TIPE2介导的DNA损伤促进丙型肝炎病毒诱发肝细胞恶变的机制研究

The balance between DNA damage and damage repair is one of the key barriers to prevent tumourgenesis, and this balance is usually employed by viruses or virus-encoded proteins to promote their own replication and oncogenic effects. According to present studies, HCV (Hepatitis C virus) and HCV-encoded proteins pose a direct threat to host genomic stability through their ability to induce DNA damage and/or prevent damage repair, however, the molecular mechanisms have thus far remained unclear. In our previous study, TIPE2（Tumor necrosis factor (TNF)-α induced protein 8-like 2 ）was found obvious down-regulated in liver tissue of HCV positive hepatocellular carcinoma (HCC) patients. Directly knocking down TIPE2 by lentivirus infection resulted in a significant alteration in the DNA damage response，including the decrease of γ-H2AX and pChk1. On the other hand, we also found HCV-encoded NS5A can interact and promote the degradation of TIPE2. In this study, the association of HCC and TIPE2 will be further confirmed in liver tissue from more HCV positive HCC patients and cell culture derived infectious HCV (HCVcc). Further more, the molecular mechanism of how HCV promote TIPE2 degradation and subsequently affect DNA damage/repair pathway will also be investigated. The results from this proposal will reveal the new function of TIPE2 in tumourgenesis in addition to its immune regulation, and also provide new insight into the mechanisms of HCV pathogenesis and tumorigeness.

丙型肝炎病毒（HCV）感染可诱导DNA损伤并抑制损伤后修复，这是引起肝细胞癌变的重要因素，具体机制尚不十分清楚。我们前期在HCV阳性肝癌病人肝组织及肝癌细胞系中检测到免疫调节因子TIPE2表达显著降低，提示TIPE2可能与HCV感染导致的肝细胞肝癌密切相关。利用慢病毒感染建立TIPE2沉默细胞株，经羟基脲处理后检测发现DNA 损伤/修复通路中γ-H2AX 、pChk1等明显下调，细胞克隆形成能力增强。另外，我们还发现HCV 非结构蛋白NS5A可与TIPE2相互作用。本课题拟在前期研究基础上，利用HCV阳性肝癌病人标本及HCV细胞培养病毒（HCVcc）进一步确认TIPE2在HCV感染诱发肝癌中的作用；并分别在细胞、动物与病人层面揭示HCV通过调控TIPE2蛋白表达进而影响DNA损伤/修复通路的分子机制。研究结果有助于了解HCV感染致肝癌发生的新机制，并将拓展对TIPE2新功能的认识。

丙型肝炎病毒（HCV）感染与肝细胞肝癌的发生密切相关，其中的分子机制仍未完全阐明。TIPE2 (Tumor necrosis factor-α-induced protein 8-like 2) 是近年被鉴定出来的新型免疫调节分子，在免疫稳态调节和肿瘤发生中均扮演重要角色。我们推测TIPE2也可能在HCV感染后肝细胞癌变过程中发挥作用。基于此，本项目从临床上收集10例HCV阳性的肝癌病人肝癌组织和癌旁组织标本，利用Western blot检测TIPE2表达；利用免疫共沉淀、GST-pull down 和免疫荧光激光共聚焦考察HCV编码蛋白与TIPE2的相互作用；利用细胞系和裸鼠模型探讨肿瘤形成情况和内在分子机制。研究结果发现，与癌旁组织相比，肝癌组织中TIPE2表达明显降低。HCV编码的非结构蛋白NS5A与TIPE2存在相互作用，且NS5A可促进TIPE2蛋白降解，可能是以蛋白酶体依赖的方式。利用shRNA在肝细胞系中干扰掉TIPE2后可提高克隆形成能力，加快划痕愈合时间；同样，干扰TIPE2的细胞在裸鼠体内的肿瘤形成时间加快，瘤体积增大。机制研究表明，TIPE2对DNA损伤反应通路存在调控作用，干扰TIPE2可引起基因组不稳定，过表达可逆转这一现象并抑制肿瘤形成。综上HCV NS5A 通过相互作用下调TIPE2，引发DNA损伤，从而促使肝细胞癌变。这一研究丰富了对HCV致病机理的研究，也为HCV相关的肝细胞肝癌治疗提供了新方向。