臺灣博碩士論文加值系統

一段富鳥嘌呤的核苷酸序列有可能形成四股螺旋結構，此結構我們又稱為 G-quadruplex。G-quadruplex在細胞內扮演許多角色，除了會出現端粒上而抑制端粒酶的活性，在基因轉錄以及轉譯也具有調控功能。其中，我們經軟體分析下原致癌基因Wnt-1啟動子上具有一段富鳥嘌呤的核苷酸序列。CD光譜測試確認此段序列可以形成同方位形式的G-quadruplex，在NMR光譜中，證實有鉀離子下Wnt-1啟動子能夠形成G-quadruplex。進一步，我們使用四股螺旋結構穩定化合物3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC)和3,6-bis(4 -methyl-2-vinylpyrazinium iodine) carbazole (BMVC4)做為抑制Wnt-1的標的，它們能夠提升Wnt-1啟動子G-quadruplex約20 oC，而在Luciferae報告基因的調控測試結果也看到BMVC和BMVC4皆具有抑制Wnt-1啟動子表現能力。進一步我們也發現BMVC和BMVC4不但抑制了Wnt-1的表現，Wnt-1調控路徑中-catenin、MMP-7和Survivin的表現量也受到抑制。在細胞遷移以及侵入實驗中觀察到BMVC和BMVC4具有抑制腫瘤細胞具有的遷移以及侵入的能力。由以上實驗結果，我們建立了另一新方式去抑制Wnt-1路徑的進行而達到抑制癌細胞的轉移能力。

G-quadruplex is a four-stranded DNA structure formed by G-rich sequence. Formation of G-quadruplex structure was shown to inhibit telomerase activity and affect gene expression at both transcription and translation levels. A G-quadruplex forming sequences was identified at the promoter of proto-oncogene Wnt-1. CD spectra analysis revealed that the sequence was capable of forming parallel form G-quadruplex. And NMR spectra also showed the sequence was capable of forming G-quadruplex stracture in the presence of potassium ion. The expressions of Wnt-1 upon G-quadruplex ligand treatments were first analyzed. We found two G-quadruplex stabilizers 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC) and 3,6-bis(4 -methyl-2-vinylpyrazinium iodine) carbazole (BMVC4) stabilized G-quadruplex structure by increasing the melting temperature by as many as ~20oC. Both BMVC and BMVC4 repressed the expression of Wnt-1 through stabilization of G-quadruplex structure at the promoter region. The reporter assay showed that BMVC and BMVC4 could inhibit Wnt-1 promoter expression. And next we found both BMVC and BMVC4 inhibited the expression of Wnt-1 downstream genes -catenin, Survivin and MMP-7. The cellular effects of BMVC and BMVC4 treatments were next determined. Consequently, BMVC and BMVC4 inhibited the migration and invasion abilities of cancer cells. Together our results support a novel mechanism that G-quadruplex stabilizers BMVC and BMVC4 inhibit cancer cell metastasis through repressing the expression of Wnt-1 expression.

目 錄 ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙2
縮寫表 ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙5
中文摘要 ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙8
英文摘要 ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙9
一、緒 論 ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙10
二、材料方法 ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙21
三、實驗結果 ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙26
四、討 論 ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙32
五、參考文獻 ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙40
六、附 圖 ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙45

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