黑色素瘤是皮膚癌最致命的形式並且每年的發生率在全世界逐年攀升。在黑色素瘤當中最主要的兩種突變基因分別是NRAS及BRAF，大約佔了總突變比率的75%。相對於BRAF的突變已經擁有了許多專一性的抑制劑作為治療藥物，NRAS到目前為止尚未發展出專一有效的治療應用。多數NRAS突變的黑色素瘤患者對於藥物的治療反應都不太理想，而且在很短的時間內就會產生抗藥性。因此，研發有效且精準的治療來針對NRAS突變的黑色素瘤是非常迫切需要的。在我們先前的研究中發現了NRAS有可能是微小核醣核酸microRNA-708這個腫瘤抑制microRNA的潛在目標；另一方面，我們的研究也發現糖皮質醇可以透過誘導microRNA-708的產生來達到抑制卵巢癌轉移的效果。因此，為了測試microRNA-708以及糖皮質醇在黑色素瘤NRAS突變的治療應用，我們用NRAS突變的黑色素瘤細胞株SK-MEL-2來觀察microRNA-708對於細胞功能上的影響，並發現在透過microRNA-708降低NRAS表現後，跟NRAS信號途徑有關的兩個下游信號蛋白ERK和AKT的磷酸化也同時被下調，並抑制了SK-EML-2細胞在增生、移動、入侵以及存活的能力，而這些功能都跟腫瘤的增生和轉移能力十分有關。接著我們也想了解是否能將先前的研究應用於具有NRAS突變的黑色素瘤，同樣利用糖皮質醇誘導NRAS突變的黑色素瘤內源性microRNA-708表現量，在我們的結果當中也證實了在SK-MEL-2上的確可以透過糖皮質醇誘導microRNA-708表現的提升，同時抑制NRAS的表現量及其下游的訊號傳遞。總體而言，我們的研究提供了一個可能可以用於治療黑色素瘤NRAS突變的方式，並且希望未來可以運用在同樣是NRAS突變所導致的其它癌症中。

Melanoma is the deadliest form of skin cancer and cases continue to rise worldwide every year. Aberrant RAS-RAF signaling pathway contributes to the cancer progression in up to 75% of melanoma patients, especially in those with BRAF or NRAS mutation. In contrast to BRAF mutated melanoma, currently there is no targeted therapies specific for NRAS mutated melanoma. Most patients with NRAS mutated melanoma respond to treatments poorly, and drug resistances emerge over a remarkably short period of time. Therefore, developing an effective treatment precisely for NRAS mutated melanoma is urgently needed. Our preliminary data indicate that NRAS is a novel target of a tumor suppressive microRNA, microRNA-708. On the other hand, our previous study indicates that microRNA-708 is induced by glucocorticoids (GCs) to suppress ovarian cancer metastasis. In order to test the therapeutic potential of microRNA-708 or GCs in NRAS mutated melanoma, we examined the cellular functions mediated by miR-708 in NRAS mutated melanoma cell line, SK-MEL-2. After reducing the expression of NRAS through overexpressed microRNA-708 in SK-MEL-2 cells, the phosphorylation level of two downstream signaling proteins, ERK and AKT, were both downregulated, and we found that cell proliferation, migration, invasion, and colony formation were collectively reduced in SK-MEL-2 cells with microRNA-708 overexpression. Those functions are critical for tumor growth and distant metastasis. To determine the effect of glucocorticoid induced microRNA-708 expression in NRAS mutated melanoma, we applied dexamethasone on SK-MEL-2. As a result, we found that dexamethasone not only induced the expression of microRNA-708, but also reduced the expression of NRAS and its two downstream signaling pathways, which are critical for cell proliferation and survival. Overall, our data may provide an alternative approach to treat patients with NRAS mutated melanoma and hope that it can be applied to other NRAS mutated cancers in the future.

摘要 1
Abstract 3
致謝 5
Contents 7
Table of Figures 9
Introduction 10
Malignant Melanoma 10
Current strategies in treating malignant melanoma 11
MAPK signaling pathway in malignant melanoma 12
MicroRNAs in cancer 14
MicroRNA-708 as the tumor suppressor during cancer development 16
Motivation and Aim 17
Material and methods 18
Cell culture and transfection 18
Western blot analysis 19
Luciferase reporter assay 21
Quantitative Real-time RT-PCR 22
Cell proliferation 24
Cell migration and invasion 24
Soft agar colony formation 25
Result 26
NRAS is the direct target of miR-708 26
Depletion of NRAS by miR-708 reduces the cell proliferation and colony forming ability in NRAS mutated melanoma cells 27
Both migration and invasion are reduced by miR-708 in NRAS mutated melanoma cells through a NRAS independent signaling pathway 28
Treatment of dexamethasone depletes NRAS protein expression through induction of miR-708 expression 29
Treatment with dexamethasone suppresses anchorage independent cell growth in NRAS mutated melanoma cells 30
Discussion 31
Figures 35
Resource Table 51
Table-1 53
Reference 54

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