肝癌是台灣人癌症死因的第二位，B肝病毒感染是肝癌的主要危險因素之一。目前已知的B肝病毒基因型多達8種，台灣又以B及C型為主。臨床證據顯示，感染B肝病毒基因型C相較於基因型B，罹患肝癌和肝硬化的風險增加2.5到3倍，而年輕肝癌患者卻是感染B肝病毒基因型B較多。已知性激素與B肝病毒的活化有關，雄性激素受體活化B肝病毒增強子I，而雌性激素受體則抑制B型肝炎病毒增強子I的活性。此外，在與B肝病毒相關的肝癌中，端粒酶啟動子是B肝病毒插入的熱點，導致端粒酶過表現。本研究中，我們利用斑馬魚和細胞系統，研究了B型與C型肝病毒基因與性別差異和致癌性之間的關係。我們首先生成了轉基因斑馬魚，其中B肝病毒基因型B或基因型C的增強子驅動人類端粒酶啟動子或斑馬魚肝臟特異性啟動子表現，以過表現斑馬魚端粒酶。這些轉基因魚株旨在比較基因型B和基因型C增強子與肝癌發生的關聯性。我們還設計了mCherry和tert的2A結構，以直接觀察端粒酶的表現位置和強度。此外，我們進行了體外細胞培養實驗，研究了雄激素受體和雌激素受體對基因型B或基因型C的增強子與轉錄活性的差異。在此實驗中，我們並未重現先前觀察到的雄性激素強化增強子下游基因表現的現象，同時重現了雌性激素受體使下游基因表現減弱的現象。
我們實驗室此前已建立了多個代表不同亞型的轉基因斑馬魚肝癌模型。使用Ingenuity Pathway Analysis (IPA)，我們已確定了多種潛在化學物質，包括 U0126、LY294002、NAC、MTX 和薑黃素，它們可以逆轉這些轉基因魚的基因表現。為了進一步驗證這些藥物的功效，我們處理了代表非酒精性脂肪性肝炎誘導的肝癌模型fabp10a:CD36xabcg1(KO1)和端粒酶過表現的肝癌模型fabp10a:tert。我們還通過異種移植測試了它們的抗肝癌作用。初步結果表明，薑黃素可以顯著抑制異種移植的Hep3B細胞的轉移和增殖，並輕微抑制CD36xabcg1（KO1）而非tert轉基因魚的細胞增殖相關基因表現。我們還發現 U0126、LY294002、NAC 顯著降低了 tert 轉基因魚的細胞增殖標誌物，但MTX 和薑黃素不降低tert 轉基因魚的細胞增殖標誌物。這些結果表明，不同亞型的肝癌需要精準醫療。

Hepatocellular carcinoma (HCC) ranks the second of cancer mortalities among Taiwanese individuals. Infection with the hepatitis B virus (HBV) is one of the major risk factors for HCC. Of the eight known HBV genotypes, genotypes B and C are the dominant in Taiwan. Clinical evidence suggests that individuals infected with genotype C have a 2.5 to 3 times higher risk of developing HCC and cirrhosis compared to those infected with genotype B. Moreover, there is an higher prevalence of genotype B among young HCC patients. It is known that sex hormones play a role in the activation of the HBV, the androgen receptor binds and activates the enhancer I of the HBV, while the estrogen receptor inhibits the activity of the enhancer I of the HBV. Furthermore, in HBV-related HCC, the insertion of the virus into the telomerase promoter is a hotspot of HBV integration sites and accounts for telomerase overexpression.
In this study, we investigated the relationship between the B or C genotype of the HBV on gender differences and carcinogenicity using zebrafish and HCC cell lines. Initially, we generated transgenic zebrafish with overexpression of telomerase driven by either the enhancer of genotype B or genotype C of the hepatitis B virus, using either the human telomerase promoter or the zebrafish liver-specific promoter. These transgenic fish were designed to compare the association of the B and C genotype enhancers with the occurrence of HCC. We also designed a 2A structure with mCherry and tert to directly observe the expression sites and intensity of telomerase. Additionally, we conducted in vitro cell culture experiments to study the differences in transcriptional activity between the enhancers of genotype B and genotype C in the presence of androgen and estrogen receptors.
