的重要线索。.通过对文献的分析，我们共找到817个HBV整合宿主靶基因，其中肝癌组中有513个，癌旁组中有304个。进一步分析发现，上述宿主靶基因中有75个出现了两次或两次以上整合，这些基因被定义为重复整合靶基因（recurrently targeted genes, RTGs），其中50个靶基因仅在癌组织中被重复整合，11个基因仅在癌旁组织中被重复整合，肝癌组织中重复整合靶基因出现的频率显著高于癌旁组织。通过对RTGs进行功能注释分析发现，这些基因所富集的功能条目与癌症的发生发展密切相关，包括细胞凋亡负调控、转录调控以及癌症信号通路。其后，25例HBV感染相关肝癌组织的aCGH结果显示，RTGs中有54%的基因在5例或以上的组织中存在拷贝数变异。为寻找新的肝癌相关候选基因，我们根据75个RTGs的整合位点位置、aCGH结果以及文献检索结果，挑选12 个基因做进一步的表达分析。实时定量 PCR 结果显示，MLL4 和 TUFT1肝癌组织中表达水平显著高于癌旁组织，而 FGF9、UBXN8 和 DGKγ则在肝癌组织中显著下调。UBXN8是本研究首次发现的RTGs，其功能尚未见报道。进一步扩大样本量发现，UBXN8在43%的肝癌组织中呈现低表达，UBXN8低表达肝癌患者的五年生存率显著低于非低表达组，提示UBXN8对于患者预后有重要的临床价值。体外实验证实UBXN8仅在具有野生型p53的肝癌细胞系中发挥抑癌基因的作用，具有野生型p53的HepG2和SK-Hep-1细胞中外源表达UBXN8可以上调p53和p21的表达，从而抑制细胞周期进程和细胞的迁移。.综上所述，本研究为进一步认识HBV相关肝癌发生发展的分子机制、发现和研究新的肝癌相关基因提供了新的研究途径和思路。