臺灣博碩士論文加值系統

大約三分之一的癌症包含黑色素瘤，其細胞素激活的蛋白激酶
(MAPK/ERK, Mitogen-Activated Protein Kinases/ Extracellular signal-Regulated Kinase) 信號傳導途徑失去調控。微型核糖核酸 (MicroRNAs, micro ribonucleic acid) 為內源性產物，並且可以抑制特定致癌信號傳導途徑中多個目標蛋白。因此已經成為應用於癌症治療中具有吸引力的策略。先前實驗室的微型核糖核酸微陣列 (microarray) 研究，在黑色素瘤細胞中找出了22個與MAPK/ERK信號傳導途徑有關的microRNA。然而，這些MAPK/ERK信號傳導途徑有關的microRNA，在黑色素瘤細胞中的抑制癌症活性致病特性及如何參與在致癌信號傳導途徑仍不明確。
  22個與MAPK/ERK信號傳導途徑有關的microRNA中，我們利用GEO (Gene Expression Omnibus) database的分析近一步篩選出9個與黑色素瘤檢體有相關的microRNA。最後的實驗數據發現，除了miR-192-3p及miR-592以外，其他的候選microRNA對黑色素瘤細胞的生長與移動有不同程度的影響。
更深入的研究，我們探討了miR-524-5p和miR-596在黑色素瘤的機制。發現miR-524-5p藉由抑制BRAF和ERK2的表現量調為控MAPK信號傳導的活性。由於BRAF和ERK2是MAPK信號傳導途徑中主要的蛋白質，因此miR-524-5p的過度表現，可以有效抑制MAPK/ERK信號傳導途徑、腫瘤增殖和黑素瘤細胞遷移。
miR-596是另一個有潛力的microRNA，其表現量在黑色素瘤中比痣低。此外，miR-596的過度表現可有效抑制MAPK/ERK信號傳導途徑、黑色素瘤細胞增殖、遷移和侵襲、並增加細胞凋亡。新的發現指出，miR-596不僅透過MEK1三端非翻譯區 (3’UTR, untranslated region) 來抑制MAPK/ERK信號傳導途徑，還可以透過MCL1和BCL2L1三端非翻譯區來增加細胞凋亡。這是第一個發現到miR-596是一個重要的抑癌microRNA，可以同時調節黑色素瘤的存活和死亡。
在應用方面，microRNA combination是一個新的基因治療方式，希望透過抑制致癌途徑中更多的目標，以克服原有無法達成的挑戰。恰巧此議題在黑素瘤細胞中鮮少被研究。我們的初步數據指出，由miR-524-5及miR-596兩種microRNAs組成的microRNA combination在黑素瘤細胞中未顯示有協同效應但有些微的加成效應。
在本研究結果中，闡述了microRNA在MAPK信號傳導途徑中扮演著重要的角色。

MAPK/ERK pathway is deregulated in one-third of all human cancers, including melanoma. MicroRNAs have become an attractive strategy to apply in cancer therapy because that they are endogenous products and can repress several targets in a specific oncogenic signaling pathway. Previous results from microRNA microarray identified 22 candidate MAPK-related microRNAs in melanoma cells. However, the possible tumor-suppressive activities and clinic pathological characteristics of candidate microRNAs in melanoma had poorly evaluated and how microRNAs involved in the oncogenic signaling pathway remains unclear.
Among 22 candidates, we narrowed down nine microRNAs (miR-192-3p, miR-218-2-3p, miR-518d-5p, miR-518f-5p, miR-520e, miR-524-5p, miR-567, miR-592 and miR-596) are linked to melanoma by GEO database analysis. We further found that all of them can control the melanoma cell growth and migration with different efficiency except miR-218-2-3p and miR-592.
In further investigation, we delved into the mechanism of miR-524-5p and miR-596. miR-524-5p represses in MAPK signaling pathway through regulating the levels of BRAF and ERK2. Since BRAF and ERK2 are the main components of MAPK signaling, the overexpression of miR-524-5p effectively inhibits MAPK/ERK signaling, tumor proliferation, and melanoma cell migration.
Another potential microRNA, expression of miR-596, is lower in melanoma than in nevi tissues. In addition, miR-596 overexpression effectively inhibits MAPK/ERK signaling, melanoma cell proliferation, migration, and invasion as well as increases cell apoptosis in vitro. Our novel findings showed that miR-596 not only negatively regulates the MAPK/ERK signaling pathway via the MEK1 3’ untranslated region (UTR) but also increases apoptosis via the MCL1 and BCL2L1 3’UTRs. This is the first finding illustrating that miR-596 is an important tumor-suppressive microRNA that can regulate both the survival and death of melanoma.
In microRNA application, combinatorial microRNA therapeutics is a new frontier in gene therapy that has the ability to overcome the challenges by targeting multiple components of key oncogenic pathways. The issue of microRNA combination was rarely investigated in melanoma cells. In our preliminary data indicate that combination of miR-524-5p and miR-596 did not show a synergistic but lightly additive effect in melanoma cells.
These results from my research studies should provide a research resource to further elucidate the regulatory roles of microRNAs in MAPK signaling pathway.

