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研究生:葉希文
研究生(外文):His-Wen Yea
論文名稱:探討PPP2R2C在非小細胞肺腺癌上所扮演的角色
論文名稱(外文):The functional analysis of PPP2R2C in non-small-cell lung carcinoma
指導教授:陳健尉陳健尉引用關係
學位類別:碩士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
畢業學年度:96
語文別:中文
論文頁數:42
中文關鍵詞:去磷酸化酵素2ASrc激酶蛋白磷酸酯酶PP2A複合物中B調節亞基B1亞家族
外文關鍵詞:PP2ASRC kinasePPP2R2C
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近年來,台灣的肺癌病例有顯著增加的趨勢,在女性的癌症病例中因肺癌而死亡的人數已高居首位。因此了解腫瘤生成及轉移的機制成為當務之急。進年來在台灣,肺癌早已躍居女性癌症死亡率第一位,因此為了找出肺腺癌轉移及侵入的機制是非常重要的。先前利用微陣列技術以及微陣列-比較基因組雜交技術,交叉分析肺腺癌侵入/轉移細胞株 CL1-0和CL1-5,篩選出可能與腫瘤轉移相關的基因。結果篩選出一個功能仍未被完全定義的基因PPP2R2C。該基因隸屬於PP2A調控次單元的其中一員,但因該基因的功能仍不確定,因此我們計劃將pEF6/V5-PPP2R2C送入CL1-5細胞株中,觀察當大量表現PPP2R2C後,會大幅降低CL1-5的侵入、移動、增生以及細胞聚落形成的能力。蛋白質交互作用為細胞中蛋白質執行功能的重要環節,一方面利用免疫沉澱法,發現細胞內生性的c-SRC會與PPP2R2C有交互作用。而大量表現PPP2R2C會抑制c-SRC活化以及Ras-ERK1/2的磷酸化程度。另一方面,利用GST 沉降試驗來尋找其他可能與PPP2R2C有交互作用的蛋白。由質譜分析結果顯示,eukaryotic translation elongation factor 1 gamma (EEF1G)為參與細胞內tRNA的運送,可能會與PPP2R2C有交互作用。總合以上的結果PPP2R2C可能參與調控細胞的侵犯以及轉移機制,本研究提供更多明確的方向供後續研究,期望能發展新的抗癌治療標的。
Recently, lung cancer has dominantly increased and ranked up to the first place among Taiwanese women died from cancers. Therefore, understanding the tumorigenesis and metastasis in lung cancer is very important. Previously we have identified ppp2r2c, a gene for regulatory subunit of the PP2A complex, by using microarray and array-CGH in lung cancer cell lines that is with various invasive abilities. In order to clarify the role of PPP2R2C in NSCLC, we stably transfect human CL1-5 lung cancer cells with pEF6/V5-PPP2R2C and found decreased invasive activity in the resulting clones compared to control cells. In addition, compare to control cell, cell that overexpresses PPP2R2C decreased proliferation and colony formation rate in soft agar. To address the celluar function of PPP2R2C, we examined the known biochemical regulators of invasion and proliferation. In immunoprecipitation experiment, endogenous c-SRC co-immunoprecipitate with PPP2R2C-V5. Additionally, overexpression PPP2R2C decreased c-SRC activityand markedly suppressed the activation of Ras-ERK1/2 in highly invasive CL1-5. On the other hand, we have identified the interacting proteins of PPP2R2C through affinity purification-based proteomics to sort out the candidate proteins. The result revealed that eukaryotic translation elongation factor 1 gamma (EEF1G) could interact with PPP2R2C. EEF1G encodes a subunit of the elongation factor-1 complex which’s function is known as enzymatic delivery of aminoacyl tRNAs to the ribosome. This study gives us a new annotation of PPP2R2C in cancer metastasis and invasion mechanism and that PPP2R2C might be a promising therapeutic target for cancer metastasis and cell growth.
第一章 序論
第一節 前人研究 1
一、肺癌 1
二、腫瘤細胞之生成 2
三、癌細胞轉移 3
四、比較型基因體雜交分析技術 5
五、蛋白質磷酸化 7
六、PP2A結構及次單元 7
七、PP2A的調節功能 8
八、c-SRC酪氨酸激酶家族之結構及功能 9
九、c-Src酪氨酸激酶的調控機制 10
第二節 研究目的和策略架構 11
第二章 材料與方法
第一節 細胞培養 13
第二節 細胞冷凍保存及解凍 13
第三節 構築載體 13
第四節 勝任細胞製備 14
第五節 轉型作用 14
第六節 細菌質體DNA抽取 15
第七節 細胞染色體DNA萃取 15
第八節 共轉染作用 16
第九節 細胞RNA萃取 16
第十節 即時定量反轉錄聚合酶連鎖反應 17
第十一節 細胞總蛋白質抽取 18
第十二節 蛋白質定量 19
第十三節 蛋白質凝膠電泳分析 19
第十四節 西方墨點法 20
第十五節 免疫螢光染色及纖維型肌動蛋白(F-actin)染色 21
第十六節 細胞基質侵襲力分析(matrixgel gel invasion) 21
第十七節 細胞遷移分析(cell migration assay) 22
第十八節 細胞增生分析 22
第十九節 細胞非貼附性分析 23
第二十節 純化GST-PPP2R2C蛋白 23
第二十一節 GST-pull down 分析 24
第二十二節 分離萃取核質蛋白 24
第三章 結果 26
第一節 PPP2R2C在mRNA表現量以及蛋白表現量在CL1-0高於CL1-5 26
第二節 PPP2R2C基因構築 26
第三節 PPP2R2C進行穩定表現細胞株加藥篩選 26
第四節 穩定大量表現PPP2R2C會改變CL1-5細胞型態並降低細胞移動以及侵犯能力 27
第五節 穩定大量表現PPP2R2C會降低CL1-5細胞增生以及貼附能力 28
第六節 穩定大量表現PPP2R2C會降低CL1-5非貼附生長能力 28
第七節 PPP2R2C蛋白主要在細胞質中表現 28
第八節 以GST沉澱分析與PPP2R2C有交互作用的蛋白 29
第九節 c-SRC與PPP2R2C有交互作用 29
第十節 大量表現PPP2R2C會抑制CL1-5內生性SRC以及ERK的磷酸化程度 30
第十一節 EEF1G與PPP2R2C有交互作用 31
第四章 討論 32
第五章 結論 36
參考文獻 37
實驗結果圖表 43
附表及附圖 68
附錄 80
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