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  1. NTU Theses and Dissertations Repository
  2. 醫學院
  3. 生物化學暨分子生物學科研究所
Please use this identifier to cite or link to this item: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55870
Title: "分析WNT-1, BMP2及EGFR啟動子之G-quadruplex形成序列"
Analyzing the role of G-quadruplex-forming sequence on WNT-1, BMP2 and EGFR promoter
Authors: Shao-Jung Yen
顏劭容
Advisor: 林敬哲(Jing-Jer Lin)
Keyword: 四股螺旋結構,
G-quadruplex,BMVC,BMVC4,
Publication Year : 2014
Degree: 碩士
Abstract: 四股螺旋結構 (G-quadruplexs) 是由含有許多重複鳥糞嘌呤 (Guanine) 的DNA序列所形成, G-quadruplexs二級結構容易穩定的在端粒和一些啟動子區域的原致癌基因上形成,我們發現WNT1啟動子上帶有一段G-rich的序列,利用可穩定G-quadruplex結構的化合物BMVC和BMVC4,這兩種G-quadruplex stabilizers藥物可以幫助WNT1啟動子上的G-quadruplex形成,並抑制下游WNT1基因的表現,為了進一步了解是否抑制的現象是經由G-quadruplex的結構,我們利用點突變的序列做in vitro (CD and NMR)的分析以及螢光reporter的分析,來看wild-type和mutants的WNT1啟動子活性,從實驗結果中,的確G-quadruplex stabilizers可以抑制wild-type WNT1 promoter的活性,且顯示是屬於dose-dependent的方式,而mutants則不受藥物影響是因為不會形成G-quadruplex結構,除此之外,為了了解是否藥物影響Wnt1蛋白的表現是因改變蛋白質的結合,在ChIP分析中,Nucleolin結合到G-quadruplex的量有增加,顯示WNT1啟動子在藥物的處理下會形成穩定的G-quadruplex結構,而抑制Wnt1蛋白的表現。另外我們想知道是否藥物可以可以影響到其他啟動子G-quadruplex的形成,例如BMP2和EGFR啟動子,從微陣列基因分析BMP2的表現會被抑制,因此我們想知道透過G-quadruplex stabilizers抑制Bmp2是否可使EMT過程轉變,實驗指出Bmp2及EMT現象不受到G-quadruplex stabilizers影響,由於藥物無法作用到BMP2啟動子而使G-quadruplex結構無法形成;EGFR啟動子帶有兩段GFS,亦有機會形成G-quadruplex,因此我們利用螢光reporter分析GFS的功能為何,以分析是否藥物可以影響到EGFR的表現,在NMR氫圖譜證明-137 bps和-376 bps的GFS在in vitro下有形成G-quadruplex的訊號,從repoter分析結果中,藥物處理下可增加-137 bps GFS的EGFR啟動子之活性。
DNA sequences with tandem guanine repeats tend to from G-quadruplex (G4) structure. G-quadruplex is easily formed in telomere and the promoter regions of many proto-oncogenes. Previously our lab has found a G-rich sequences located at the WNT1 promoter is capable of forming G-quadruplex. The expression of Wnt1 was repressed by G-quadruplex stabilizers. To determine whether the repression is G-quadruplex structure dependent, we first analyzed mutations on these G-rich sequences using CD and NMR. Mutants that failed to form G-quadruplex were identified. I then established a luciferase reporter assay to monitor the WNT1 promoter expression. Our results revealed that wild-type WNT1 promoter can be suppressed by G-quadruplex stabilizers in a dose-dependent manner. Mutants that failed to form G-quadruplex structure were not repressed by drugs. The binding of nucleolin is also tested to check whether the repression of expression by BMVC is due to a change in protein binding. Using ChIP assay, we found BMVC increased the binding of nucleolin to WNT1 promoter, suggesting that the G-quadruplex structure was formed at the WNT1 promoter upon drug treatment. Thus, our results indicated that formation of the G-quadruplex structure at WNT1 promoter repressed its expression. We also identified G-qaudruplex-forming sequences on BMP2 and EGFR promoters. From microarray results, we found BMP2 is repressed by G-quadruplex stabilizers. Our results showed Bmp2 is not repressed due to G-quadruplex structure cannot be formed in cell level, suggesting our drugs cannot target to BMP2 promoter. We determine two G-qaudruplex-forming sequences on EGFR promoter functional roles by luciferase reporter assay and the ability to form G-quadruplex in vitro. And it can induce -137 GFS EGFR promoter activity in G-quadruplex stabilizers treament.
URI: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55870
Fulltext Rights: 有償授權
Appears in Collections:生物化學暨分子生物學科研究所

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