Previously, our laboratory has established several transgenic zebrafish HCC models representing different subtypes. Using Ingenuity Pathway Analysis (IPA), we have identified several potential chemical substances, including U0126, LY294002, NAC, MTX and curcumin, which can reverse the gene expression in these transgenic fish. To further validate the efficacy of these drugs, we treated fabp10a:CD36xabcg1(KO1) represents non-alcoholic fatty liver disease induced HCC, and fabp10a:tert HCC models with telomerase overexpression driven by the liver-specific promoter. We also tested their anti-HCC effects through xenograft experiments. Preliminary results indicate that curcumin significantly inhibits the migration and proliferation of Hep3B cells in xenografts and mildly suppresses the expression of cell proliferation-related genes in CD36xabcg1(KO1) transgenic fish, but not in tert transgenic fish. We also found U0126, LY294002, NAC significantly reduced the cell proliferation markers in tert transgenic fish but not MTX and curcumin. Those results indicated that precision medicine is required for different subtypes of HCC.

致謝 I
中文摘要 II
Abstract III
Glossary VII
Chapter 1 Introduction 1
1.1 Hepatocellular Carcinoma (HCC) Prevalence and Its Impact 1
1.1.1 Understanding HCC and HBV Infection: Global Perspectives and Vaccination Impact 1
1.1.2 Treatment Options and Therapeutic Advancements for HCC: Tailoring Strategies for Optimal Outcomes 2
1.2 Hepatitis B Virus 3
1.2.1 Insights into Hepatitis B Virus Genomes and Genotypes: Implications for Clinical Variability and Sex Disparity in HBV-Related Outcomes 3
1.2.2 HBV Genome Integration in Chronic Infection: Implications for HCC Development and Regulatory Element Modulation 4
1.3 Telomerase and TERT: Implications in Carcinogenesis and Telomere Maintenance in HCC 5
1.4 Zebrafish Models for Human Diseases 6
1.4.1 Zebrafish as a Powerful Model Organism for Unraveling Disease Mechanisms and Drug Screening 6
1.4.2 Zebrafish HCC Models: Unraveling Carcinogenesis and Replicating Human Liver Cancer Progression for Therapeutic Insights 7
1.4.3 Augmenting Zebrafish HCC Model with HBV Enhancer for Deeper Insights into Tumor Persistence 8
Specific Aims 10
Chapter 2 Materials and Methods 12
2.1 Cell culture and transfection 12
2.2 Luciferase assay 13
2.3 Dual-Glo® Luciferase Assay 13
2.4 HBV genome sequencing 14
2.5 Gateway cloning-BP reaction 14
2.6 Gateway cloning-LR reaction 15
2.7 Site-directed mutagenesis 16
2.8 Microinjection 17
2.9 Zebrafish husbandry 18
2.10 Zebrafish larvae feeding 18
2.11 Genotyping for Zebrafish 18
2.12 Zebrafish larvae drug treatment 19
2.13 Staining of zebrafish larva for cellular senescence detection 20
2.14 RNA extraction 21
2.15 Reverse Transcription Polymerase Chain Reaction 22
2.16 Real-time quantitative PCR (qPCR) 22
2.17 DNA sequence analysis 23
2.18 Statistical analysis 23
Chapter 3 Results 24
3.1 In-vitro Cell Culture to Investigate Sexual Differences and Higher Prevalence of Genotype B among Young Patients 24
3.2 Generation of Transgenic Zebrafish Expressing Tg(HBV(B/C)-TERT/fabp10a:tert; myl7:EGFP) 28
3.3 Establishment of Transgenic Zebrafish Line Overexpressing tert Specifically in the Liver 29
3.4 Generation of Transgenic Constructs Tg(HBV(B/C)-TERT/fabp10a:tert-2A-mCherry;myl7:EGFP) 29
