| 摘要: | 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. |
