| dc.description.abstract | The liver is the largest organ in the body; it regulates many physiological functions such as lipid and carbohydrate synthesis, transformation, detoxification, excretion and storage functions. Previous studies have demonstrated liver development mechanisms are highly conserved in vertebrate. Identification of genes involved in the formation of the liver and liver diseases regulatory pathways provide diagnostic and therapeutic foundation. Hepatocellular carcinoma (HCC) is a chronic progressive process, from chronic inflammation, to steatosis, fibrosis and cirrhosis eventually developed into liver cancer. Hepatitis B virus (HBV) infection is a major risk factor in liver cancer. Hepatitis B virus X antigen (HBx protein) can enhance colony formation and cell transformation in vitro cell lines, and can induce liver cancer in mice. Human cancers including liver cancer often associated with p53 mutations, and aflatoxin (AFB1) is a very common carcinogenic factor. Using albumin promoter driven expression of HBx protein in a mouse model, I have shown up-regulation of four common regulators including Src, Edn1, Bmp4, and Bmp7 are associated with HCC formation. Because of the many advantages of zebrafish, the zebrafish has become an emerging model for human diseases; zebrafish animal models provide an in-vivo model for test the oncogenicity of candidate genes, and a high throughput drug screening platform.
In this dissertation, I focus on three parts: first, using transgenic zebrafish to investigate the synergistic effect between HBx protein and aflatoxin on liver disease and cancer formation. Second, by establishing HBx transgenic fish in p53 mutant background, and src transgenic fish, explore the role of HBx, src and p53 mutant in the formation of HCC and their synergy. Third, through edn1 transgenic fish, discover the role of edn1 in hepatocarcinogenesis.
I found that HBx and AFB1 synergistically promoted steatosis as indicated by histopathological examinations and the increased expression of lipogenic factors, enzymes, and genes related to lipid metabolism. Moreover, treatment of AFB1 in HBx transgenic fish accelerated the development of liver hyperplasia and enhanced the expression of cell cycle related genes. PCNA was co-localized with active caspase 3 protein expression in HBx zebrafish liver samples and human HBV positive HCC samples by double fluorescence immunostaining. Finally, I found that in human patients with liver disease, the significant glycogen accumulation in the inflammation, cirrhosis stage, and all cases of hepatocellular and cholangiocellular carcinoma showed a moderate cytoplasmic accumulation of glycogen. Our data demonstrated a synergistic effect of AFB1 and HBx on the regulation of lipid metabolism related genes and cell cycle/division-related genes which might contribute to enhanced steatosis and hyperplasia at 5.75 months.
Liver-specific expression of HBx in wild-type zebrafish caused steatosis, fibrosis and glycogen accumulation. However, HBx induced tumorigenesis was observed only in p53 mutant fish in association with up-regulation and activation of src tyrosine kinase pathway. Furthermore, overexpression of src in the p53 mutant zebrafish also caused hyperplasia, HCC, and sarcomatoid HCC, which is accompanied with increased levels of the signaling proteins p-erk, p-akt, myc, jnk1 and vegf. Increased expression levels of lipogenic factors and the genes involved in lipid metabolism and glycogen storage were detected at the earlier stage of hepatocarcinogenesis of the HBx and src transgenic zebrafish. Up-regulation of the molecules in cell cycle, tumor progression and other molecular hallmarks of human liver cancer were found at later stages of both the HBx and src transgenic in the p53 mutant zebrafish. Together, our study demonstrates that HBx and src overexpression in the p53 mutant both induced hepatocarcinogenesis in zebrafish, which both mimic human HCC formation and provide potential in vivo platforms for drug screening to find therapy for human liver cancer.
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 migration in xenotransplantation assays, and was accompanied by the up-regulation of migration related genes. Lipid metabolism-related genes were up-regulated at five months, may be related to the formation of fatty liver. The cell cycle, proliferation, tumor metastasis-related genes were up-regulated at 11-month, correlated to the formation of hyperplasia and HCC. Using xenotransplantation method, I found stable overexpression EDN1 in 293T cells can enhance cell migration. Using tissue array of ten different malignant human tumors, I found EDN1 up-regulated in breast cancer, brain cancer, liver cancer, prostate cancer and kidney cancer, and down-regulated in stomach cancer. Additionally, miR-1 was found to inhibit the expression of EDN1, and I observed an inverse correlation between EDN1 and miR-1 in HCC patients. In conclusion, our data suggest that EDN1 plays an important role in HCC progression and is regulated by miR-1.
Our long term goal is to understand the molecular mechanisms of liver cancer, and the development the therapeutic means for liver cancer treatment. The use of zebrafish as a model of chronic liver disease and HCC can be used as drug screening platform. With chemical libraries and zebrafish animal models, I hope to find out prevention and treatment method for liver disease and liver cancer induced by HBx protein and other carcinogenic factors. Our findings imply that, through the use of zebrafish cancer model, I can not only engage molecular mechanisms, I can also use it to screen small molecule drugs to treat cancer. The established transgenic zebrafish liver disease and cancer models should provide a cooperation platform for the translational research of liver cancer study in Taiwan. | en_US |