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dc.description.abstractLung cancer is the most common cause of cancer deaths in the world, and the incidence is increasing each year. Since 1982, cancer has been the major cause leading to mortality for cancer patients that domestic ten major causes of the death ranked. In the previous studies, we have identified and characterized a novel tumor/invasion suppressor gene HLJ1 (human liver DnaJ-like protein, also called DNAJB4) in a lung cancer cell line model with different invasion capability (CL cell lines). HLJ1 is a member of Hsp40 family, and heat shock proteins are commonly involved in the essential defense mechanism for protein folding and assists cell against stress. According to the previous studies, over-expression of HLJ1 protein in lung cancer cell could reduce the abilities of proliferation, colony formation, invasion and migration. However, the suppressive mechanism of HLJ1 is unclear. Therefore, the objective of this study is to investigate and characterize the proteins interacting with HLJ1. To obtain the novel HLJ1-binding protein, the different density of tumor cell culture is employed to mimic metastatic characteristics in vitro. We have identified two proteins, p54nrb and PSF, that can interact with HLJ1 in high cell culture density by immunoprecipitation assay and MALDI-TOF analysis. The complex of HLJ1 and p54nrb was confirmed in vitro and in vivo by co-immunoprecipitation, GST pull-down and mammalian two-hybrid assays. In addition, the results of GST pull-down assay indicated that p54nrb can interact with HLJ1 through HTH motif. We also found that the expressions of both HLJ1 and p54nrb are increased in A549, CL1-0, CL1-5 and CL1-5F4 cancer cells while are culturing in high cell density. The results of immunofluorescent and cytosolic/nuclear protein fraction revealed that p54nrb is mainly located in the nucleus. Furthermore, we find that over-expression of p54nrb protein in highly invasion lung cancer cell line, CL1-5, can reduce cancer cell proliferation, colony formation, invasion and migration. On the other hand, p54nrb is decreased when most cells are in DNA synthesis phase. Once over-expression of p54nrb in CL1-5 cell line, the cell cycle would arrest in G1 phase to reduce cancer cell proliferation. Investigating the novel molecular mechanism about HLJ1-p54nrb interaction is important, it may not only provide a new insight to understand the pathway of metastasis and growth regulation but also be the new target molecule for anti-cancer therapy in the future.en_US
dc.description.abstract近來全球肺癌人數有顯著增加之趨勢,自 1982 年起癌症已成為國內十大死因排名的第一位。在實驗室先前的研究中,利用肺腺癌模式細胞株(CL 系列)及 cDNA 微陣列(microarray)技術,篩選到一個具有抑制肺癌細胞轉移及侵入能力的基因 HLJ1 (human liver DnaJ-like protein,又稱DNAJB4)。HLJ1 屬於熱休克蛋白 40 家族中的一員,熱休克蛋白主要參與蛋白質的折疊與抵抗外界壓力保護細胞。當大量表現 HLJ1 時,會大幅降低癌細胞的增生、聚落形成、侵入和移動能力。然而 HLJ1 是透過何種機制來抑制癌細胞轉移及侵入能力,目前尚不清楚。因此,本研究藉由細胞生長疏密之不同(細胞培養密度差 10 倍),來模擬細胞移動能力的差異,再以免疫沉澱法分析與 HLJ1 有交互作用,且在疏密培養條件下有差異的蛋白質。經由質譜儀分析鑑定,結果顯示 HLJ1 與 p54nrb (non-POU domain containing, octamer-binding)和 PSF (Homo sapiens splicing factor proline/glutamine-rich polypyrimidine tract binding protein)具有交互作用,而且當細胞培養密度高時,PSF 及 p54nrb 蛋白質和 HLJ1 的結合表現量會增加。進一步分別以免疫共沉澱法、GST (Glutathione-S-transferase)沉澱法以及哺乳動物細胞蛋白交互作用分析法,在細胞內及細胞外的情形下,證實 HLJ1 與 p54nrb 會形成蛋白複合體;並利用 GST 沉澱法發現 p54nrb 主要可能是透過其 HTH 區域與 HLJ1 結合。而在 A549、CL1-0、CL1-5 及 CL1-5F4 四株肺癌細胞株中也發現,在細胞疏密差十倍情況下,p54nrb 和 HLJ1 的蛋白表現量會隨著細胞密度增加而增加。在分離細胞核內/外蛋白及免疫螢光染色中也發現 p54nrb 會位於細胞核中。此外,本研究也發現,當大量表現 p54nrb 蛋白於高侵襲性的肺癌細胞 CL1-5,會降低癌細胞的增生、貼附性及非貼附性細胞聚落形成、移動及侵入能力。而在觀察細胞週期中發現,當大部份細胞於 DNA 合成時期時,p54nrb 蛋白表現量會下降;當大量表現 p54nrb 蛋白於 CL1-5 細胞時,則會使得細胞停留於 G1 間期的時間顯著地延長,進而抑制 CL1-5 細胞的生長。未來將進一步探討 HLJ1-p54nrb 蛋白複合物可能參與的調控機制,藉此研究提供一個可能抑制癌細胞生長及轉移的方法,並應用在癌症的治療上。zh_TW
