2013-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/657390摘要：微核醣核酸(miRNAs或miRs)為內生性不具編碼蛋白功能的RNA小片段，負責轉錄後之基因調控，可做為抑癌基因或致癌基因。此外，miRNAs 在維持腫瘤幹細胞的功能及上皮間質轉化 (EMT) 過程中亦扮演著關鍵角色。過去研究指出，人類黑色素細胞癌中有一群細胞，在以無血清配方的幹細胞培養液培養時，會形成懸浮性且細胞聚集成團的黑色素癌細胞球。這些聚集成團的細胞已被證實有自我再生、多重分化、促進腫瘤發生的類幹細胞特性，並負責維持腫瘤的持續生長、轉移及增強化學藥物抗性。雖然我們已知道 miRNAs 在調控黑色素細胞癌腫瘤形成及腫瘤進展的重要性，但目前尚沒有針對類幹細胞黑色素癌細胞球的miRNA 標誌的相關研究。我們假設和黑色素細胞癌轉移相關的 miRNA 標記也可能呈現在具有類幹細胞高轉移特性的黑色素癌細胞球中。因此，本計畫的研究目的是以類幹細胞的黑色素癌細胞球為模式，探討可促進黑色素細胞癌進展的單一微核醣核酸或微核醣核酸群，及其相關分子調控機制。我們使用A2058人類黑色素細胞癌細胞株進行培養研究，利用全基因微核醣核酸陣列分析，比較懸浮性聚集球狀細胞和貼盤細胞 miRNA 表現情形之差異，發現一群特定的 miRNA 有達八倍以上顯著的增加。我們發現在人類第19對染色體上有46個 miRNA 的微核醣核酸叢集處 (C19MC)，有33個 miRNA 在黑色素癌細胞球中有高度表現的狀況，其中包括 miR-519d，這些特定的 miRNA 表現可能會促進惡性黑色素細胞癌的擴展。因此，我們將本計畫分成三個部分，首先在第一年的研究，我們會以電腦程式預測及生化方法確認 miR-519d 及其目標蛋白 EphA4 的交互作用。在第二年的研究，我們會進行功能性的探討，瞭解 miR-519d 在 EphA4 的分子訊息傳遞如何調控黑色素細胞癌的腫瘤進展。在第三年，我們將研究人類第19對染色體上的微核醣核酸叢集區的表觀遺傳如何調控黑色素癌細胞球生成及黑色素細胞癌轉移。我們也會以臨床黑色素細胞癌組織樣本驗證C19MC微核醣核酸的表現及啟動子CpG島甲基化之情況。此三年計劃將讓我們瞭解新穎的C19MC微核醣核酸機制如何調控黑色素細胞癌的惡性侵略度。<br> Abstract: MicroRNAs (miRNAs or miRs) are endogenous, small non-coding RNAs that regulatepost-transcriptional gene expression and can work as either tumor suppressors oroncogenes. Moreover, miRNAs are crucial for cancer stem cell function as well asepithelial to mesenchymal transition (EMT). It has been found that a subset of cells inhuman melanomas can form nonadherent melanospheres under serum-free stem cellmedium. These sphere-forming cells have been demonstrated to have the stemcell-like capabilities including self-renewal, multi-differentiation, enhancedtumorigenecity, and are responsible for maintaining continuous tumor growth,metastasis, and chemoresistance. Despite the important role of miRNA in melanomaformation and progression, to date there have been no reports of miRNA expressionprofiling in stem-like melanospheres. We hypothesized that microRNA signaturesof metastatic risk found in metastatic melanoma may work through similarmechanisms in stem-like melanospheres. Therefore, the aim of the study is to usethe melanosphere model to determine single miRNAs or miRNA clusters with primaryimportance given to melanoma progression and their molecular regulatorymechanisms. Through a genome-wide microRNA array based platform to screen fordifferentially expressed miRNAs between nonadherent sphere-forming and adherentparental A2058 melanoma cells, we discovered a characteristic set of miRNAs withsignificant (up to 8-fold) upregulation. A high-level expression involving 33 of 46miRNAs in the chromosome 19q13.41 microRNA cluster (C19MC), includingmiR-519d, was found in sphere-forming melanoma cells. This characterized miRNAsignatures may foster the progression of malignant melanoma. In this 3-year proposal,we will first use computational and biochemical approaches to identify andcharacterize the interaction of miR-519d and its predicted target protein EphA4.In the second year, we will perform functional characterization of the role ofmiR-519d by targeting EphA4 signaling pathway in melanoma progression. In thethird year, we will decipher the epigenetic regulation of C19MC in melanosphereformation and melanoma metastasis. The expression of miRNAs in C19MC as well asits methylation status of CpG islands in the promoter region will be validated inclinical melanoma samples. This 3-year project will unravel novel molecularmechanisms related to how C19MC miRNAs control melanoma aggressiveness.