臺灣博碩士論文加值系統

據世界統計，卵巢癌為女性癌症致死率的第九位，而卵巢癌的高致死率是由於它相當容易復發。特別是轉移型式的復發，或稱遠處復發(復發的位置在遠處而非原發位)。轉移型復發也是卵巢癌的主要致死的原因，因此，此類復發卵巢癌將是未來研究相當重要的課題。這裡我們將焦點放在微型核醣核酸microRNAs (小的非編碼型態的核醣核酸)，它的功能主要是去抑制它下游目標基因及其蛋白質的表現。近年來，有許多研究指出微型核醣核酸的異常表現與癌症形成有關。因此，在本研究中，我們認為微型核醣核酸的不正常表現可能與轉移型復發的卵巢癌有關，並且進一步探討其作為生物標誌或治療標地的可能性。我們一開始檢視兩位病人的臨床檢體(分別包含原發腫瘤及復發腫瘤)的microRNA表現圖譜，在合併分析後發現microRNA-150在復發卵巢腫瘤中異常上升，更進一步檢視22對臨床檢體的表現，我們發現到其中16對的復發腫瘤microRNA-150也呈現上升現象。並且藉由GEO資料庫的查詢發現到miR-150的表現量與病人的早期復發及死亡率呈現正相關。接著，探討microRNA-150的功能，我們在microRNA-150表現量較低的BG1卵巢癌細胞株過度表現microRNA-150發現到可以增加細胞移行及侵襲的能力，反之在microRNA-150表現量高的SKOV3則可抑制其移行及侵襲力。此外，利用cDNA microarray分析microRNA-150可能影響的下游目標基因，並結合microRNA目標基因預測軟體及文獻搜尋，我們發現MYB (v-myb myeloblastosis viral oncogene homolog)的mRNA層級會明顯受到microRNA-150的調控，並且可能與細胞移行有關。更進一步探討其目標基因-MYB與細胞移行和侵襲力的影響，發現到在過度表現MYB後會減低細胞移行及侵襲力。並且在臨床檢體中也發現microRNA-150和 MYB 呈現負相關。這些結果證明miR-150的異常上升可能與轉移型復發的卵巢癌的進程有關。miR-150會促進癌細胞的轉移及侵襲能力，極有可能是透過調控MYB。

Ovarian cancer is the ninth leading cause of global cancer-related deaths. It is associated with high mortality due to high recurrence rates, especially metastatic recurrence. Metastatic recurrence is the main feature of advanced ovarian cancer and a direct cause of death. It is important to identify biomarkers and therapeutic targets for early detection and treatment of metastatic recurrence of ovarian cancer. The focus of our study is on microRNAs (miRNAs), which are small (~22 nt) non-coding RNAs that negatively regulate downstream target genes. They control a wide range of biological functions and their abnormal expression may cause cancer. Thus, we hypothesized that microRNA dysregulation are involved in the recurrence of ovarian cancer. We used microarrays to analyze global microRNA expression in the primary and recurrent tumors of 2 patients. We found that microRNA-150 was upregulated in recurrent tumors in comparison to primary tumors. To verify the relationship between miR-150 expression and cancer recurrence in ovarian cancer, we collected primary and recurrent tumors from 22 clinical patients. miR-150 was upregulated in 16 of these patients. Analysis of 2 Gene Expression Omnibus databases revealed a positive association between miR-150, shorter survival time, and early recurrence. Next, we explored the function of miR-150 in ovarian cancer recurrence in vitro. Expression of miR-150 positively correlated with cell migration; overexpression of miR-150 in BG-1 ovarian cancer cells promoted migration and invasion but did not influence proliferation. In contrast, inhibition of miR-150 in SKOV3 reduced cell migration and invasion. To identify the target genes of miR-150-mediated invasion and migration, we used whole gene expression microarrays. Expression signatures were analyzed with 5 microRNA target prediction software programs, which identified MYB (v-myb myeloblastosis viral oncogene homolog) as a target of miR-150 regulation that is also associated with cell motility and invasion. These results suggest miR-150 upregulation mediates the progression of metastatic recurrence of ovarian cancer. miR-150 promotes tumor cell invasion and migration by targeting the MYB pathway. In the future, we will directly examine the function of miR-150 in metastasis in vivo.