dc.description.tableofcontents中文摘要...............i 英文摘要..............ii 目次...................iv 圖表目次........................vii 縮寫字對照表.............................ix 第一章 序論............................1 第一節 前人研究.................1 一、肺癌..........................1 二、癌細胞侵入(Invasion)/轉移(Metastasis)........4 三、熱休克蛋白.....................5 四、細胞核蛋白.................9 第二節 研究目的與策略架構...................13 第二章 實驗材料與方法....................16 第一節、細胞株..............................16 第二節、細胞培養..................16 第三節、細胞冷凍保存及解凍..................16 第四節、細胞蛋白質之萃取及定量分析................17 第五節、免疫共沉澱.......................17 第六節、蛋白質凝膠電泳分析..........................18 第七節、質譜儀分析(MALDI-TOF) ...............18 第八節、西方墨點法........................19 第九節、細胞 RNA 之抽取............................19 第十節、反轉錄聚合酶連鎖反應...........................20 第十一節、DNA 片段回收純化及 DNA 黏接反應.........21 第十二節、質體 DNA 之抽取..........................22 第十三節、勝任細胞之製備.............................22 第十四節、轉型作用.....................23 第十五節、菌種甘油保存法...................23 第十六節、大量抽取質體 DNA.....................23 第十七節、哺乳動物細胞蛋白質交互作用分析.............24 第十八節、癌細胞侵襲能力之分析....................26 第十九節、細胞增生分析...........................27 第二十節、細胞同步化(Cell Synchronization)及流式細胞分析技術(Flow cytometery) ..........28 第二十一節、GST 沉澱法........................29 第二十二節、免疫螢光染色(Immunofluorescence) ........31 第二十三節、冷光報導基因分析(Luciferase reporter gene assay) .....31 第三章 實驗結果..........................33 第一節、免疫沉澱分析與 HLJ1 結合之蛋白質群....33 第二節、探討 HLJ1 與 p54nrb 之間結合作用....33 第三節、探討 HLJ1 與 p54nrb 之間結合作用在哺乳動物細胞內是否也會形成蛋白複合物....34 第四節、分析 HLJ1 與 p54nrb 之間結合情形....34 第五節、即時定量聚合酶連鎖反應(Real-time quantitative PCR)分析肺腺癌細胞株 CL1-0、CL1-5 之 p54nrb 之 RNA 表現量..35 第六節、分析 HLJ1、p54nrb 在 A549、CL1-0、CL1-5 及 CL1-5F4 四株肺癌細胞株疏密不同情況下的表現量...35 第七節、HLJ1、p54nrb 在 CL1-0、CL1-5 細胞之核內/核外的表現量,及 p54nrb 在細胞的分布情況...35 第八節、將 p54nrb 大量表現在 CL1-0 和 CL1-5 細胞株中..36 第九節、以 MTT 分析大量表現 p54nrb 對於 CL1-0 和 CL1-5 細胞株生長之影響..36 第十節、大量表現 p54nrb 對於 CL1-0 和 CL1-5 細胞株侵襲之影響..37 第十一節、大量表現 p54nrb 對於 CL1-5 細胞株移動能力之影響37 第十二節、大量表現 p54nrb 對於 CL1-5 細胞株聚落形成之影響 (Colony formation assay)..38 第十三節、將細胞同步化(Cell Synchronization)後,收取不同時間點觀察 p54nrb 與 HLJ1 表現的差異..39 第十四節、將大量表現 p54nrb 的 CL1-5 細胞同步化(Cell Synchronization)後,收取不同時間點觀察細胞週期的變化..40 第十五節、暫時轉染 HLJ1 於大量表現 p54nrb 的 CL1-5 單株細胞觀察其侵入能力之影響..40 第十六節、增幅 p54nrb 基因六個不同的片段,並構築至 pGEX-4T1,以 GST 沉澱法分析 p54nrb 主要與 HLJ1 結合區域..40 第十七節、暫時轉染 p54nrb 的區域片段於 CL1-0 及 CL1-5 細胞株觀察其侵入能力之影響..41 第十八節、冷光報導基因分析(Luciferase reporter gene assay)當大量表現 p54nrb 對於 E-cadherin 及 Slug 啟動子活性之影響..42 第四章 討論....43 第五章 結論.........49 參考文獻..................50 實驗結果表圖...57 附表與附圖..83 附錄..95zh_TW
dc.title研究腫瘤抑制蛋白 HLJ1 與 p54nrb 蛋白形成複合物對人類肺腺癌細胞遷移及侵入之影響zh_TW
dc.titleStudy of protein complex formation between a novel tumor suppressor HLJ1 and nuclear protein p54nrb on human lung adenocarcinoma cell migration and invasionen_US
dc.typeThesis and Dissertationzh_TW
item.openairetypeThesis and Dissertation-
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