Table of contents

中文摘要 i
Abstract iii
Acknowledgement v
List of tables xi
List of Abbreviations xii
Chapter 1: Literature review 1
1.1 Introduction to cancer 1
1.1.1 Definition of cancer 1
1.1.2 Prevalence and incidence of cancer 2
1.2 Introduction to melanoma 2
1.2.1 Genetic background in melanoma 4
1.2.2 Dysregulated pathways in melanoma 4
1.2.3 Melanoma therapy 10
1.2.4 Drug resistant in Melanoma 11
1.3 The mechanism and application of microRNA 11
1.3.1 Biogenesis and mechanism of microRNA 11
1.3.2 microRNA dysregulation in disease 12
1.3.3 microRNA application in disease 13
1.4 Significances and purpose 14
Chapter 2: Materials and Methods 28
2.1 Cell lines and reagents 28
2.2 GEO data analysis 28
2.3 Oligonucleotides and plasmids 29
2.4 Tissue specimens 31
2.5 In situ hybridization (ISH) 31
2.6 Transfection 32
2.7 RT-qPCR 32
2.8 microRNA array card 32
2.9 Luciferase reporter gene assay 33
2.10 Immunoblotting 34
2.11 Alamar blue assay 34
2.12 Soft agar assay 35
2.13 Colony formation assay 35
2.14 Annexin V assay 35
2.15 Caspase3/7 activity assay 35
2.16 Migration assay 36
2.17 Wound healing assay 36
2.18 xCELLigence Real-Time Cell Analysis (RTCA): migration and invasion 37
2.19 Xenograft 37
2.20 Statistical analysis 38
Chapter 3: Identify MAPK-related microRNAs in melanoma cells 39
3.1 Introduction 39
3.2 Results 40
3.2.1 Screen microRNAs which is related to MAPK signaling pathway by GEO data 40
3.2.2 Overexpression of MAPK-related microRNAs reduce MAPK/ERK signaling pathway activity 40
3.2.3 MAPK-related microRNAs decrease cancer cell proliferation 41
3.2.4 MAPK-related microRNAs induce cell apoptosis 42
3.3 Discussion 43
Chapter 4: miR-524-5p represses MAPK signaling pathway through regulating the level of BRAF and ERK2 in melanoma 53
4.1 Introduction 53
4.2 Results 54
4.2.1 The expression level of miR-524-5p is decreased in melanoma cells with highly MAPK signaling 54
4.2.2 The level of miR-524-5p is associated with MAPK/ERK pathway 55
4.2.3 BRAF and ERK2 is a directly targets of miR-524-5p 56
4.2.4 miR-524-5p modulates the activity of MAPK/ERK pathway, cell cycle and apoptosis proteins 57
4.2.5 miR-524-5p represses cell proliferation, growth and migration 58
4.2.6 miR-524-5p slows the progression of melanoma in mice 59
4.3 Discussion 60
Chapter 5: miR-596 decrease MAPK signaling pathway by regulating MEK1 and enhance apoptosis pathway through targeting MCL1 and BCL2L1 in melanoma 79
5.1 Introduction 79
5.1.1 Copy number alteration 79
5.1.2 Apoptosis dysregulation in cancer 79
5.2 Results 80
5.2.1 miR-596 levels are repressed in melanoma cells with elevated MAPK/ERK activity 80
5.2.2 miR-596 decreases cell proliferation and growth in melanoma cells 82
5.2.3 miR-596 enhances apoptosis ability in melanoma cells 83
5.2.4 miR-596 inhibits cell migration and invasion in melanoma cells 83
5.2.5 miR-596 attenuates the activity of the MAPK/ERK and anti-apoptosis pathways 84
5.2.6 miR-596 mediates cell survival by directly inhibiting MEK1, MCL1, and BCL2L1 expression through their 3’UTRs 85
5.3 Discussion 86
Chapter 6: The effect of microRNA combination in melanoma cells 104
6.1 Introduction 104
6.2 Results 105
6.2.1 Screen the effective microRNA combination in melanoma cells 105
6.2.2 The effect of proliferation in melanoma cells treated with microRNA combination 106
6.2.3 The effect on apoptotic pathway in melanoma cells treated with microRNA combination 107
6.3 Discussion 107
Chapter 7: Concluding remarks and future direction 116
7.1 Conclusion remarks 116
7.2 Future direction 118
References 121
Appendix A. List of primers described in this thesis. 134
Appendix B. List of plasmids described in this thesis. 136
Appendix C. Tumor samples used in GISTIC analysis from TCGA . 154
Appendix D. 367 melanoma samples used in GISTIC analysis: 20 significant focal amplifications were found. 155
Appendix E. 367 melanoma samples used in GISTIC analysis: 34 significant focal deletions were found. 156
Appendix F. Publication list. 157

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