探討gankyrin誘導肝癌的機制並透過斑馬魚基因轉殖技術提高omega-3脂肪酸以調控腫瘤生長

Mechanism of gankyrin-induced liver cancers and control of tumor growth in elevated omega-3 fatty acids by transgenic technology of zebrafish

黃信傑(Shin-Jie Huang)

指導教授 ： 吳金洌

共同指導教授 ： 陳志毅(Jyh-Yih Chen)

國立臺灣大學/生命科學院/漁業科學研究所/博士(2017年)

https://doi.org/10.6342/NTU201703637

摘要

肝癌是人類重要的臨床科學問題，也是全球第二大癌症死亡原因。常見的兩種原發性肝癌分別是肝細胞癌（hepatocellular carcinoma，HCC）和肝內膽管癌（intrahepatic cholangiocarcinoma，ICC）。新的研究顯示，在肝癌發展的過程中，腫瘤微環境內的慢性肝臟炎症是個重要的過程並會促進腫瘤惡化。因此，治療慢性肝炎即為預防肝癌的一種可能途徑。在人類健康和發育中，包括ω-6（n-6）和ω-3（n-3）脂肪酸在內的多元不飽和脂肪酸（PUFAs）有著許多關鍵的生物功能，如參與脂質循環及發炎等功能。幾項最近的研究也顯示，食用特定的多元不飽脂肪酸與降低HCC 和ICC 的發生風險是有關的。然而，目前用來研究肝癌和PUFAs 之間調控機制與療效的模式動物仍相當有限。因此，建立這種肝臟疾病動物模式，對於開發新療法所需的基礎和轉譯研究顯得相當重要。我們的目標是探討調節脂肪酸組成是否可以改善gankyrin 所誘發的肝癌生長。由於n-3 PUFAs 的攝取量可能會隨膳食補充差異而有顯著變化，因此我們建立基轉斑馬魚來消除由飼料所引起的飲食干擾因素並進一步增加n-3 多元不飽和脂肪酸含量。
在這裡，我們建立了幾種模式動物，包含一種致癌蛋白gankyrin，肝癌基轉魚和四種單一不飽和脂肪酸合成基因基轉魚，以了解脂肪酸組成變化與肝臟疾病間的相互調控關係。首先，我們試著建立的一種新的斑馬魚肝癌模型，通過致癌蛋白gankyrin 的過量表達於肝細胞來誘導自發的HCC 和ICC 產生，而無須癌細胞接種或藥物處理。在7至12 個月齡時，我們發現gankyrin 基轉魚會發生持續性肝細胞損傷，伴隨著脂肪肝，膽汁鬱積，膽管炎，肝纖維化，肝細胞癌（HCC）和肝內膽管癌（ICC）發生。在7 個月時，經由組織學分析顯示轉基因魚中約有63％（n = 40）的肝細胞癌發生；在12 個月時，於仍存活的魚中有36％（n = 30）的比例產生似ICC 的腫瘤。