臺灣博碩士論文加值系統

microRNAs (miRNAs) 藉由負調節標的基因的表達而在細胞增生、死亡、分化及早期發育等過程中扮演重要角色。目前在不同的癌症研究中皆發現miRNAs的表現異於正常組織；且其特殊的表達特徵(miRNA signature)可應用於癌症分類與預後結果上。神經母細胞瘤 (NB) 是兒童常見腫瘤之一，迄今我們仍不清楚miRNA在此癌症中所扮演的角色。因此本研究目的即在探討神經母細胞瘤組織中是否存在不一樣的miRNA表達模式且與癌症之間有何關連性存在。首先，我們利用Q-PCR方式偵測162個miRNA，Dicer和Drosha (miRNA生成相關基因 )在66個癌組織樣本中的表現情形。結果顯示，在愈惡性的樣本中miRNAs的表達相對較低；利用PAM的分析方式可以找出27個miRNAs而將高危險樣本從中區分出來。此外我們亦發現在高危險的樣本中Dicer或Drosha基因的較低表現可能是與大部分miRNAs的低表現及較差的存活率結果有關。值得注意的是，在不帶有MYCN基因放大 (gene amplification) 的樣本，低表現的Dicer可當作一獨立的預後因子(HR = 9.6，p = 0.045，n = 52)，這是該類病人首次有生物標誌預後因子的發表。此外使用類神經網路軟體(PNN) 選擇 15 個生物標誌物(包括 Dicer 和 Drosha的表現，診斷年齡與 12 miRNAs 表現)的組合，我們能夠將所有病人區分成 4 個不同模式而有效的預測臨床結果。另外，在細胞株實驗中，我們亦證明減少細胞內Dicer或Drosha的表現會促使細胞生長，更加證明Dicer、Drosha和miRNAs在神經母細胞瘤癌化過程中扮演重要角色。
另一方面，我們進一步探討特定、單一的miRNA，miR149*，在癌細胞株中的影響。上述研究發現，miRNA-149 在高度惡性神經母細胞瘤樣本中表現顯著偏低，然而在細胞株內異位表達(ectopic expressing) miR-149並未發現任何異常；反而是表現miR-149的同源 miRNA- miR-149 *會抑制神經母細胞瘤細胞株-Be2C與子宮頸癌細胞株-HeLa的細胞增長和誘導細胞凋亡的發生。藉由TargetScan線上平台分析，基因Akt1、 E2F1，和 b-Myb被預測可能為miR-149*的目標基因。隨後西方點漬法與RT-PCR結果顯示，在細胞轉染而大量表現miR-149*後，Akt1、 E2F1和 b-Myb 的蛋白表現量與mRNA表達程度皆明顯下降。進一步藉由reporter assay我們證明除了b-Myb外，Akt1 和 E2F1 是miR-149*直接作用的目標基因，並且進而找出在這些基因3’UTR上被miR-149*直接作用的位置點。同時我們也觀察到，降低細胞中Akt1或E2F1的表現也會導致類似於miR-149*所造成的細胞凋亡情形。這些結果顯示，miR-149*造成的細胞凋亡有可能是經由降低Akt1和E2F1的表現而達成的。重要的是，分析原始神經母細胞瘤樣本(n = 56) 亦顯示miR-149*的表現與E2F1的表現量呈現顯著負相關性 (p = 0.026)。此外，使用reporter檢測，過量表達miR-149(藉以模擬細胞內miR-149表現較優勢的狀態)並不能壓制miR-149*對目標基因的抑制作用。這意味著，in vivo狀態下miR-149*造成的細胞凋亡影響並不會受其同源miR-149的優勢表現而被抑制。綜合上述結果，我們的研究不僅提供第一個證據證明 Akt1 是一個 miRNA 的直接目標基因，並且也顯示miR-149 *可藉由壓制Akt1 和 E2F1的表達而被視為一個細胞凋亡miRNA(pro-apoptotic miRNA)。
由於miRNA在調節基因表達上的重要性陸續披露，使得該領域成為當前熱門的研究焦點；藉由以上的研究結果將有助我們更加瞭解miRNA在生物體內的功能與在癌化過程中扮演的角色，且證明miRNA具有可應用於臨床診斷與腫瘤治療上的潛力。

