Overexpression of endothelin 1 triggers hepatocarcinogenesis in zebrafish and promotes cell proliferation and migration through the AKT pathway

Jeng-Wei Lu; Chung-Yi Liao; Wan-Yu Yang; Yueh-Min Lin; Shiow-Lian Catherine Jin; Horng-Dar Wang; Chiou-Hwa Yuh

doi:10.1371/journal.pone.0085318

Overexpression of endothelin 1 triggers hepatocarcinogenesis in zebrafish and promotes cell proliferation and migration through the AKT pathway

PLoS One. 2014 Jan 8;9(1):e85318. doi: 10.1371/journal.pone.0085318. eCollection 2014.

Authors

Jeng-Wei Lu¹, Chung-Yi Liao², Wan-Yu Yang³, Yueh-Min Lin⁴, Shiow-Lian Catherine Jin⁵, Horng-Dar Wang⁶, Chiou-Hwa Yuh⁷

Affiliations

¹ Institute of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan, Miaoli, Taiwan ; Department of Life Sciences, National Central University, Jhongli City, Taoyuan, Taiwan.
² Institute of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan, Miaoli, Taiwan ; Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan.
³ Institute of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan, Miaoli, Taiwan.
⁴ Department of Pathology, Changhua Christian Hospital, Changhua City, Changhua County, Taiwan ; Department of Medical Technology, Jen-Teh Junior College of Medicine, Nursing and Management, Miaoli, Taiwan.
⁵ Department of Life Sciences, National Central University, Jhongli City, Taoyuan, Taiwan.
⁶ Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan.
⁷ Institute of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan, Miaoli, Taiwan ; Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan ; Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan.

Abstract

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Carcinoma, Hepatocellular / etiology
Carcinoma, Hepatocellular / genetics*
Carcinoma, Hepatocellular / metabolism
Carcinoma, Hepatocellular / pathology
Cell Cycle
Cell Movement
Cell Proliferation
Endothelin-1 / antagonists & inhibitors
Endothelin-1 / genetics*
Endothelin-1 / metabolism
Fatty Liver / complications
Fatty Liver / genetics
Fatty Liver / metabolism
Fatty Liver / pathology
Gene Expression
HEK293 Cells
Humans
Liver Cirrhosis / complications
Liver Cirrhosis / genetics
Liver Cirrhosis / metabolism
Liver Cirrhosis / pathology
Liver Neoplasms / etiology
Liver Neoplasms / genetics*
Liver Neoplasms / metabolism
Liver Neoplasms / pathology
MicroRNAs / genetics*
MicroRNAs / metabolism
Phosphatidylinositol 3-Kinases / genetics
Phosphatidylinositol 3-Kinases / metabolism
Phosphoinositide-3 Kinase Inhibitors
Protein Kinase Inhibitors / pharmacology
Proto-Oncogene Proteins c-akt / antagonists & inhibitors
Proto-Oncogene Proteins c-akt / genetics*
Proto-Oncogene Proteins c-akt / metabolism
Unfolded Protein Response / genetics
Zebrafish
Zebrafish Proteins / antagonists & inhibitors
Zebrafish Proteins / genetics*
Zebrafish Proteins / metabolism

Substances

Endothelin-1
MicroRNAs
Mirn1 microRNA, zebrafish
Phosphoinositide-3 Kinase Inhibitors
Protein Kinase Inhibitors
Zebrafish Proteins
edn1 protein, zebrafish
Proto-Oncogene Proteins c-akt

Grants and funding

This research was supported by grants from the National Research Program for Genomic Medicine (NRPGM) (99-3112-B-400-010) and the National Science Council (NSC) (101-2321-B-400-017) to CHY. Additional funding from the National Health Research Institute provided to CHY is gratefully acknowledged. The fellowship and funding to Jeng-Wei Lu and Dr. Chiou-Hwa Yuh, respectively, from the Liver Disease Prevention & Treatment Research Foundation (Taiwan) is also highly acknowledged. The authors would like to thank the Taiwan Zebrafish Core Facility at NTHU-NHRI for providing the fish lines and resources; TZeTH is supported by a grant from the NSC (101-2321-B-400-014). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.