Liu Zhikui, Tu Kangsheng, Wang Yufeng, Yao Bowen, Li Qing, Wang Liang, Dou Changwei, Liu Qingguang, Zheng Xin
Cell Physiol Biochem. 2017;44(5):1856-1868. doi: 10.1159/000485821. Epub 2017 Dec 11.
BACKGROUND/AIMS: Hypoxic microenvironment, a common feature of hepatocellular carcinoma (HCC), can induce HIF-1α expression and promote the epithelial-mesenchymal transition (EMT) and invasion of cancer cells. However, the underlying molecular mechanisms have not fully elucidated.
HCC cells were cultured under controlled hypoxia conditions or normoxic conditions. Transwell assays were used to examine the migration and invasion capacity. HIF-1α siRNA, cyclopamine (a SMO antagonist) and GLI1 siRNA were used to inhibit HIF-1α transcription or Hh signaling activation.
In present study, we first observed a strongly positive correlation between HIF-1α and GLI1 expression in HCC tissues. Then, we showed that hypoxia significantly promoted EMT process and invasion of HCC cells, associated with activating the non-canonical Hh pathway without affecting SHH and PTCH1 expression. HIF-1α knockdown mitigated hypoxia-induced SMO and GLI1 expression, EMT invasion of HCC cells. Moreover, the SMO inhibitor or GLI1 siRNA also reversed the hypoxia-driven EMT and invasion of HCC cells under hypoxia condition. Here, we show that non-canonical Hh signaling is required as an important role to switch on hypoxia-induced EMT and invasion in HCC cells. In addition, we found that hypoxia increased ROS production and that ROS inhibitors (NAC) blocked GLI1-dependent EMT process and invasion under hypoxic conditions. To determine a major route of ROS production, we tested whether nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) is involved in hypoxia-induced ROS production. NOX4 expression was found to be increased at both mRNA and protein levels in hypoxic HCC cells. Furthermore, siRNA-mediated knockdown of NOX4 expression abolished hypoxia induced ROS generation and GLI1-dependent activation and invasion of HCC cells.
Our findings indicate that hypoxia triggers ROS-mediated GLI1-dependent EMT progress and invasion of HCC cells through induction of NOX4 expression. Thus, hypoxia-driven ROS mediated non-canonical Hh signaling may play an important role in the initiation of EMT and provides a potential marker for cancer prevention and treatment.
背景/目的：缺氧微环境是肝细胞癌（HCC）的一个常见特征，可诱导缺氧诱导因子-1α（HIF-1α）表达，并促进癌细胞的上皮-间质转化（EMT）和侵袭。然而，其潜在的分子机制尚未完全阐明。
将肝癌细胞在可控的缺氧条件或常氧条件下培养。采用Transwell实验检测迁移和侵袭能力。使用HIF-1α小干扰RNA（siRNA）、环杷明（一种 smoothened（SMO）拮抗剂）和GLI1 siRNA抑制HIF-1α转录或Hh信号激活。
在本研究中，我们首先观察到肝癌组织中HIF-1α与GLI1表达之间存在强正相关。然后，我们发现缺氧显著促进肝癌细胞的EMT进程和侵袭，这与激活非经典Hh信号通路相关，而不影响音猬因子（SHH）和patched 1（PTCH1）表达。敲低HIF-1α可减轻缺氧诱导的SMO和GLI1表达以及肝癌细胞的EMT侵袭。此外，SMO抑制剂或GLI1 siRNA也可逆转缺氧条件下缺氧驱动的肝癌细胞EMT和侵袭。在此，我们表明非经典Hh信号在开启缺氧诱导的肝癌细胞EMT和侵袭中起重要作用。此外，我们发现缺氧会增加活性氧（ROS）的产生，并且ROS抑制剂（N-乙酰半胱氨酸（NAC））可在缺氧条件下阻断GLI1依赖的EMT进程和侵袭。为了确定ROS产生的主要途径，我们检测了烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶4（NOX4）是否参与缺氧诱导的ROS产生。发现在缺氧的肝癌细胞中，NOX4的mRNA和蛋白水平均升高。此外，siRNA介导的NOX4表达敲低消除了缺氧诱导的ROS生成以及肝癌细胞的GLI1依赖的激活和侵袭。
我们的研究结果表明，缺氧通过诱导NOX4表达触发ROS介导的GLI1依赖的肝癌细胞EMT进程和侵袭。因此，缺氧驱动的ROS介导的非经典Hh信号可能在EMT启动中起重要作用，并为癌症预防和治疗提供了一个潜在标志物。
