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Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21764
標題: 腫瘤抑制蛋白HLJ1與beta-肌動蛋白形成複合物對人類肺腺癌細胞遷移及侵入能力之探討
Study of protein complex formation between a novel tumor suppressor HLJ1 and beta-actin on human lung adenocarcinoma migration and invasion
作者: 戴廷浩
Dai, Ting-Haw
關鍵字: 熱休克蛋白;heat shock protein;肌動蛋白;actin
出版社: 分子生物學研究所
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摘要: 
Lung cancer has become the most common cause of cancer deaths in the world, and it gets worse each year. In previous studies, a novel tumor suppressor gene, HLJ-1, was found by utilizing microarray technology. In invasion/metastasis cell line model CL1-0, CL 1-1, CL 1-5, CL 1-5 F4 (invade ability and increase in order), HLJ-1 expression was associated with repressing lung cancer cell metastasis and invasion. HLJ1 (human liver DnaJ-like protein, also called DNAJB4), a member of Hsp 40 family, mainly participates in protein folding and assists cell against stress. When overexpressing HLJ1 protein, it reduces lung cancer cell abilities of proliferation, colony formation, invasion and migration. However, it is not yet clarified that how HLJ-1 suppresses lung cancer cell metastasis and invasion. Moreover, protein-protein interactions make further meaningful phenomena or situation in cell. Thus, we proposed HLJ-1 suppresses several lung cancer cell abilities through interacting with other proteins. We discovered HLJ1 interacts with heat shock protein 70 (HSC70), and beta-actin (ACTB) by immunoprecipitation and MALDI-TOF analysis in different cell lines. Furthermore, the complex of three components was also confirmed by co-immunoprecipitation and was found getting more in CL1-0 cell. ACTB is not only cell''s skeleton protein, but also control cell movement. By polymerizing ACTB or not, the cancer cell can stretch out lamellipodium and filopodium to make metastasis; when the cancer cell invades the basis membrane, reorganization of ACTB is necessary to modulate cell''s shape. So we wondered the complex of HLJ-1 and ACTB plays which role in lung cancer cell regarding metastasis and invasion. In immunofluorescent and immunoprecipitation experiments, we found HLJ1/HSC70/ACTB complex mainly in the cytoplasm, but little in the nucleus. In addition, we utilized mammal two hybrid assay and identified HLJ1/HSC70/ACTB complex in the cell. Meanwhile, it was also identified that HLJ-1 interacts with the C terminal of ACTB through J domain. When overexpressing J domain of HLJ1 to compete the interaction of ACTB, we found cancer cell invasion increases; when overexpressing C terminal of ACTB to compete interaction of HLJ1, we found cancer cell invasion also increases. Furthermore, with transfecting EYFP-ACTB plasmid, we analyzed ACTB polymerization affected by HLJ1 or not. We found ACTB polymerization increases in HLJ-1 silenced cell line. In this research, we found that HLJ1 forms a complex with HSC70, ACTB and mainly in cytoplasm. HLJ1 interacts with ACTB through J domain to influence ACTB polymerization, and suppress the cancer cell invasion ability. Follow-up we will further confirm how HLJ1 regulate ACTB polymerization, and make use of the research we can offer a regulate and control metastasis and invasion mechanism of cancer cell; perhaps this can apply on the treatment of cancer in future.

肺癌為國人癌症十大死因之一，且有逐年增加之趨勢。在本實驗室先前的研究中，利用肺腺癌CL1-0、CL 1-1、CL 1-5、CL 1-5F4 (侵入能力依序增加)作為模式細胞株，並利用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新基因HLJ1 (human liver DnaJ-like protein,又稱DNAJB4)。HLJ1屬於熱休克蛋白40家族中的一員，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(proliferation)、聚落形成(colony formation)、侵入(in vitro invasion)和移動(in vitro migration)能力。HLJ1是透過何種機制來抑制癌細胞轉移、侵入能力，目前尚不清楚。然而，許多的蛋白是透過與其他蛋白交互作用來執行其功能。因此，本實驗藉由偵測與HLJ1結合之蛋白，來探討HLJ1所形成之複合物對肺腺癌細胞轉移及侵入有何影響。我們利用不同轉移能力的細胞株進行免疫沉澱並以質譜技術分析蛋白質可能身分，結果顯示HLJ1與HSC70、ACTB具有交互作用，且在CL1-0細胞株中有較高的表現量。以共免疫沉澱進行交叉實驗也確認HLJ1、HSC70與ACTB形成一複合物。ACTB不但為細胞骨架蛋白，也是細胞移動的重要蛋白。藉由ACTB的聚合重組，癌細胞可伸出絲狀偽足及片狀偽足增加癌細胞的轉移能力；當癌細胞進行變形運動侵入基底膜時，也須ACTB的重組使細胞形態改變。因此我們進一步探討HLJ1與ACTB結合在肺腺癌細胞轉移及侵入中扮演何種角色。以免疫螢光染色發現HLJ1/HSC70/ACTB複合物主要存在於細胞質，少部份會進入細胞核內。而分離核內/外蛋白並進行免疫沉澱，也證實此複合物確實在細胞質較多。另外，我們利用哺乳動物細胞蛋白交互作用也證明在細胞內HLJ1/HSC70/ACTB複合物確實存在，且HLJ1會透過J domain與ACTB的C端結合。進一步大量表現HLJ1的J domain來競爭HLJ1與ACTB的結合區域，發現癌細胞的侵入能力會增加；當大量表現ACTB的C端來競爭ACTB與HLJ1的結合區時，癌細胞的侵入能力也會增加。另外，我們轉染構築EYFP-ACTB的質體並以免疫沉澱法分析HLJ1對ACTB聚合的影響。由結果發現靜默HLJ1的細胞株，其ACTB的聚合會增加。總結而言，HLJ1會與HSC70、ACTB形成一複合物且主要位於細胞質。HLJ1可能透過J domain與ACTB結合，而影響ACTB的聚合，進而抑制肺腺癌細胞的侵入能力。後續我們將進一步探討HLJ1如何調控ACTB聚合的機制。藉由此研究我們可提供一個調控癌轉移及侵入的機制，或許在未來可應用於癌症的治療上。
URI: http://hdl.handle.net/11455/21764
其他識別: U0005-2508200620280200
Appears in Collections:分子生物學研究所

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