乙肝病毒调控DcR3在肝细胞肝癌发生过程中的作用及机制研究

The regulation of host genes, especially the cancer related gene by HBV and its coding protein plays an important role in the development of HCC. DcR3 is a member of tumor necrosis factor receptor superfamily. It has been implicated in tumorigenesis through its abilities to competetitvely bind to FasL, TIGHT, and TL1A, modulate immune responses and induce angiogenesis. DcR3 expression is elevated in many kinds of tumors cells. In previous study, we found that DcR3 expression increased in sera of CHB patients, and positively associated with HBV DNA and ALT. Also by comparing the expression of DcR3 in HepG2 and HepG2.2.15 cells, which were stably transfected with the 1.3-fold HBV genome, we found that DcR3 expression levels were much higher in the HepG2.2.15 cells than in the HepG2 cells. Thus, we speculate that HBV through regulation DcR3 expression participates in the development of HCC. Here, we use overexpression and siRNA method to elucidate the effect of DcR3 on the biological functions of liver cells; By overexpression HBV and its coding protein to confirm the regulation of DcR3 by HBV.; Through the dual luciferase report system, EMSA and reverse blocking technology, to clarify the molecular mechanism of HBV regulation DcR3, providing new insights into mechanisms for HBV related HCC development.

乙肝病毒（HBV）及其编码蛋白对宿主基因尤其是肿瘤相关基因的调控在HCC发生中具有重要意义。诱骗受体3（DcR3）是肿瘤坏死因子超家族的一员，通过竞争性结合FasL、LIGHT和TL1A，抑制细胞凋亡，逃避免疫监视，促进血管形成，与肿瘤的发生、发展密切相关，在多种恶性肿瘤中高表达。本课题组前期研究发现，CHB病人血清DcR3表达升高，且与HBV DNA及ALT等强相关，整合HBV基因组的HepG2.2.15细胞DcR3表达明显高于HepG2细胞，提示HBV通过调控DcR3参与HCC发生。本课题拟通过过表达及封闭DcR3，验证DcR3对肝细胞生物学功能的影响；通过过表达HBV及编码蛋白，证实HBV对DcR3的调控；通过双荧光素酶报告系统、EMSA及反向阻断技术，阐明HBV调控DcR3的分子机制，为HBV相关HCC发病机制提供新的思路。

本课题前期研究发现，慢性乙型肝炎（chronic hepatitis B, CHB）患者外周血诱骗受体 3（decoy receptor 3, DcR3）表达升高，且与乙肝病毒 （HBV）DNA 和ALT强相关。DcR3是肿瘤坏死因子受体超家族新成员，能够竞争性结合FasL、LIGHT和TL1A，阻断相关信号转导通路，调节细胞增殖、抑制细胞凋亡，在一些疾病尤其是恶性肿瘤中高表达，与肿瘤的发生、发展密切相关。本课题以这些发现为基础，利用已收集的乙肝病人标本，首先检测了不同程度乙肝病人血清和肝癌组织中DcR3和Fas的表达以及相关性，进一步揭示了DcR3和Fas在乙肝肝癌发生发展中的作用。其次通过设计和构建DcR3小干扰RNA重组慢病毒（LV-shDcR3，shDcR3），感染肝癌细胞HepG2和Huh-7，结果发现肝癌细胞HepG2和Huh-7中DcR3的mRNA和蛋白表达水平与正常肝细胞相比明显升高，差异具有统计学意义。细胞上清中DcR3的分泌水平也明显升高。与空白组和感染对照组相比，感染shDcR3组，肝癌细胞中DcR3的表达显著降低，细胞生存率明显降低，早期凋亡细胞比例明显升高，凋亡相关蛋白表达水平明显升高。与感染前相比，肝癌细胞感染shDcR3后，TRAIL和FasL的表达水平明显升高。进一步以HepG2为对象，深入研究发现TRAIL联合shDcR3对诱导Huh7细胞凋亡具有明显的协同效果，其机制可能是一方面促进了caspase凋亡通路，另一方面抑制了NF-Kb依赖的cFLIP的表达。项目研究成果在《检验医学》《世界华人消化杂志》《Oncotarget》等期刊发表。