核苷酸切除修复因子XPD通过靶向HBx阻遏HBV整合

HBV integration is associated with the onset and development of hepatocellular carcinomas, and the mechanisms of HBV integration remains far from understood. Studies have shown that inhibition of DNA damage repair can increase the frequency of HBV integrations. HBx, expressed by HBV, interferes with liver cell DNA damage repair through a variety of pathways, implying HBx can promote HBV integration. Our previous studies have found that the nucleotide excision repair - transcription factor TFIIH subunit XPD can down-regulate the expression of HBx ,therefore, we hypothesize that "XPD can inhibit HBV integration through down-regulating the expression of the HBx ". This research will further our preliminary studies, by gene transfection in hematoma cell and normal liver cell lines, Southern-blotting, to elucidate the mechanisms of HBV integration and confirm our hypothesis. From the perspective of the interaction between product of HBV virus and host factors, this study will explore the mechanisms of HBV integration from another view. Previous studies focused on trans-activation of HBx, its role in HBV integration has not been documented, and this study focuses on the role of HBx in HBV integration. This study will enrich the carcinogenic theory of HBx, providing a new strategy for HBV-DNA integration intervention, prevention of genomic instability due to HBV-DNA integration and sequent occurrence of hepatocellular carcinomas, and providing potential targets for clinical interventions.

HBV整合与肝细胞癌（HCC）的发生、发展密切相关，有关HBV整合的确切机制尚未完全明了。研究发现，抑制DNA损伤修复能促进HBV整合，而HBV的表达产物HBx能抑制肝细胞DNA损伤修复，提示HBx具有促进HBV整合的作用。本课题组前期研究发现，着色性干皮病基因D（XPD）能下调HBx的表达。据此，我们提出"XPD通过下调HBx的表达，解除HBx的DNA损伤修复抑制作用，从而抑制HBV整合"的假说。为证实这一假说，我们将在前期工作基础上，采用基因共转染技术在肝癌细胞系、正常肝细胞中研究XPD对DNA损伤修复的促进作用及对HBV整合的抑制作用，并探讨其机制。研究HBx在HBV整合的作用将丰富HBV的致癌机制理论，XPD通过下调HBx的表达阻遏HBV整合将为干预乙肝病毒整合、预防HBV-DNA整合引发的基因组不稳定及HCC的发生提供一种全新的策略，从而为临床提供新的干预靶点。

肝细胞癌（hepatocellular carcinoma，）的发病机制复杂多变，乙肝病毒（hepatitis B virus，HBV）整合与的发生密切相关，HBV致癌机制有待进一步阐明。为了探讨HBV的致癌机制及可能的干预措施，本课题通过基因共转染、Southern-blot、Western-blot 等细胞分子学技术，研究了着色性干皮病基因D（xeroderma pigmentosum D，XPD）对HBV-DNA整合的阻遏作用，并探讨了其分子机制。在肝癌细胞株HepG2.2.15中，通过对X基因、XPD的敲低/入，研究了HBx蛋白在HBV-DNA整合中的促进作用及XPD对HBx蛋白的抑制作用；在正常肝细胞LO2中，通过XPD和X基因的敲入，研究了XPD在HBV-DNA整合中的抑制作用，证实XPD通过下调HBx的表达，影响了ATM-Chk2-P53信号通路介导的DNA损伤修复过程，进而抑制HBV-DNA整合。通过本研究，我们证实1.HBV能整合到细胞基因组，其可能的机制是通过HBx介导；2.XPD通过ATM-Chk2-P53信号途径影响HBx进而阻遏HBV的整合。同时还发现，HBV整合过程中HBx能影响很多肿瘤相关信号通路的改变（如mTOR通路，NF-κB通路），XPD能通过影响细胞自噬发挥抑制肝癌发生的作用。综合上述结果，本研究为探讨HBx在肝癌发生过程中的作用及XPD抑制HBV整合作用的机制提供了理论基础，同时对于乙肝相关性肝癌的发病机制提供了线索。项目资助发表SCI论文3篇，待发表两篇。项目投入经费50万元，支出44.3375万元，各项支出基本与预算相符。剩余经费5.6625万元，剩余经费计划用于本项目研究后续支出。