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標題: | 表皮生長因子促進 CTEN 基因表達及磷酸化之調控機制 Study of the regulatory mechanism of EGF-induced upregulation and phosphorylation of CTEN |
作者: | Chih-Hsuan Lin 林芷萱 |
指導教授: | 廖憶純(Yi-Chun Liao) |
關鍵字: | 基因調控,表皮生長因子,轉錄因子,磷酸化, CTEN,EGF,p300,Sp1,phosphorylation, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 表皮生長因子受體 (EGFR) 訊息傳導的活化能促進 CTEN 基因表達,並增強乳癌細胞的遷移能力與侵犯性,但其調控 CTEN 基因表達之機制目前尚未清楚。我們先前的研究證實,在人類子宮頸癌細胞株 HeLa 中,EGFR的活化會透過 MEK/ERK訊息傳導路徑,藉由組蛋白乙醯轉移酶p300調控 CTEN 啟動子上組蛋白的乙醯化,並促進 p300 與 CTEN 啟動子的結合,進而誘導 CTEN 的表現。本論文進一步研究發現,將 p300 受 ERK2 磷酸化之胺基酸位點突變,會降低 p300 與 CTEN 啟動子的結合,並減少 EGF 誘導 CTEN 之 mRNA及蛋白質表現量,證實 EGFR 訊息傳導活化,經由調控 p300 的磷酸化而影響與 CTEN 啟動子的結合,進而促進 CTEN 基因表達。後續針對與 p300 有結合能力且在 CTEN 啟動子上有預測結合位之轉錄因子 Sp1 進行深入探討,結果證實 HeLa 細胞中 p300 及 Sp1 確實有交互作用,且 EGF 能增進 Sp1 與 CTEN 啟動子的結合。另一方面,也證實 EGFR 能透過 MEK/ERK 及 PI3K 訊息傳導路徑,誘導 CTEN 蛋白質 Thr347、Ser350、Ser386 位點之磷酸化,初步結果發現 CTEN 磷酸化與否並不影響 HeLa 細胞之增生能力,磷酸化的CTEN 與促進腫瘤發生的相關性有待更深入的探討。 CTEN gene expression is upregulated by the activation of epidermal growth factor receptor (EGFR), and CTEN promotes cell migration and invasion in breast cancer. However, the mechanism of EGFR-induced CTEN gene expression is still unclear. On the basis of our previous studies, we have demonstrated that EGFR activation induces histone acetylation on CTEN promoter by histone acetyltransferase p300 through MEK/ERK signaling pathway in the human cervical cancer cell line, HeLa. EGF stimulation also recruits p300 binding to CTEN promoter and results in up-regulation of CTEN expression. In this study, p300 with mutations on the amino acid residues that can be phosphorylated by ERK2 was constructed. We discovered that recruitment of the mutant p300 toward CTEN promoter ia decreased and the levels of CTEN mRNA and proteininduced by EGF are reduced in the presence of the mutant p300. It indicates that EGFR signaling can regulate the recruitment of p300 toward CTEN promoter and further activate CTEN gene expression through phosphorylating p300. In addition, we investigated whether the Sp1 transcription factor, which has putative binding site on CTEN promoter, can interact with p300 and bind to CTEN promoter. Our results have shown that after EGF stimulation, the recruitment of Sp1 to CTEN promoter is enhanced. Furthermore, we demonstrated the interaction between p300 and Sp1 in HeLa cell. On the other hand, we confirmed that EGFR can induce the phosphorylation of T347, S350, S386 sites on CTEN protein via MEK/ERK pathway and PI3K pathway. Our preliminary result has shown that CTEN phosphorylation has no effect on the proliferation of HeLa cell. However, the correlation between CTEN phosphorylation and tumorigenesis warrants further investigation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77857 |
DOI: | 10.6342/NTU201703908 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科技學系 |
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