ICC 是人類第二常見的原發性肝癌，也是首次在這腫瘤模式動物中發生。我們進一步研究了補體系統的中心分子補體 C3 的作用，發現於2,3,4 和6 個月時的基轉魚肝臟中C3a 和C3b mRNA 的表達水平持續下降，蛋白質分析也顯示表現量的降低。這些研究結果表明，gankyrin 可以在持續性肝損傷的背景下促進肝細胞的惡性轉化，這種轉變可能與補償性增殖和持續發炎的微環境有關。我們觀察到的補體C3 減少可用來允許已經癌轉化的細胞逃避其所誘發的免疫反應。在這裡，我們建立了一個優秀的斑馬魚肝癌模型，將有助於研究腫瘤發生的分子機制。接著，我們藉由斑馬魚模式動物來探討三種去飽和酶(FADS)和長鏈不飽和脂肪酸延長酶(ELOVL)在不飽和脂肪酸合成時的效能。我們建立了四個轉基因品系，即Fadsd5 和Fadsd4，Fadsd6 和Elvol5a 單基因基轉斑馬魚，並互相雜交產生雙基因基轉斑馬魚。最終產生三種雙轉基因斑馬魚Fadsd5 /Fadsd6、Fadsd6 / Elvol5a 和Fadsd6 / Fadsd4。脂肪酸分析結果顯示，不僅是單一PUFA合成基因基轉魚或雙PUFA 合成基因基轉魚均增強了n-3 PUFAs 的合成能力。在Fadsd6/Elvol5a 雙轉基因斑馬魚中delta-6 去飽和酶和延長酶的表達將20：5n-3 和22：6n-3含量分別增加至1.27 和2.64 倍。類似地，在Fadsd5 / Fadsd6 雙轉基因斑馬魚中共表達的delta-5 和delta-6 去飽和酶分別將20：5n-3 和22：6n-3 含量分別提高到1.3 和2.87 倍。在Fadsd5 / Fadsd6 雙轉基因斑馬魚中共同表達delta-6 和delta-4 去飽和酶分別使20：5n-3 和22：6n-3 含量分別增加1.5 倍和2.29 倍。
最後，我們研究了Gankyrin 和Elvol5a 雙轉基因魚所引起的脂肪酸組成變化與肝臟疾病之間的關聯。結果表明，雙轉基因魚能顯著降低肝細胞的凋亡和過度增生現象，於12個月時腫瘤發生率從60％降至32％。與對照組相比，gankyrin 基轉魚中的二十碳五烯酸（EPA）和花生四烯酸（ARA）的比值由1.4 降低至0.7 倍，而Gan-Tet-Off/ Elvol5a雙轉魚可以進一步降低百分之五十的ARA 含量。總結，這些初步結果提供了一個相當好的關聯，顯示了控制EPA 和ARA 這兩種脂肪酸間比例，與總體生存率及癌症發病率有關。這些結果可作為一種新穎的策略，在飲食中增加n-3 不飽和脂肪酸的攝取量，可用來預防及調控腫瘤生長。總結而言，由於n-3 脂肪酸在癌症中的作用已經在我們的模型中得到證實，因此可以推斷，進一步透過轉錄組和脂質組學，可以深入了解脂質代謝機制與n-3 LC- PUFA 在肝病的潛在治療效用。

