HBx上调TGF-β/TβR-II通路易化肝细胞癌对索拉菲尼耐药的研究

Recently, the role of inflammatory microenviroment in tumor drug resistance has been widely recognoized.Our previous study demonstrated that the overexpression of hepatitis B virus HBx protein in hepatocellular carcinoma (HCC) was associated with sorafenib-resistance in HCC,and co-immunoprecipitation assays revealed HBx could complex with TGF-β1 receptor--TβR-II.Further study showed that the overexpression of HBx could down-regulate the anti-tumor ability of sorafenib in invasion/proliferation of HCC cells.The results obtained thus far led us to investigate whether HBx might mediate the TGF-β receptor signaling to control the response to cancer drug-sorafenib in HCC.To probe this, we herein first employ tissue microarrays(TMAs) to detect the HBx expression in large sample of tissues in HCC patients who received cancer drug-sorafenib, accompanied with in vitro and in vivo studies in other HCC cell lines,to determine whether the HBx expression in HCC to be associated with sorafenib-resistance. Then, we will examine structurally and functionally whether HBx could act on TGF-β1/TβR-II system by assays of RNA interference, laser confocal microscopy, co-immunoprecipitation, Western blot,.etc. We further employ vsh-RNA interference to selectively knockdown the expression of HBx and/or TβR-II in HCC cell lines to determine potential functional mechanism of HBx in TGF-β1/TβR-II system to control the sorafenib-resistance in HCC. Lastly, we will verify those significance above in nude mice model. We thus, for the first time to our knowledge, investigate that, under inflammatory microenviroment of chronic hepatitis B infection, HBx’s vital role in regulation of TGF-β1/TβR-II signaling, and further, we probe the signal.pathways involved in its role in sorafenib-resistance in HCC.

炎性微环境与肿瘤耐药已受到极大关注。我们前期研究发现乙肝病毒HBx蛋白高表达与肝癌对索拉非尼耐药正相关，免疫共沉淀分析发现TGF-β1之受体TβR-II可与HBx形成复合物，且后者高表达可抑制索拉非尼对肝癌的抗肿瘤作用、促进细胞侵袭增殖。据此，我们推测并探讨HBx可能上调TGF-β/TβR-II通路并易化肝癌对索拉菲尼耐药。本研究对服用索拉非尼的大样本肝癌病人肿瘤组织检测并结合体内外实验，明确HBx表达与肝癌对索拉非尼耐药的关系；免疫共沉淀后的质谱分析、western blot以及激光共聚集等研究并结构上明确HBx与TβR-II相互作用。在慢病毒干扰HBx和（或）TβR-II的情况下，功能上探讨HBx对TGF-β/TβR-II的影响及其抑制索拉菲尼抗肿瘤作用的可能机制；最后体内实验证实。本研究首次探讨乙肝慢性炎性微环境下HBx对TGF-β/TβR-II的影响，及其诱导索拉菲尼耐机制。

前期研究发现乙肝病毒HBx蛋白高表达与肝癌对索拉非尼耐药正相关，HBx沉淀富集的蛋白中，首次发现有TβR-II存在。据此我们推测HBx蛋白可能通过TGF-β1/TβR-II通路而上调肝癌对靶向药物索拉非尼耐药性而降低其疗效。本研究通过大样本（n=120）的HBV相关肝癌中HBx表达检测验证分析，构建慢病毒感染HBx高、低表达的HepG2系列稳定细胞株并结合体外细胞侵袭、增殖和索拉菲尼IC50凋亡实验等，明确了HBx表达与肝癌对靶向治疗药物索拉非尼耐药正相关。进而在干扰HBx表达的情况下，通过免疫共沉淀结合质谱分析以及western blot明确证实TβR-II在肝癌细胞中可直接或间接与HBx蛋白形成复合物，结构上明确HBx可偶联TβR-II。我们随后进一步在功能上实验分析HBx蛋白能否参与TGF-β/TβR-II通路发现：在TGF-β1作用下行CCK8细胞增殖和Transwell侵袭试验显示在HBx受到干扰下（HBx低表达），TGF-β1对肝癌细胞的增殖和侵袭能力无明显作用，而在HBx未受到干扰（HBx过表达）的细胞中，加入TGF-β1具有促进细胞增殖和提高侵袭能力作用，提示在干扰HBx后，TβR-II与HBx之间存在作用分离情况，提示两者存在密切功能关系。随后，凋亡实验和免疫印迹分析发现HBx高表达可导致肝癌细胞耐药性增加，与肝癌对索拉菲尼耐药密切相关；行耐药相关蛋白检测分析HBx高表达HepG2细胞中多药耐药蛋白TGF-β1的受体TβR-II蛋白表达量明显增高，提示HBx上调可以通过TGF-β1/TβR-II通路在肝癌细胞中导致索拉菲尼耐药发生，即导致肝癌细胞对索拉菲尼药物产生更强的耐药性。最后从基础研究回归到临床：大宗临床病例调查分析验证了肝癌中HBx表达与索拉非尼耐药正相关。本项目研究提示：HBx高表达是肝癌对索拉非尼耐药密切相关的分子预测指标，并可能成为提高索拉非尼敏感性的潜在治疗靶点，有潜在的较大临床应用价值。