Overexpression of Endothelin 1 Triggers Hepatocarcinogenesis in Zebrafish and Promotes Cell Proliferation and Migration through the AKT Pathway

Hepatocarcinogenesis commonly involves the gradual progression from hepatitis to fibrosis and cirrhosis, and ultimately to hepatocellular carcinoma (HCC). Endothelin 1 (Edn1) has been identified as a gene that is significantly up-regulated in HBx-induced HCC in mice. In this study, we further investigated the role of edn1 in hepatocarcinogenesis using a transgenic zebrafish model and a cell culture system. Liver-specific edn1 expression caused steatosis, fibrosis, glycogen accumulation, bile duct dilation, hyperplasia, and HCC in zebrafish. Overexpression of EDN1 in 293T cells enhanced cell proliferation and cell migration in in vitro and xenotransplantation assays and was accompanied with up-regulation of several cell cycle/proliferation- and migration-specific genes. Furthermore, expression of the unfolded protein response (UPR) pathway-related mediators, such as spliced XBP1, ATF6, IRE1, and PERK, was also up-regulated at both the RNA and protein levels. In the presence of an EDN1 inhibitor or an AKT inhibitor, these increases were diminished and the EDN1-induced migration ability also was disappeared, suggesting that the EDN1 effects act through activation of the AKT pathway to enhance the UPR and subsequently activate the expression of downstream genes. Additionally, p-AKT is enhanced in the edn1 transgenic fish compared to the GFP-mCherry control. The micro RNA miR-1 was found to inhibit the expression of EDN1. We also observed an inverse correlation between EDN1 and miR-1 expression in HCC patients. In conclusion, our data suggest that EDN1 plays an important role in HCC progression by activating the PI3K/AKT pathway and is regulated by miR-1.肝癌发生通常涉及从肝炎逐步进展为纤维化、肝硬化，最终发展为肝细胞癌（hepatocellular carcinoma, HCC）。内皮素1（Endothelin 1, Edn1）已被证实为小鼠HBx诱导的肝细胞癌中显著上调的基因。本研究中，我们借助转基因斑马鱼模型与细胞培养体系，进一步探究了Edn1在肝癌发生中的作用。斑马鱼体内的肝脏特异性Edn1表达可引发脂肪变性、纤维化、糖原蓄积、胆管扩张、增生以及肝细胞癌。在293T细胞中过表达EDN1，可在体外实验与异种移植实验中增强细胞增殖与迁移能力，同时伴随多个细胞周期/增殖相关及迁移特异性基因的上调。此外，未折叠蛋白反应（unfolded protein response, UPR）通路相关介质，如剪接型XBP1、ATF6、IRE1及PERK，其RNA与蛋白水平的表达均出现上调。当使用EDN1抑制剂或AKT抑制剂处理时，上述上调效应被抵消，EDN1诱导的迁移能力也随之消失，这提示EDN1的作用是通过激活AKT通路，增强未折叠蛋白反应，进而激活下游基因的表达。相较于GFP-mCherry对照转基因鱼，Edn1转基因鱼体内的磷酸化AKT（p-AKT）水平显著升高。研究发现微小RNA miR-1可抑制EDN1的表达；我们还在肝细胞癌患者样本中观察到EDN1与miR-1的表达呈负相关。综上，本研究数据表明，EDN1可通过激活PI3K/AKT通路在肝细胞癌进展中发挥重要作用，且其表达受miR-1的调控。