Netrin-1 Induces Epithelial-Mesenchymal Transition and Promotes Hepatocellular Carcinoma Invasiveness
Netrin-1 Induces Epithelial-Mesenchymal Transition and Promotes Hepatocellular Carcinoma Invasiveness
复制标题
Netrin-1 诱导上皮间质转化并促进肝细胞癌侵袭
DOI：
10.1007/s10620-013-3016-z
复制
发表时间：
2014-06-01
影响因子：
3.1
通讯作者：
Fu, Yu
中科院分区：
文献类型：
--
作者：
Yan, Wei;Han, Ping;Fu, Yu
缺氧常见于实体瘤中，并与肿瘤进展和不良临床结果相关。我们阐明了低氧应激下释放的netrin-1诱导上皮间质转化（EMT）促进肝细胞癌（HCC）细胞侵袭的机制。通过免疫组化或蛋白质印迹法检测HCC结直肠癌（DCC）中netrin-1及其依赖性受体UNC5H和缺失的表达。 HepG2细胞在21％O-2（常氧）或1％O-2（缺氧）中培养24小时。通过ELISA检测缺氧细胞中netrin-1的释放。通过蛋白质印迹检查E-钙粘蛋白和波形蛋白的表达。使用倒置显微镜或共聚焦显微镜来显示细胞形态或细胞骨架重排。 Transwell小室分析缺氧诱导的细胞侵袭。通过实时PCR评估细胞因子IL-8和IL-10 mRNA水平。HCC组织和细胞系中netrin-1的表达增加。大多数 HCC 细胞系中依赖性受体 UNC5H 和 DCC 均减少。缺氧诱导 netrin-1 以时间依赖性方式释放。研究发现缺氧 HCC 细胞中 EMT 诱导发生的过程依赖于 netrin-1 的细胞外释放。此外，netrin-1 的过度表达导致常氧肿瘤细胞诱导 EMT。在 netrin-1 过表达的细胞中发现细胞骨架重排发生并且细胞侵袭增加。最后，重组人netrin-1处理后IL-8和IL-10的mRNA也增加。这些结果表明，在缺氧的HCC细胞中，netrin-1激活下游信号通路，诱导EMT激活，随后产生多种炎症介质，进而促进癌症侵袭。
Hypoxia is often found in solid tumors and is associated with tumor progression and poor clinical outcomes. We elucidated the mechanism by which netrin-1 released under hypoxic stress can induce epithelial-mesenchymal transition (EMT) to promote invasion in hepatocellular carcinoma (HCC) cells.The expression of netrin-1 and the dependent receptors UNC5H and deleted in colorectal cancer (DCC) in HCC was examined by immunohistochemistry or western blot. The HepG2 cells were cultured in 21 % O-2 (normoxia) or 1 % O-2 (hypoxia) for 24 h. The release of netrin-1 from hypoxic cells was detected by ELISA. Expression of E-cadherin and vimentin were examined by western blot. Inverted microscopy or confocal microscopy was used to show the cell morphology or cytoskeletal rearrangements. Cell invasion induced by hypoxia was analyzed by Transwell chamber. Cytokine IL-8 and IL-10 mRNA levels were assessed by real-time PCR.The expression of netrin-1 was increased in HCC tissue and cell lines. The dependent receptors UNC5H and DCC were decreased in most HCC cell lines. Hypoxia induced netrin-1 release in a time-dependent manner. EMT induction was found to occur in hypoxic HCC cells in a process that was dependent on the extracellular release of netrin-1. Moreover, overexpression of netrin-1 resulted in EMT induction in normoxic tumor cells. Cytoskeletal rearrangements were found to occur and cell invasion was increased in cells with netrin-1 overexpression. Lastly, mRNA of IL-8 and IL-10 were also increased after recombinant human netrin-1 treatment.These results suggest that in hypoxic HCC cells, netrin-1 activates downstream signaling pathways to induce EMT activation with subsequent production of multiple inflammatory mediators which in turn promotes cancer invasion.