Aberrant miRNA expression has been reported in various tumors and shown to be useful for cancer classification and prognostication. Neuroblastoma (NB) is a common childhood tumor and accounts for 15% of pediatric cancer deaths; so far whether dysregulation of miRNAs play a role in NB tumorigenesis remain unclear. To investigate the microRNA (miRNA) profile and the role of Dicer and Drosha in NB, we have analyzed the expression of 162 human miRNAs, Dicer and Drosha in 66 NB tumors by real-time PCR methods. We found global downregulation of miRNA expression in advanced NB and identified 27 miRNAs that can clearly distinguish low- from high-risk patients. Furthermore, expression levels of Dicer or Drosha were low in high risk NB tumors, which accounted for global downregulation of miRNAs in advanced disease and correlated with poor outcome. Notably, for patients with MYCN non-amplified tumors, low expression of Dicer can serve as a significant and independent predictor of poor outcome (HR=9.6, p=0.045, n=52). This is the first biomarker shown to be predictive of outcome for NB patients with MYCN-non-amplified tumors. In addition, using Plausible Neural Networks (PNN) to select a combination of 15 biomarkers which consist of 12 miRNAs signature, expression levels of Dicer and Drosha and age at diagnosis, we were able to segregate all patients into 4 distinct patterns which were highly predictive of clinical outcome. In vitro studies also showed that knockdown of either Dicer or Drosha promoted the growth of NB cell lines. Our results revealed that a combination of 15 biomarkers could delineate risk groups of NB and serve as a powerful predictor of clinical outcome. Moreover, our findings of growth promotion by silencing Dicer/Drosha implied their potential use as therapeutic targets for neuroblastoma.
On the other hand, we further explored effects of specific miRNA-miR149* in caner cell lines. miR-149* is cognate of miR-149, which was identified as the least expressed miRNA in the advanced tumor from our NB-miRNA signature study. Here, we identified Akt1 and E2F1 to be two direct targets and b-Myb to be an indirect target of miR-149* by reporter assays and Western blot analyses. Ectopic expression of miR-149* induced apoptosis in Be2C, a neuroblastoma cell line, and in HeLa cells. Silencing of Akt1 or E2F1 expression also led to similar apoptotic changes in these two cell lines, suggesting that the pro-apoptotic effects of miR-149* were exerted by repressing Akt1 and E2F1 expressions. Importantly, analysis of primary neuroblastoma samples revealed a significant inverse correlation of miR-149* with E2F1 expressions (p=0.026). Interestingly, using the reporter assays, excess miR-149 introduced by transfection to simulated its preponderance in the in vivo condition, could not overcome the repressive function of miR-149* on the target genes. This implies that the pro-apoptotic function of miR-149* may not be dampened by its predominant cognate, miR-149, in vivo. These results not only provided the first evidence that Akt1 is a direct target of miRNA but also demonstrated that miR-149* is a pro-apoptotic miRNA by repressing the expression of Akt1 and E2F1.
Taken together, our findings have not only provided new miRNA-based biomarkers to improve current risk stratification system and outcome prediction, but also identified potential molecular targets, such as Dicer, Drosha, and miR-149* for the treatment of NB.

Contents.............. I
Table contents III
Figure contents IV
Appendix contents VI
Abstract-Chinese VII
Abstract-English X
Chapter 1. Introduction 1
1.1 microRNA overview 1
1.2 microRNA and cancer 3
1.3 miRNA and PI3K/Akt signaling pathway 5
1.4 Neuroblastoma 6
1.5 Scientific Rationale and Specific Aims 10
Chapter 2. Material and Methods 12
2.2 Real-time PCR quantification of miRNAs and Computational Analysis 13
2.3 PNN analysis 14
2.4 Real-time RT-qPCR of genes 15
2.5 shRNA design and transfection 15
2.6 Western Blot 16
2.7 Cell proliferation and soft-agarose colony forming assay 16
2.8 Apoptosis assay 17
2.9 miRNA array 18
2.10 Plasmid construction 19
2.11 miRNA oligo, transfection and luciferase reporter assay 20
2.12 Statistical analysis 20
Chapter 3. Results and Discussion-Part 1 22
3.1 Global downregulation of miRNA expression profile in advanced NB tumor 22
3.2 miRNA signature can discriminate high-risk NB patients from low-risk group 22
3.3 Downregulation of Dicer and Drosha in advanced NB 24
3.4 Lower expression of Dicer or Drosha is associated with shorter survival 26
3.5 Expression of Dicer is an independent predictor for survival in NB patients without MYCN amplification 26
3.6 Identification of an unique signature of 12 miRNAs, Dicer, Drosha and age at diagnosis that can delineate clinical risk group by PNN analysis 27
3.7 Knockdown Dicer or Drosha can promote NB cell proliferation and transformation 29
3.8 Discussion 30
Chapter 4. Results and Discussion-Part 2 36
4.1 miR149* induces apoptosis 36
4.2 miR-149* represses expression of Akt1, E2F1, and b-Myb 37
4.3 Akt1 and E2F1 are the direct targets of miR-149* 38
4.4 Akt1 and E2F1 are involved in the miR-149*-induced apoptosis 40
4.5 A significant inverse correlation of miR-149* with E2F in primary NB samples 41
4.6 Excess miR-149 can not neutralize the inhibitory effects of miR-149* in vitro 42
4.7 Discussion 43
Chapter 5. Conclusion 46
Chapter 6. References 48
Tables and Figures 59
Appendixes 85
Abbreviation 90

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