肝細胞癌(HCC)是最常見的肝癌類型，且在全世界統計下癌症死亡率高居第四名。造成肝癌的風險因子有很多，主要可以分成三類，包括：病毒(慢性B型或C型肝炎)、毒素(過量酒精或黃麴毒素攝取)、代謝(肥胖、糖尿病及非酒精性脂肪肝)。肝癌患者通常被診斷出來時已為晚期肝癌，在治療策略上的選擇上包括：手術切除、肝臟移植、局部消融及放射治療效果都不盡理想。肝癌末期患者使用標靶藥物，治療效果很有限，平均可延長壽命三到四個月。因此了解肝癌中遺傳驅動因子致癌機制，可以幫助進一步發展標靶治療。有高達百分之六十的肝癌病患發現了端粒酶 (Telomerase, TERT)基因啟動子區突變，會導致端粒酶的過量表現並促進癌細胞的生長，但是，尚無斑馬魚端粒酶過度表達肝癌模型。這項研究的目的是建立端粒酶過度表達轉基因斑馬魚，研究在肝細胞中過度表達斑馬魚端粒酶是否可以促進肝癌的形成及其機制，我們希望可以使用該模型篩選抗肝癌治療藥物。
在這項研究當中，我們建立了在肝臟過量表達端粒酶逆轉錄酶(tert)的轉基因斑馬魚，並通過分子及組織病理學的方式研究了肝癌的形成。使用即時核酸定量的方式分析，我們觀察在受精後15天，端粒酶逆轉錄酶轉基因魚中細胞增殖基因(ccne1/cdk1/cdk2)相較於野生種斑馬魚都有顯著性的上升。此外，與肝癌形成有密切相關的β-catenin 下游基因(ccnd1/myca/mycb)也顯著的上升。通過蘇木精-伊紅染色分析，轉基因斑馬魚在細胞分裂及三核比例都有顯著增加，但這些現象在30天時都下降了。為了了解30 天時癌症形成的減少，我們觀察在15及30天中端粒酶逆轉錄酶過量表達也誘導p53表現上升。我們假設p53表現上升減少了過量表達端粒酶逆轉錄酶引發的肝癌形成，這現象也與臨床中觀察到肝癌同時帶有TERT及p53突變相似。為了驗證這一假設，我們將轉基因魚跟tp53突變的魚配種，進一步進行驗證。本次研究當中，我們轉基因斑馬魚模型提供了更加接近臨床肝癌的遺傳背景，未來據此研究更多遺傳變異所導致肝癌形成的機制。

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and is the fifth leading cause of cancer mortality worldwide. Major risk factors for HCC include viral infection (chronic Hepatitis B and Hepatitis C), substance (alcohol consumption and aflatoxin B1 exposure), and metabolic disorder (diabetes, obesity, and non-alcoholic fatty liver disease). Patients with liver cancer are usually diagnosed as advanced liver cancer. The choice of treatment strategy includes: surgical resection, liver transplantation, local ablation and radiotherapy are not satisfactory. Target therapy for the advanced HCC patients have limited therapeutic effects and only can extend their lifespan by three to four months on average. Therefore, understanding the carcinogenic mechanism of genetic drivers in HCC can help further develop targeted therapy. The top genetic alteration for HCC is telomerase reverse transcriptase (TERT) promoter mutations affecting around 60% of all HCC patients, which leads to overexpression of telomerase and promotes cancer development. However, there is no zebrafish telomere overexpression liver cancer model. The purpose of this study is to establish telomerase overexpression transgenic zebrafish, and to study whether overexpression of zebrafish telomerase in liver cells can promote the formation of liver cancer and its mechanism. We hope that we can use this model to screen anti-liver cancer therapeutic drugs.
In this study, we generated liver-specific tert overexpression transgenic zebrafish and investigated the cancer formation by molecular and histopathological analysis. Using real-time quantitative PCR, we found the cell proliferation markers (ccne1, cdk1, cdk2) were upregulated in tert transgenic fish as early as 15 day-post-fertilization (dpf). Moreover, β-catenin was associated with HCC, the downstream genes (ccnd1, myca, mycb) were also significantly upregulated. Furthermore, the mitotic figures and trinucleated cells were significantly increased in tert transgenic fish using histopathological analysis by H&E staining. However, these phenomena declined in 30 dpf. To further understand the reduction of cancer at 30 dpf, we found that the tert also induces the up-regulation of p53 mRNA. We hypothesized increment of tp53 diminished the tert overexpression induced HCC, this phenomenon coherent the fact that TERT and p53 mutations are concurrent in HCC. To test this hypothesis, we cross the tert transgenic fish with p53 mutant, and further validate the cancer formation. Our transgenic fish model supplies a clinically relevant background upon which to study how other driver mutations function in HCC.

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