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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 生物醫學研究所 > 依資料類型分類 > 碩博士論文 >  探討致癌基因14-3-3zeta分泌至胞外之機轉及其與EGFR交互作用對Cyclin D1表現之影響

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/152674

標題: 探討致癌基因14-3-3zeta分泌至胞外之機轉及其與EGFR交互作用對Cyclin D1表現之影響
Study of the secretion machenism of 14-3-3zeta and the effect of its interaction with EGFR on Cyclin D1 expression
作者: 柳右弼
Liu, You-Pi
Contributors: 陳健尉
生物醫學研究所
關鍵字: 致癌基因;表皮生長因子接受器
14-3-3zeta;EGFR
日期: 2012
Issue Date: 2013-11-18 11:04:39 (UTC+8)
Publisher: 生物醫學研究所
摘要: 14-3-3zeta蛋白是一個被廣泛研究的蛋白,並且被定義為致癌基因。然而在先前並無探討到其分泌方式以及分泌後之功能,因此本篇研究中主要來探討14-3-3zeta蛋白的分泌以及其在癌症中所扮演之角色。首先,我們利用了純化之條件培養液來確認14-3-3zeta蛋白的分泌,接著利用帶有GFP之14-3-3zeta蛋白來進行免疫螢光染色,並發現了14-3-3zeta蛋白在肺癌細胞中可藉由細胞的內吞作用來送入細胞質與細胞核內。由於細胞膜上的表皮生長因子接受器 (EGFR) 在肺癌研究中扮演了非常重要的角色,我們接著探討14-3-3zeta與EGFR之關聯。藉由免疫沈澱法,我們確認了在高侵襲能力之肺癌細胞株中,14-3-3zeta與EGFR是有交互作用的,同時藉由免疫螢光染色,我們也確認了EGFR可與帶有GFP之14-3-3zeta蛋白一同被運送。在先前的研究中發現,14-3-3zeta會影響細胞週期蛋白Cyclin D1 (CCND1) 的表現,因此我們推測14-3-3zeta進核之後可能去調控Cyclin D1基因的表現。藉由冷光報導基因的實驗,我們發現14-3-3zeta可藉由與Cyclin D1啟動子上的TCF-4及EGFR結合序列結合,進而直接影響Cyclin D1 基因的表現。為了探討被分泌的14-3-3zeta蛋白之功能,我們利用含有14-3-3zeta蛋白之條件培養液來處理細胞,發現細胞型態會轉變為較類似神經細胞的細長型態,另一方面上皮細胞與間葉細胞轉型過程的因子-鈣黏著素 (E-cadherin) 的表現也會隨之下降。藉由西方墨點法的分析,我們可以發現14-3-3zeta主要是藉由微泡的分泌來被運送到周圍環境中,並且能被其他的表皮細胞所吞入。綜合以上結果,我們推論在肺癌細胞中,14-3-3zeta蛋白能藉由微泡被分泌出細胞,被其他細胞吞入後,能與表皮生長因子接受器一同被運送到細胞核內,進而影響上皮細胞與間葉細胞轉型過程的產生;另一方面,也會結合至細胞週期蛋白Cyclin D1的啟動子上,並影響其表現。
14-3-3zeta protein is well-known in many cellular processes and has been identified as an oncogene. However, 14-3-3zeta has not been reported previously to be a secretable protein and its secretion involved in tumor biology is still unclear. Therefore, the objective of this study is to investigate the secretion machenism of 14-3-3zeta and its effect on lung cancer progression. First, secreted 14-3-3zeta of lung cancer cells was detected by purification of the conditioned medium. Next, an immunofluorescent staining showed that the purified GFP-tagged 14-3-3zeta was taken up and transported into the cytosol and nucleus of lung cancer cells. Uptake of secretable protein by the cells is major through endocytosis. Owing to intracellular EGFR plays an important role in lung cancer, we first examined the interaction between 14-3-3zeta and EGFR. A co-immunoprecipitation assay showed that 14-3-3zeta bound to EGFR in a highly invasive lung cancer cell line. An immunofluorescent staining also revealed that GFP-tagged 14-3-3zeta might be co-localized with EGFR. In previously study, 14-3-3zeta will regulate Cyclin D1 (CCND1) expression. Therefore, we speculate that 14-3-3zeta could be transported to nucleus then binding to cyclin D1 promoter. By using luciferase assay we found that 14-3-3zeta would regulate cyclin D1 promoter activity through TCF-4 and EGFR binding site. We also observed that cobble-like appearance of CL1-0 cells was replaced by a neuron-like morphology in presence of the conditioned medium from 14-3-3zeta transfectants; also, the EMT marker E-cadherin was down-regulated. Furthermore, we discovered that 14-3-3zeta protein could be secreted through microvesicle (MVs) then be uptaken by other epithelia cells. Taken together, we propose that 14-3-3zeta could be secreted with MVs and taken up by other cancer cells. While uptake by cells, 14-3-3zeta could interact with EGFR to induce EMT, as well as be transported to nucleus to regulate cyclin D1 expression.
Appears in Collections:[依資料類型分類] 碩博士論文

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