蒽环类抗生素通过RFX1引起HBV再激活的机制研究

Hepatitis B virus (HBV) reactivation is well documented in previously resolved or inactive HBV carriers who receive cancer chemotherapy. With increasing life expectancy and the global increasing in cancer, chemotherapy induced reactivation of hepatitis B is likely to become an increasing problem. Anthracycline antibiotics is one of the most common chemotherapy drugs inducing HBV reactivation. However, the exact mechanism of HBV reactivation induced by anthracycline antibiotics is not fully understood. Regulatory factor X-box 1 (RFX1) is one of the DNA damage effectors, and takes part in the repair of DNA damage. Meanwhile, RFX1 is one of the most important transcription factors which transactivate HBV transcription and promote HBV replication through the EP site in HBV enhancer I region and the analogue of EP site in HBV core promoter region. Our previous research found that doxorubicin and epirubicin could promote HBV replication and RFX1 expression in parallel, but for HBV with EP site mutation, the effect is much weaker. We hypothesized that anthracycline antibiotics may enhance the transcription activity of HBV enhancer I and core promoter through promoting RFX1 expression, and then promote HBV replication. In this study, we will investigate the influence of anthracycline antibiotics on the transcription activity of HBV enhancer I and core promoter, verify that RFX1 takes part in the regulation of HBV replication by anthracycline antibiotics, and then elucidate the new mechanism of anthracycline antibiotics upregulating HBV transcription and replication. The present study will identify a novel DNA damage-HBV replication pathway in liver cancer cell, which may be a potential target for prevention in HBV reactivation and efficacy in tumor therapy.

蒽环类抗生素是常用的肿瘤化疗药物。使用蒽环类抗生素可引起处于免疫控制状态的慢性HBV感染者、甚至既往感染者的HBV再激活，但其机制尚未充分阐明。调节因子RFX1是一种DNA损伤效应蛋白，参与DNA损伤修复。研究表明，RFX1可与HBV 增强子I区的EP位点和核心启动子区的类EP位点结合，反式激活HBV转录，进而促进HBV复制。我们的前期研究发现，蒽环类抗生素的代表性药物阿霉素及其衍生物表阿霉素在体外不仅促进HBV复制，还能上调RFX1表达，且二者变化趋势一致。但将HBV增强子I区的EP位点突变后，阿霉素对HBV复制水平的促进作用明显减弱。据此推测，蒽环类抗生素可通过上调RFX1表达，增强HBV增强子I的转录活性，进而促进HBV复制。本课题将探索蒽环类抗生素通过RFX1引起肿瘤患者HBV再激活的新机制，为靶向预防肿瘤化疗过程中HBV再激活、提高肿瘤疗效提供依据。

包括阿霉素在内的蒽环类抗生素可直接促进乙型肝炎病毒（HBV）复制，但其机制尚未充分阐明。我们在本研究中发现调节因子（RFX1）可介导阿霉素对HBV复制的促进作用，并揭示了阿霉素通过RFX1促进HBV复制的机制。我们首先发现HBV复制水平和RFX1表达水平在阿霉素处理后同步增加，并且阿霉素可显著增加RFX1与HBV增强子I的结合水平。当内源RFX1被敲减后，阿霉素促进HBV复制的能力显著减弱。位于HBV增强子I中的EP序列可介导RFX1与HBV增强子I的结合，当EP序列突变后，阿霉素促进HBV复制的能力也显著减弱。由此可见，阿霉素主要通过上调RFX1的表达水平，增加RFX1与HBV增强子I的结合水平，进而增强HBV增强子I的转录活性，促进HBV复制。此外，我们发现在A～D基因型HBV基因中存在两种相对保守的EP序列，而无论含有哪种EP序列，阿霉素均可促进HBV复制。.据报道，阿霉素可上调转录因子SOX4表达，而SOX4可通过与HBV基因组中的AACAAAG序列结合促进HBV复制，提示SOX4可能也介导了阿霉素对HBV复制的直接促进作用。然而，我们通过序列比对发现，AACAAAG序列仅在少数HBV病毒株中存在，也就是说，大部分HBV病毒株的基因组中不含有该序列。因此，本研究进一步探讨了SOX4在调控HBV复制中的作用及其机制。结果发现，SOX4仅对少数含有AACAAAG序列的HBV病毒株的复制水平起到促进作用，而抑制大部分HBV病毒株的复制。进一步研究发现，SOX4可下调肝细胞核因子4α（HNF4α）的表达水平，而HNF4α可直接促进HBV复制；当内源HNF4α敲减后，SOX4对HBV复制的抑制作用显著减弱。由此可见，SOX4一方面通过与AACAAAG序列结合促进少数HBV病毒株复制，另一方面通过下调肝细胞核因子4α（HNF4α）的表达来抑制HBV复制。.总之，本研究部分揭示了包括阿霉素在内的蒽环类抗生素促进HBV复制，进而引起HBV再激活的分子机制，为寻求抗HBV再激活的潜在药物靶点提供理论依据。