HCV core/β-catenin诱导肝前体细胞向肝癌干细胞分化的分子机制研究

Transdifferentiation of hepatic progenitor cells (HPCs) into hepatic cancer stem cells (HCSCs) is an important cause of primary hepatocellular carcinoma (HCC), however the transdifferentiation mechanism underlying HPCs and HCSCs remains unclear. Wnt signaling pathway is activated in HCC and plays an important role in the maintenance of HCSCs, however, whether HCV could induce the differentiation of HPCs into HCSCs through activating Wnt is unknown. Our preliminary data in core-expressing HPCs indicated: 1) HCV core enhanced the migration of HPCs; 2) the differentiation of HPCs was obviously arrested; 3) the expression of biomarkers for HCSCs were significantly increased; 4) nuclear translocation of β-catenin was obviously observed; 5) β-catenin was co-localized with HCV core at nuclei. Based on our preliminary data, the present study is aimed to investigate whether HCV core could bond with β-catenin and hence directly activate Wnt/β-catenin signaling pathway in core-expressing HPCs. Moreover, by ChIP, RNAi and some other techniques, we will further explore the mechanism of HCV core/β-catenin induced upregulation of EpCAM and microRNA-181 expression and finally illustrate the molecular mechanism involved in the transdifferentiation of HPCs into HCSCs. Our study will provide new insights into the pathogenesis of HCV-associated HCC and may be helpful in developing an effective measure of prevention and treatment against HCC.

肝前体细胞向肝癌干细胞分化是原发性肝癌(HCC)发生的重要致病因素之一，但具体机制不明。Wnt信号不仅在HCC中处于活化状态，而且参与维持肝癌干细胞特性，HCV能否通过活化Wnt信号诱导肝前体细胞向肝癌干细胞分化，目前无相关报道。我们前期实验发现HCV核心蛋白（HCV core）促进肝前体细胞迁移；介导细胞分化受阻；上调肝癌干细胞标志物表达；诱导β-catenin核转位，并且二者在胞核内存在共定位。本课题拟在此基础上，从感染HCV core的肝前体细胞入手，分析HCV core能否与β-catenin相互作用，直接活化Wnt信号。并通过ChIP、基因沉默等技术探讨HCV core/β-catenin调控其下游靶基因，诱导肝前体细胞向肝癌干细胞分化的分子机制。项目的实施为阐明HCV相关HCC的发病机制提供新的理论依据，为临床优化肝癌的预防与治疗策略提供新的手段。

HCV慢性感染及肝前体细胞向肝癌干细胞分化是原发性肝癌发生的重要致病因素。且Wnt/β-catenin通路活化参与维持肝癌干细胞特性，但HCV能否通过活化Wnt通路诱导肝前体细胞向肝癌干细胞分化尚不清楚。我们采用细胞PAS染色、ICG摄取、细胞增殖、迁移、侵袭实验、RT-PCR、Western blot、免疫荧光及siRNA干扰技术验证HCV核心蛋白（HCV Core）诱导肝前体细胞向肝癌干细胞的分化，分析HCV Core诱导肝前体细胞向肝癌干细胞的分化的相关信号通路。进一步采用基因重组技术构建Epcam、microRNA181启动子区报告质粒，分析HCV Core对Epcam、microRNA181启动子活性的调控作用；通过ChIP、IP实验研究HCV Core调控Epcam、microRNA181基因的分子机制。通过体内实验进一步研究HCV Core对肝前体细胞成瘤能力的影响，并采用H&E染色和免疫组化方法检测裸鼠肝脏原位移植瘤模型中Core、β-catenin、Epcam以及CK19的表达。结果发现，在HCV Core诱导分化的肝前体细胞中，HCV Core抑制成熟肝细胞标志物Alb、CK18的表达，糖原储存能力、靛氰绿摄取能力显著下降（P<0.05），且上调肝癌干细胞标志物EpCAM、CD133、CD44等表达。另外，HCV Core增强β-catenin活性及表达水平，促使β-catenin向核内聚集，上调其下游靶基因EpCAM、CyclinD1、C-myc的表达，沉默β-catenin后，Wnt/β-catenin通路下游靶基因表达明显受到抑制，糖原储存能力部分恢复。深入分析发现：HCV Core可以增强β-catenin、microRNA181结合于Epcam启动子区，同时Core与β-catenin形成复合物，结合于EpCAM启动子区，促进EpCAM的表达。因此，在HP14.5细胞中证实：HCV Core能与β-catenin/ microRNA181直接结合，形成转录复合物，促进Epcam的转录，诱导肝前体细胞向肝癌干细胞分化。体内实验证实HCV Core促进肝前体细胞向肿瘤细胞分化，沉默β-catenin后，可以明显抑制Core促肿瘤形成，充分说明Core促肿瘤效应依赖β-catenin的参与。为阐明HCV感染相关肝癌的发生发展提供新的理论依据。