Contents
中文摘要 I
Abstract III
Acknowledgements V
Contents VII
Abbreviation XII
Introduction 1
1. Ovarian cancer and recurrence 1
2. Ovarian cancer metastasis 1
3. MicroRNA 2
3.1 Biogenesis of microRNA 2
3.2 Target gene repression 2
4. MicroRNA in cancer 3
5. MicroRNA as an metastatic regulator 4
Rationale and specific aims 5
Material and methods 6
1. Clinical samples 6
2. Cell culture 6
3. In situ hybridization 7
4. Transfection 8
5. Drug treatment 8
6. MicroRNA real-time PCR 8
7. Western blotting 9
8. Lentivirus production for shRNA and miR-150 overexpression 9
9. Real-time quantitative PCR 10
(1) Reverse transcription to cDNA 10
(2) Real-time PCR 10
10. Wound healing migration assay 11
11. Transwell invasion assay 11
12. Cell proliferation assay 11
13. Plasmid construction 12
14. Luciferase reporter assay 13
15. MicroRNA target prediction software 13
16. Analysis of GEO database 13
17. Statistical analysis 13
Results 14
1. MiRNA-150 expression in recurrent ovarian cancer 14
2. MiR-150 promotes cell migration and invasion 15
3. Identification of miR-150 target genes contributing to cell migration and invasion 15
4. miR-150 directly suppresses expression of MYB 16
5. Overexpression of MYB suppressed cell migration and invasion. 16
6. MiR-150 promotes cell motility and invasiveness by targeting MYB 16
7. Expression of miR-150 and MYB in clinical specimens of ovarian cancer 17
8. Characterization of BG1 cells overexpressing miR-150 by lentivirus 17
9. Characterization of SKOV3 cells overexpressing miR-150-specific sponge 17
Discussion 18
1. The previous study of miR-150 18
2. The upstream of miR-150 18
3. Others potential targets of miR-150 mediating cell migration and invasion 19
4. MYB downstream mechanism 19
5. Therapeutic potential of miR-150 in ovarian cancer 20
Conclusion 22
Reference 23
Tables 28
Table 1. Top 50 of upregulated miRNA signatures in two clinical patients with recurrent ovarian cancer. 28
Tables 2. Clinical characteristics of 22 ovarian cancer patients evaluated for miR-150 expression 29
Tables 3 . shRNA used in this study 30
Tables 4. Primers used in this study 31
Tables 4.1 Primers used in real-time PCR 31
Tables 4.2 Primers used in MYB-3’UTR cloning 31
Table 5. Gene expression profiles of miR-150 mediating metastatic signatures 32
Figures 33
Figure 1. miR-150 was upregulated in recurrent ovarian cancer. 33
Figure 2. miR-150 was upregulated in the recurrent tumors of 16 patients with ovarian cancer. 34
Figure 3. Expression of miR-150 correlated with shorter survival time and early recurrence 35
Figure 4. Overexpressing miR-150 did not affect BG1 sensitivity to chemotherapeutic drugs 36
Figure 5. Inhibiting miR-150 did not affect SKOV3 sensitivity to chemotherapeutic drugs 37
Figure 6. miR-150 did not affect cell growth 38
Figure 7. miR-150 expression correlates with cell migration 39
Figure 8. miR-150 promoted cell migration and invasion. 40
Figure 9. Inhibiting miR-150 reduced cell migration and invasion. 42
Figure 10. Identification of putative miR-150 targets 44
Figure 11. MYB expression is regulated by miR-150 45
Figure 12. miR-150 represses MYB expression by directly targeting its 3′UTR. 46
Figure 13. miR-150 expression negatively correlated with the expression level of MYB. 47
Figure 14. Overexpression of MYB reduced cell migration and invasion in SKOV3. 48
Figure 15. MYB knockdown promotes cell migration and invasion. 50
Figure 16. The motility and invasive ability were recovered if miR-150 was inhibited in conjunction with MYB in SKOV3 cells. 52
Figure 17. Expression of miR-150 negatively correlates with the MYB level in clinical specimens. 53
Figure 18. Functional characterization of BG1 cells overexpressed miR-150 by lentivirus. 54
Figure 19. miR-150 overexpression suppressed the expression of its potential target gene in BG1 cells. 55
Figure 20. Expression of miR-150 positively correlates with MYC level in clinical specimens 56
Figure 21. Stably expressing miR-150 sponge reduced cell motility and invasive ability. 57
Figure 22. miR-150 sponge upregulated the expression of MYB. 58
Appendix 59
Appendix I : Reagents 59
Appendix II : Equipment 61
Curriculum Vitae 62

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