3.5 Exploring the Efficacy of Curcumin and LY294002 (PI3K inhibitor) in Inhibiting Cancer using Zebrafish Model 31
3.6 Unveiling Novel Therapeutic Approaches using IPA in a TERT-Induced HCC Model 34
Chapter 4 Discussion 36
4-1 Implications and Considerations for Transgene Stability 36
4-2 Comprehensive Analysis of Transgenic Organisms for Spontaneous Knockout Understanding 37
4-3 Reevaluating HBV Enhancer Functionality: Unveiling Discrepancies and Navigating New Avenues 38
4-4 Evaluating Drug Efficacy in HCC Prevention and Therapy: Differential Effects on Proliferation and Senescence 39
4-5 Unraveling Drug Mechanisms and Future Prospects for HCC Treatment 40
Figures 43
Figure 1. The results of luciferase assays conducted in cultured cells with and without hormone treatment 43
Figure 2. Sequencing results of the six HBV genomes 45
Figure 3. Illustrates the generation of zebrafish transgenic constructs. 54
Figure 4. The generation of transgenic zebrafish expressing Tg(HBV(B/C)-TERT/fabp10a:tert; myl7:EGFP) 56
Figure 5. The genome DNA sequencing results obtained from a fin clip of the Tg(HBV(B/C)-TERT/fabp10a:tert; myl7:EGFP) specimen 57
Figure 6. The absence of fluorescence in F1 larvae of Tg(HBV(B)-TERT-tert;myl7:EGFP) and Tg(HBV(C)-TERT-tert;myl7:EGFP) 59
Figure 7. Using site-directed mutagenesis to remove the tert stop codon, attB2 site, and linker sequence 61
Figure 8. The generation of transgenic zebrafish expressing Tg(HBV(B)- fabp10a:tert; myl7:EGFP) 62
Figure 9. qPCR analysis and SA-β-gal assay for anti-HCC preventive drug treatment on Tg(fabp10a:tert;myl7:GFP) larvae 64
Figure 10. H&E analysis for anti-HCC preventive drug treatment on Tg(fabp10a:tert;myl7:GFP) larvae 65
Figure 11. qPCR analysis and SA-β-gal assay for anti-HCC therapeutic drug treatment on Tg(fabp10a:tert;myl7:GFP) larvae 66
Figure 12. H&E analysis for anti-HCC therapeutic drug treatment on Tg(fabp10a:tert;myl7:GFP) larvae 67
Figure 13. IPA result for NAC (prevention), U0126, and LY294002 (therapy) 69
Table 1. The primer list for qPCR in Tg(fabp10a:tert;myl7:GFP) transgenic zebrafish. 70
Table 2. The primer list for genotyping Tg(HBV(B)-TERT;myl7:EGFP), Tg(HBV(C)-TERT;myl7:EGFP), Tg(HBV(B)-fabp10a;myl7:EGFP), Tg(HBV(C)-fabp10a;myl7:EGFP), Tg(HBV(B)-TERT-2A-mCherry;myl7:EGFP), Tg(HBV(C)-TERT-2A-mCherry;myl7:EGFP), Tg(HBV(B)-fabp10a-2A-mCherry;myl7:EGFP), Tg(HBV(C)-fabp10a-2A-mCherry;myl7:EGFP) larvae. 71
Table 3. The primer list for sequencing the HBV genomes. 72
Appendix 73
Appendix 1. Cloning of ENI(B) and ENI(C) linked to Luciferase report gene. 73
Appendix 2. Sex hormone enhance ENI(B) but not ENI(C) in hepatoma cells. 76
Appendix 3. Cloning of HBx(B) and HBx(C). 77
Appendix 4. HBx(C) enhance ENI(C) more efficient than HBx(B) on ENI(B), and loss of activation in the presence of sex hormones. 78
Appendix 5. The structural details of the HBV(B)-TERT-Luc and HBV(C)-TERT-Luc constructs 79
Appendix 6. Significant Inhibition of Hep3B Liver Cancer Cell Proliferation and Metastasis by Curcumin at 2.5 μM. 84
Appendix 7. Effects of Curcumin on Proliferation Markers in CD36xabcg1(KO1) Transgenic Fish. 86
Appendix 8. The results of SA β-gal assay conducted on Tg(fabp10a:tert;myl7:GFP) larvae to examine the preventive effects of an anti-HCC drug. 88
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