關鍵字

斑馬魚 ； 肝內膽管癌 ； 補體C3 ； 多元不飽和脂肪酸

並列摘要

Liver cancer is a big clinical problem in humans and was estimated to be the second leading cause of cancer mortality worldwide. The two most common primary liver cancers are hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). It is now recognized that chronic liver inflammation in the tumor microenvironment is a hallmark of liver cancer and that promotes tumor progression. Thus, treatment of chronic inflammatory liver diseases is a possible approach to prevent liver cancer. Polyunsaturated fatty acids (PUFAs), including omega-6(n-6) and omega-3(n-3) fatty acids, are playing many crucial biological functions including the circulating lipids and inflammation in human health and development. Several recent studies have reported a correlation between consumption of specific PUFAs diets and reduce the risk of developing HCC and ICC. However, limited data are currently available to address the efficacy and mechanisms between liver cancers and PUFAs in animal model. As such, establishing animal models of this disease is important for both basic and translational studies that move toward developing new therapies. Our objective is to determine whether modulating the fatty acid composition could ameliorate liver cancer formation. Because of dietary intake of n-3 PUFAs may vary significantly as background from diets supplementation. Using transgenic zebrafish model can eliminate the typical confounding factors of diet and future increase omega-3 content. Here, we establish several in vivo models, including one oncoprotein gankyrin, the liver cancer transgenic model and four single PUFA synthesis gene transgenic fish, to understand the relationship of fatty acid composition change in liver diseases. First of all, we try to establish a novel zebrafish model to induce spontaneous HCC and ICC by liver-specific gankyrin overexpression without cancer cell inoculation and drug treatment. At 7 to 12 months of age, we found gankyrin transgenic fish spontaneously incurred persistent hepatocyte damage, steatosis, cholestasis, cholangitis, fibrosis and hepatic tumors. At 7 months of age, histological analysis revealed approximately 63% (n = 40) occurrence of HCC in transgenic fish. And at nearly 1 year of age, tumors that were similar to ICC developed in 36% (n = 30) of surviving fish. ICC is the second most frequent primary liver cancer in human patients and the first to develop in this tumor model. We further investigated the role of complement C3, a central molecule of the complement system, and found the expression levels of both in mRNA levels of C3a and C3b were consistently decreased in transgenic fish liver at 2, 3, 4 and 6 months, and the protein expression was decreased when measured by either Western blot or immunohistochemistry during tumorigenesis.
Together, these findings suggest that gankyrin can promote malignant transformation of liver cells in the context of persistent liver injury. This transformation may be related to compensatory proliferation and the inflammatory microenvironment. The observed decrease in complement C3 may allow transforming cells to escape coordinated induction of the immune response. Herein, we demonstrate an excellent zebrafish model for liver cancers that will be useful for studying the molecular mechanisms of tumorgenesis. Then, we exploit the zebrafish model to identify the effectiveness between three FADS and ELOVL gene’s function in PUFAs synthesis. We establish four single gene transgenic lines, namely the Fadsd5 and Fadsd4, Fadsd6 and Elvol5a transgenic zebrafish, and use to generate double genes transgenic zebrafish. We finally generate three double genes transgenic zebrafish lines, Fadsd5/Fadsd6, Fadsd6/Elvol5a and Fadsd6/Fadsd4. The fatty acid analysis revealed that not only single PUFA gene transgenic zebrafish but also double PUFA genes transgenic zebrafish exhibited enhanced ability to synthesize n-3 PUFAs. Expression of delta-6 desaturase and elongase in Fadsd6/Elvol5a double transgenic zebrafish increased the 20:5n-3 and 22:6n-3 contents to 1.27- and 2.64-fold, respectively. Similarly, co-expression delta-5 and delta-6 desaturase in Fadsd5/Fadsd6 double transgenic zebrafish increased the 20:5n-3 and 22:6n-3 content to 1.3- and 2.87-fold, respectively. And co-expression delta-6 and delta-4 desaturase in Fadsd6/Fadsd4 double transgenic zebrafish increased the 20:5n-3 and 22:6n-3 content to
1.5- and 2.29-fold, respectively.
Finally, we investigated the relationship between the fatty acids composition and liver disease caused by co-expression of Gankyrin and Elvol5a in double transgenic zebrafish. Results showed that double transgenic fish can significantly reduce apoptosis and proliferation activity in hepatocyte and had a significantly reduced tumorigenesis from 60% to 32% in 12 months old. Moreover, the ratio of e eicosapentaenoic acid (EPA) and arachidonic acid (ARA) compared significantly lower in gankyrin transgenic fish when with control fish from 1.4 to 0.7 fold, but ARA was significantly decreased 50 percent in the livers of Gan-Tet-Off / Elvol5a transgenic zebrafish compare to Gan-Tet-Off zebrafish. Collectively, these data provide fascinating preliminary observation that the balance of EPA and ARA content was related to overall survival and cancer incidence. These results give as a novel strategy that increased intake of n-3 fatty acids in our diet could help both in the prevention as well as management of tumor growth. In conclusion, since the role of n-3 FAs in cancer has been demonstrated in our model, it can be inferred that with further incorporate of transcriptome and lipidomics, knowing the underlying molecular during lipid metabolism machinery and the potential of n-3 LC-PUFAs to explore their therapeutic utility.

並列關鍵字

zebrafish ； intrahepatic cholangiocarcinoma (ICC) ； Complement C3 ； PUFA

參考文獻

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