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研究生:梁育瑞
研究生(外文):Yu-Ray Liang
論文名稱:EB病毒LMP1蛋白質調控integrinαv和integrinβ6機制之探討
論文名稱(外文):EBV LMP1 regulates integrin αv and integrin β6 expression in nasopharyngeal carcinoma
指導教授:靳應臺靳應臺引用關係蕭振仁蕭振仁引用關係
指導教授(外文):Yin-Tai JinJenn-Ren Hsiao
學位類別:碩士
校院名稱:國立成功大學
系所名稱:口腔醫學研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:英文
論文頁數:47
中文關鍵詞:鼻咽癌LMP1蛋白質細胞聯結蛋白αvβ6
外文關鍵詞:Nasopharyngeal carcinoma (NPC)Integrin αv and β6latent membrane protein 1 (LMP1)
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EB病毒感染與多種人類惡性疾病有關,如何杰金氏症和鼻咽癌,先前我們的研究指出,EB病毒在約96.4%的鼻咽癌組織中被發現,可知EB病毒在鼻咽癌的發生過程扮演重要的角色。EB病毒在NPC中以第二潛伏期存在,只有少數基因在這時期被表現,如EBNA-1, LMP1, LMP2和EBERs,LMP1為潛伏期表現的基因中首先被發現與細胞轉型和致癌能力相關。在人類表皮細胞中,LMP1可改變許多與癌細胞生長及侵犯能力有關的基因功能表現。另一方面,integrin αvβ6在所有integrin內具有表皮專一性,它能與其他細胞膜外蛋白質結合,像是fibronectin, vitronectin, tenascin和TGF-β。特別的是,integrin αvβ6並不表現於正常口腔表皮組織,而會在傷口復原和惡性細胞生長等組織重組時才會表現,因此integrin αvβ6的表現被認為會影響腫瘤的遷移和侵犯能力。目前的研究指出,在β6 promoter上可能有Ets-1及STAT3的結合位,而LMP1已被證實可調控Ets-1及STAT3的表現;並且,在B cell中LMP1也被證實可活化integrin αv promoter。依據上述,我們推測LMP1可能同時活化Integrin αv和β6的表現,進而影響NPC 細胞的遷移和侵犯能力。由我們的實驗結果顯示,雖然LMP1蛋白質在鼻咽癌細胞中能同時活化JNK、Ets-1及NF-κB 路徑的活化,但主要還是透過NF-κB 路徑而使得integrin αv和β6表現上升。我們也預測了β6 promoter上有NF-κB結合位,而Ets-1在鼻咽癌細胞中並不參與integrin β6活化,STAT3則在LMP1表現時磷酸化程度下降。除此之外,我們正在釐清integrin αv和β6的表現對鼻咽癌細胞的功能性影響。
Epstein-Barr virus (EBV) infection is associated with several human malignancies, including nasopharyngeal carcinoma (NPC). In our previous study, EBV was found in 96.4% primary NPC specimens, implying a role of EBV in tumorigenesis of NPC. EBV resides in NPC cells in a status of type II latency, with only limited genes being expressed, namely EBNA-1, LMP1, LMP2 and EBERs. LMP1 was the first EBV latent gene found to be able to transform cell lines and alter the phenotype of cells due to its oncogenic potential. In human epithelial cells, LMP1 alters many functional properties in tumor progression and invasions. Moreover, Integrins are αβ heterodimeric type I membrane proteins which are formed of 18 α and 8 β subunits to assemble into 24 distinct integrins. Integrin αvβ6 is epithelial-specific and binds to the ECM proteins, like fibronectin, vitronectin and tenascin, and also to the latency associated peptide of TGF-β. Especially, integrin αvβ6 is not expressed by healthy oral epithelia, but is upregulated during tissue remodeling, including that accompanying wound healing and inducing carcinogenesis. Integrin αvβ6 has been proposed to play an important role in tumor migration and invasion. Some studies have shown that Ets-1 and STAT3 could be regulated by LMP1, and putative Ets-1 and STAT3 binding sites on β6-promoter were considered to be important for transcriptional activation of β6 subunit. And also, LMP1 was proved to transactivate integrin αv promoter in human B cells. Taken together, we hypothesized that LMP1 can activate both αv and β6 genes to potentiate the invasion and migration abilities of NPC cells. We currently found that although LMP1 could activate Ets-1, JNK and NFκB pathways in NPC cells, the main pathway which regulated integrin αv and β6 was via NFκB pathway. Ets-1 was not involved in regulation of integrin β6 promoter, and STAT3 was downregulated in NPC cells. Furthermore, the cell functional effects of LMP1-mediated integrin αv and β6 expression will be elucidated.
中文摘要 I
英文摘要 II
誌謝 IV
CONTENTS V
ABBREVIATIONS i
INTRODUCTION 1
Nasopharyngeal carcinoma 1
Epstein-Barr virus and NPC 1
Latent membrane protein 1 and its downstream signaling pathways 3
Integrins 4
Integrin αvβ6 5
Study purpose 6
METERIALS AND METHODS 7
Materials 7
Cell lines 7
Plasmids 7
Primary antibodies 7
Secondary antibodies 8
small interfering RNAs (siRNAs) 9
Inhibitors 9
PCR Primers 9
Labwares and reagents 10
Methods 11
Transfection and inhibitor treatment 11
Protein extraction and quantification 12
Western blotting 12
RNA extraction 12
RT-PCR 13
Immunohistochemistry (IHC) staining 13
Cell growth and proliferation 14
Migration and matrigel invasion assay 14
RESULTS 15
EBV LMP1 induces integrin αv and β6 expression in NPC cell line. 15
LMP1 can activate Ets-1 in HONE-1 cell line. 15
Ets-1 is not involved in LMP1-regulated αv and β6 expression. 16
Identification of LMP1 truncation effects on integrin αv and β6. 16
LMP1-mediated signaling is not via Erk, PI3K and p38 pathways. 17
STAT3 phosphorylation was downregulated in LMP1-induced HONE-1 cell line. 17
JNK pathway is not involved in LMP1-mediated signaling. 18
NF-κB pathway plays a critical role in LMP1-induced signaling. 18
LMP1-induced cell functions in HONE-1 cell line. 19
Protein expression profiles of integrin αv and β6 in various cancer cell lines. 19
Integrin αvβ6 is expressed on NPC specimens 20
DISCUSSION 21
CONCLUSION 23
REFERENCES 24
FIGURES 31
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