2012-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/644498摘要：第二型似胰島素生長因子 (IGF-II) 傳訊RNA結合蛋白-3 (IMP-3) 乃是IGF-II傳訊RNA結合蛋白家族的一員。黑色素細胞癌是最惡性的皮膚腫瘤，它極易轉移，且對大部分的現有化學治療藥物具有抗藥性。最近發現，IMP-3在轉移的黑色素細胞癌中表現遠高於淺層黑色素細胞癌，而在良性黑色素痣則不表現，顯示IMP-3是一個新的黑色素細胞癌進展標記。微核醣核酸 (miRNAs) 是內生性的小片段RNA，它可抑制特定序列基因之表現。許多證據顯示miRNA的表現在人類癌症中出現異常，也有研究證實miRNA與黑色素細胞癌的發生與進展有關。在初步研究中，我們發現IMP-3表現量與黑色素細胞癌的移動與侵襲能力有關，藉由電腦預測，我們發現數個微核醣核酸包括let-7、miR-9、miR-98、miR-181、miR-196ab與miR-204/miR-211可以作用在IMP-3的3’UTR序列上。有趣的是，曾有報告指出在黑色素細胞癌發生與進展過程中let-7表現量會下降；我們的初步研究也發現：黑色素細胞癌淋巴結轉移處組織與原發部位組織相比，miR-211表現量顯著下降；let-7a與miR-211的表現量與黑色素細胞癌的移動與侵襲能力呈反向關係，亦即移動與侵襲能力高的癌細胞株，let-7a與miR-211的表現量明顯較低；我們同時發現， let-7a與miR-211的表現也與癌細胞的IMP-3表現量呈反向關係。這些結果顯示：微核醣核酸可能藉由調控IMP-3的表現而影響黑色素細胞癌的增生、移動與侵襲能力。因此在本計畫中，我們將探討微核醣核酸如何調控黑色素細胞癌IMP-3表現，以及微核醣核酸與IMP-3對於黑色素細胞癌增生、移動與侵襲能力的影響。第一年：(1) 探討高表現IMP-3後，對於黑色素細胞癌增生、移動與侵襲能力的影響。(2) 以轉染方式送入let-7a或miR-211，探討其對於黑色素細胞癌 IMP-3表現，以及增生、移動與侵襲能力的影響。(3) 送入針對let-7a或miR-211的抗核醣核酸後，探討其對於黑色素細胞癌 IMP-3表現量，以及增生、移動與侵襲能力的影響。第二年：(1) 藉由選殖IMP-3不同的3’端UTR，確認上述微核醣核酸與IMP-3的3’UTR結合之特異性。(2) 在高表現let-7a或miR-211的癌細胞中，送入高表現IMP-3或其下游蛋白，檢測其效應。(3) 在低表現let-7a或miR-211的癌細胞中，進行IMP-3或其下游蛋白的RNA干擾，檢測其效應。第三年：(1) 分別檢測高/低表現let-7a或miR-211或IMP-3細胞中IGF-II mRNA與蛋白、HMGA2 mRNA與蛋白的表現量。(2) IGF-II與HMGA2在IMP-3調控黑色素細胞癌增生、移動與侵襲能力中所扮演的角色。(3) 探討let-7a、IMP-3、HMGA2之間的交互作用。<br> Abstract: Insulin-like growth factor-II messenger RNA (mRNA)-binding protein-3 (IMP-3) is a member of insulin-like growth factor-II mRNA-binding protein family and is expressed during embryogenesis and in some malignancies. Melanoma is the most aggressive skin cancer, with a propensity to metastasize, and is resistant to most of the current therapeutic regimens. Recently, IMP-3 has been found to be a novel progression marker in malignant melanoma because it is expressed in metastatic melanomas significantly more than in thin melanomas but not in benign nevi. MicroRNAs (miRNAs) are endogenous ≈22-nt noncoding small RNAs, which negatively regulate gene expression in a sequence-specific manner. Increasing evidence shows that miRNA gene expression is deregulated in human cancers. Several reports also found some miRNAs are involved in melanoma development.Our preliminary results showed that IMP-3 levels were correlated with migration/invasion ability of melanoma cell lines. By using computerized prediction, we found IMP-3 could be regulated by several candidate miRNAs: let-7, miR-9, miR-98, miR-181, miR-196ab, and miR-204/miR-211. Interestingly, let-7 has been reported to be down regulated in melanoma development and progression. The levels of miR-211 were down regulated in the melanoma specimens from lymph node metastasis sites as compared to the paired specimens from the primary sites in our preliminary studies. Both let-7a and miR-211 levels were lower in those highly migratory/invasive melanoma cell lines. We also found let-7a and miR-211 levels had inverse relationship with the IMP-3 levels in different melanoma cell lines. The above results indicate that miRNAs may influence the proliferation/migration/invasion abilities of melanoma cells by regulating IMP-3 expression. In this project, we will investigate the roles of miRNAs in IMP-3 expression and their effects on melanoma cell proliferation/migration/invasion.In the 1st year, we will examine:(1) The effects of IMP-3 over-expression on the proliferation/migration/invasion of melanoma cells.(2) The effects of ectopic let-7a/miR-211 expression on IMP-3 levels as well as proliferation/migration/invasion of melanoma cells.(3) The effects of antagomir treatments for let-7a and miR-211 on IMP-3 levels as well as proliferation/migration/invasion of melanoma cells.In the 2nd year, we will perform the following experiments:(1) Confirm the binding of miRNAs to the 3’UTR of IMP-3 by using different constructs encoding the 3’UTR sequence of IMP-3 with or without mutations(2) Check the effects of IMP-3 over-expression in let-7a/miR-211 highly expressing cells(3) Check the effects of IMP-3 RNA silencing in let-7a/miR-211 low-expressing cellsIn the 3rd year, we will check:(1) The expression of IGF-II mRNA, IGF-II protein, HMGA2 mRNA, and HMGA2 protein in let-7a/miR-211/IMP-3 over-expressing cells and let-7a/miR-211/IMP-3 low-expressing cells(2) The roles of IGF-II and HMGA2 in IMP-3 regulated melanoma proliferation/migration/invasion(3) The complex interaction between let-7a, IMP-3 and HMGA2.IMP-3侵襲黑色素細胞癌微核醣核酸移動RNA結合蛋白IMP-3InvasionMelanomaMicroRNAMigrationRNA-binding protein