Liver Research Unit, Confocal Microscopy Facility of "Bambino Gesù" Children's Hospital, IRCCS, Rome, Italy.
PLoS One. 2012;7(8):e44147. doi: 10.1371/journal.pone.0044147. Epub 2012 Aug 28.
Hepatitis C Virus (HCV) infection is one of the most common etiological factors involved in fibrosis development and its progression to hepatocellular carcinoma (HCC). The pivotal role of hepatic stellate cells (HCSs) and extracellular matrix (ECM) in fibrogenesis is now certainly accepted, while the network of molecular interactions connecting HCV is emerging as a master regulator of several biological processes including proliferation, inflammation, cytoskeleton and ECM remodeling. In this study, the effects of HCV proteins expression on liver cancer cells, both pro-invasive and pro-fibrogenic phenotypes were explored. As a model of HCV infection, we used permissive Huh7.5.1 hepatoma cells infected with JFH1-derived ccHCV. Conditioned medium from these cells was used to stimulate LX-2 cells, a line of HSCs. We found that the HCV infection of Huh7.5.1 cells decreased adhesion, increased migration and caused the delocalization of alpha-actinin from plasma membrane to cytoplasm and increased expression levels of paxillin. The treatment of LX-2 cells, with conditioned medium from HCV-infected Huh7.5.1 cells, caused an increase in cell proliferation, expression of alpha-smooth muscle actin, hyaluronic acid release and apoptosis rate measured as cleaved poly ADP-ribose polymerase (PARP). These effects were accompanied in Huh7.5.1 cells by an HCV-dependent increasing of FAK activation that physically interacts with phosphorylated paxillin and alpha-actinin, and a rising of tumor necrosis factor alpha production/release. Silencing of FAK by siRNA reverted all effects of HCV infection, both those directed on Huh7.5.1 cells, and those indirect effects on the LX-2 cells. Moreover and interestingly, FAK inhibition enhances apoptosis in HCV-conditioned LX-2 cells. In conclusion, our findings demonstrate that HCV, through FAK activation, may promote cytoskeletal reorganization and a pro-oncogenic phenotype in hepatocyte-like cells, and a fibrogenic phenotype in HSCs.
丙型肝炎病毒(HCV)感染是肝纤维化发展及其进展为肝细胞癌(HCC)的最常见病因之一。肝星状细胞(HCS)和细胞外基质(ECM)在纤维化中的关键作用现在已被普遍接受,而连接 HCV 的分子相互作用网络正成为包括增殖、炎症、细胞骨架和 ECM 重塑在内的几个生物学过程的主要调节因子。在这项研究中,我们探讨了 HCV 蛋白表达对肝癌细胞的影响,包括侵袭前和促纤维化表型。我们使用了允许 JFH1 衍生的 ccHCV 感染的 Huh7.5.1 肝癌细胞作为 HCV 感染的模型。从这些细胞中提取的条件培养基被用于刺激 LX-2 细胞,这是一种 HSC 系。我们发现,Huh7.5.1 细胞的 HCV 感染降低了细胞的黏附能力,增加了细胞的迁移能力,并导致 α-辅肌动蛋白从质膜向细胞质移位,同时增加了桩蛋白的表达水平。用 HCV 感染的 Huh7.5.1 细胞的条件培养基处理 LX-2 细胞,导致细胞增殖增加、α-平滑肌肌动蛋白表达增加、透明质酸释放增加以及作为切割多聚 ADP-核糖聚合酶(PARP)的细胞凋亡率增加。在 Huh7.5.1 细胞中,这些效应伴随着 HCV 依赖性的 FAK 激活增加,FAK 激活与磷酸化的桩蛋白和 α-辅肌动蛋白物理相互作用,并增加肿瘤坏死因子-α的产生/释放。siRNA 沉默 FAK 逆转了 HCV 感染对 Huh7.5.1 细胞的所有影响,以及对 LX-2 细胞的间接影响。此外,有趣的是,FAK 抑制增强了 HCV 条件培养基中 LX-2 细胞的凋亡。总之,我们的研究结果表明,HCV 通过 FAK 的激活,可能促进肝样细胞的细胞骨架重排和致癌表型,以及 HSCs 的纤维化表型。