Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23756
標題: 表皮生長因子受體蛋白經由活化Cdk5/p35進而調控非小型細胞肺癌之細胞生長與移動
EGFR Modulates Non-Small Cell Lung Cancer Cell Proliferation and Migration through Cdk5/p35 Activation
作者: 洪慶宗
Hong, Ching-Tsung
關鍵字: non-small cell lung cancer;非小型細胞肺癌;EGFR;Cdk5/p35;表皮生長因子受體蛋白;Cdk5/p35
出版社: 生命科學系所
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摘要: 
Cdk5(Cyclin-dependent kinase 5)為CDK家族成員之一,但不具有調控細胞週期運行的功能。此外,Cdk5的專一性活化蛋白並非典型的cyclin而主要是p35蛋白。Cdk5/p35在神經系統與神經退化相關疾病當中扮演重要的角色。在近幾年的研究也發現,Cdk5/p35對於腫瘤細胞的生理調控也相當重要。肺癌一直是全世界發生率以及致死率最高的惡性腫瘤。肺癌依據病理特徵可分為小型細胞肺癌以及非小型細胞肺癌,其中又以非小型細胞肺癌患者佔多數。根據前人研究指出,Cdk5與p35表現量在惡化程度不同的非小型細胞肺癌腫瘤組織中有顯著差異。此外,在韓國族群中發現Cdk5基因促進子(promoter)序列上的單一核苷酸多型性(Single- nucleotide polymorphism,SNP)與肺癌的發生率有正相關的趨勢。但Cdk5和p35參與調控肺癌細胞生長以及生物功能的詳細機制尚未被進一步探討。本篇論文的研究目的在於探討Cdk5和p35於非小型肺癌細胞中所扮演的角色以及調控Cdk5 與p35表現的相關機制。結果顯示,增加Cdk5或是p35的表現可以刺激A549細胞的生長,反之,阻斷Cdk5或是p35的表現則抑制了A549細胞的增生。同時傷口癒合實驗(Wound healing assay)結果顯示抑制了Cdk5或是p35的表現明顯抑制了A549細胞的移動。在神經細胞中,p35的表現被發現受到ERK經由Early Growth Response-1轉錄因子路徑的調控。ERK為表皮生長因子接受器(epidermal growth factor receptor, EGFR)所活化的下游路徑之一。因此我們初步假設在肺癌細胞A549中,EGFR透過活化ERK路徑進而誘導EGR-1以及p35的表現。實驗結果顯示於不同時間點給予A549細胞表皮生長因子(EGF)刺激後,p35的蛋白與messenger RNA表現量隨著時間的上升而增加。利用離體磷酸酶活性分析法 (in vitro kinase assay)進一步檢測也發現EGF能夠促進Cdk5活性上升。為了進一步驗證,我們利用ERK inhibitor(PD98059)以及shEgr-1分別阻斷ERK的活性以及Egr-1的表現,結果顯示EGF誘導p35表現明顯因ERK以及Egr-1受到抑制而阻斷。此外,傷口癒合實驗的結果也顯示,EGF促進A549細胞移動的效應會因Egr-1以及p35的表現受到抑制而阻斷。綜合以上結果,我們發現Cdk5和p35對於非小型肺癌細胞生長與生物功能之調控的重要性,以及在非小型肺癌細胞中p35與Cdk5的表現間接受到EGFR的調控。經由以上研究結果期望能從基礎研究角度充分了解Cdk5和p35對於肺癌的重要性,並對於未來診斷及治療有所貢獻。

Cdk5(Cyclin-dependent kinase 5) belongs to CDK family but does not involve cell cycle regulation. The specific activator of Cdk5 is p35 rather than cyclin. Cdk5/p35 play an important role in nervous system and neurodegenerative disease. In the recent studies, Cdk5/p35 also play an important role in non-neuronal cells including cancer cells. Lung cancer is the leading cause of cancer death in the worldwide. According to the pathological characteristics, lung cancer can be divided into small cell lung cancer and non-small cell lung cancer. Most lung cancer patient are non-small cell lung cancer. According to the previous research, Cdk5/p35 has high expression in non-small cell lung cancer. In addition, single-nucleotide polymorphisms in the promoter of the CDK5 gene contribute to the genetic susceptibility to lung cancer in the Korean population. The mechanism of Cdk5/p35 on cell proliferation and biological function of lung cancer is still unclear. Here we verify the role of Cdk5/p35 in non-small cell lung cancer. The MTT assay results show that Cdk5 or p35 overexpression by transient transfection increased the cell proliferation of A549 cells. On the other hand, knockdown Cdk5 or p35 expression by siRNA could inhibit cell growth and cell migration of A549 cells monitored by wound healing assay. According to the reference published in 2001, ERK induces p35 through induction of Egr-1. ERK is one of the EGFR downstream signal pathways. Here we propose the mechanism of EGFR regulate p35/Cdk5 through ERK/Egr-1 pathway in non-small cell lung cancer. The results show that EGF induces p35 through ERK/Egr-1 pathway in a time-course manner. Cdk5 activity also increased under EGF treatment. Furthermore, the induction of p35 by EGF could be abolished by ERK inhibitor or knockdown Egr-1 by shRNA. The stimulation on cell migration by EGF could be inhibited by knockdown p35 or Egr-1 expression. Taken together, we demonstrate the important of Cdk5/p35 in cell proliferation and cell migration of non-small cell lung cancer. We also demonstrate the regulation of p35/Cdk5 by EGFR through ERK/Egr-1 pathway. Base on these experimental results, we hope to understand the roles of Cdk5/p35 in lung cancer from the view points of basic research. These findings in the future can contribute to the diagnosis and treatment of lung cancer.
URI: http://hdl.handle.net/11455/23756
其他識別: U0005-2007201114304800
Appears in Collections:生命科學系所

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