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研究生:林晏宏
研究生(外文):Yan-Hong Lin
論文名稱:轉錄因子POU3F2對tNOX蛋白的調控
論文名稱(外文):The transcription factor POU3F2 modulates tNOX expression
指導教授:闕斌如
指導教授(外文):Pin-Ju Chueh
口試委員:林宜玫楊肇基
口試委員(外文):Yi-Mei LinZhao-Ji Yang
口試日期:2016-07-25
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:36
中文關鍵詞:轉錄因子
外文關鍵詞:POU3F2tNOX
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癌症是十大死因之首,對癌症的治療方式也成為大家十分關注的議題,而癌症的治療可分為三種方式,一、手術治療,二、放射線治療,三、標靶治療。標靶治療不同於另外兩種方法,能針對癌細胞進行抑制減少對正常細胞的影響。標靶治療針對著癌細胞中突變的蛋白進行抑制,而這些突變的蛋白相較於正常的蛋白,一些是失去了功能另一些則是額外得到了功能,這些突變導致細胞生長失去控制或者細胞不易死亡。因此標靶則治療針對這些蛋白進行抑制,藉此達到抑制癌細胞生長而導致癌細胞的死亡。然而,並非所有的癌細胞都能被標靶藥物抑制,因此需要尋找更多可能的標靶蛋白。
因此突變的蛋白或是只在癌細胞中大量表現的蛋白其轉錄或者與其他蛋白的交互作用與常成為人們研究的重點,而實驗室研究的蛋白tNOX蛋白也具有成為標靶蛋白的特性,tNOX只在癌細胞中表現,在過去的研究也被發現能促進癌細胞的增生、移動與轉移,甚至在癌細胞因辣椒素的刺激引發細胞凋亡時也能觀察到tNOX蛋白的減少,而對辣椒素刺激較不敏感的癌細胞株在將tNOX抑制後,也因為辣椒素的刺激而產生了細胞凋亡。從過去的研究可以得知對癌細胞而言tNOX是一個重要的蛋白,然而對於tNOX蛋白的轉錄調控研究甚少,因此我們想了解tNOX蛋白的上游的轉錄因子進而了解tNOX的調控。而在實驗室先前對tNOX promoter的研究中發現了POU2F1的結合位,而在過去對POU2F1的研究中發現POU2F1在各種細胞中廣泛表現的蛋白,但是他的結合位卻也可以被同家族的POU3F2結合,而POU3F2被歸類為oncoprotein,過去的研究中也發現到POU3F2與POU2F1對於相同的基因會有不同的調控結果。於是當我們在tNOX的上游發現POU2F1的結合位時便想知道是否POU3F2會調控tNOX的表現,而在實驗中當POU3F2被大量表現於TMK細胞時,tNOX的表現並無明顯改變,而當我們在AGS細胞中表現 POU3F2時,tNOX蛋白的表現隨著POU3F2的增加而增加,反之當我們將A375細胞中的POU3F2抑制時,tNOX蛋白的表現隨之減少。細胞中ERK與P-AKT與細胞增生相關的蛋白的表現量減少,而SIRT1的表現量也在tNOX減少後減少,SIRT1能去除P53的乙醯化避免細胞走向凋亡,由這些實驗看來POU3F2能調控 tNOX 的轉錄表現,改變tNOX在細胞中的表現量而影響癌細胞的生長與對抗細胞凋亡的能力。


Because cancer is the top ten caused death, the treatment is concern of every one of us. The treatment of cancer can be divided into three classes, surgical treatment, radiation therapy, and target therapy. Target therapy is different from other therapies. It can inhibit cancer cells proliferation but not normal cells by targeting a specific protein in cancer cells. Generally, these protein targets in cancer cells mutation proteins that may either gain of function or lose of function to resilt in uncontrolled cell growth. Those mutation proteins enhance cell proliferation and/or reduce cell death. Therefore, therapeutic agents that induces cell death by targeting those mutated proteins may help in cancer management. However most of cancer target therapies still have their limitations, therefore, it is important for us to find new target proteins.
In our laboratory, we have identified that tNOX is only expressed in cancer cells and enhances cell proliferation , migration and metastasis. Therefore, it is important to understand the transcriptional regulation of this tumor associated tNOX protein.
POU binding sites have been found in tNOX promoter region. In previous studies, POU2F1 is found to be a ubiquitous protein but other POU protein POU3F2 can recognize this POU2F1 binding site to modulate protein expressions. POU3F2 is classified to oncoprotein. POU3F2 and POU2F1 can cause different outcomes in the same gene. We decided to investigate whether POU3F2 regulates expression of tNOX. In this study, when we overexpressed POU3F2 in TMK cells, the tNOX expression did not change. However, tNOX protein increased when POU3F2 was expressed in AGS cells. On the other hand, we knocked down POU3F2 in A375 cells and found a reduction in tNOX expression. Subsequently, ERK and P-AKT were reduced which are important for cell proliferation regulation when tNOX were down regulated. Similarly, SIRT1 a deacetylase for p53 that is important for inhibiting apoptosis, was reduced when tNOX was downregulated which can deacetylated P53 and prevent apoptosis also showed the amount of decrease as the reduction of tNOX. Together, We propose that POU3F2 regulates expression of tNOX and affects cell survival and proliferation.


中文摘要……………..…………………………………………………………...…..i
英文摘要……………………………………………..………….…………….…..…iii
目錄……………………..……………………………………….…………….…...…v
壹、緒論………………….……………………………………………………………1
一、癌症………….………………………………………………………………1
二、tNOX蛋白….………………………………………….……………………1
三、POU蛋白….……………………….……………………………………….4
貳、研究動機….………………………………………………………………………8
参、實驗材料……………………………………………………………………..…..9
肆、實驗方法
一、細胞培養與繼代 (Cell culture and subculture)………………………..12
二、細胞冷凍與解凍 (Cell culture and subculture) ………………….……12
三、質體DNA的中量抽取 (Plasmid Midi purification) …………………….13
四、細胞轉染 (Cell transfection) ………………….……………………….13
五、西方墨點法 (Western blotting) ………………….………………………14
伍、實驗結果
一、 從tNOX上游promoter中找到POU2F1的結合位……………………15
二、 TMK細胞中表現POU3F2蛋白…………………………………………15
三、 AGS細胞中表現POU3F2蛋白…………………………………………16
四、 抑制A375細胞的POU3F2蛋白對tNOX的影響………………….…17
五、 抑制POU3F2後導致tNOX表現量下降對A375細胞的影響…….…17
陸、討論
柒、結果圖表
圖一、tNOX的promoter 中POU2F1的結合位置與tNOX gene前1200bp
序列…………………..……………………………………………… …22
圖二、TMK細胞中表現POU3F2對tNOX沒有影響………………………23
圖三、AGS細胞中表現POU3F2促進tNOX的表現………………………24
圖四、AGS細胞中表現POU3F2但減少組數……………………………..…25
圖五、在AGS細胞轉染POU3F2後看tNOX改變後會有改變的蛋白表現.26
圖六、細胞中POU3F2對其他蛋白的影響,但POU3F2無法穩定表現..27
圖七、將A375細胞的POU3F2用siRNA抑制………………………………28
圖八、A375中用兩種shPOU3F2抑制POU3F2表現造成tNOX表現量下
降……………………………………………………………………….29
圖九、在A375細胞中抑制POU3F2後看與細胞增生相關的蛋白以及tNOX
表現改變後SIRT1的改變…………………………………………….30
捌、參考文獻………………………………………………………………………..32


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