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標題: 過量表現 gelsolin 對 MDA-MB-231人類乳癌細胞內肌動蛋白絲重組和細胞增生的影響
Effects of gelsolin overexpression on actin filament reorganization and cell proliferation in MDA-MB-231 human breast cancer cells
作者: 邱冠穎
Chiu, Kuan-Ying
關鍵字: breast cacner
actin binding proteins gelsolin
cell proliferation
肌動蛋白結合蛋白 gelsolin
出版社: 生命科學系所
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摘要: Gelsolin (GSN)是肌動蛋白結合蛋白(ABP)的成員之一,主要藉由切割和鍵結來調控肌動蛋白絲動態的變化。近年來許多研究都在探討肌動蛋白絲動態的變化與肌動蛋白結合蛋白在惡性腫瘤中所扮演的角色。研究指出:GSN在腫瘤細胞中為促進因子或是抑制因子,可能與腫瘤的階段與轉移的能力有關;然而GSN對於腫瘤細胞增生的影響仍然不是很明確。因此本論文利用過量表現GSN的人類乳癌細胞株MDA-MB-231做為模式,探討GSN在乳癌細胞中對肌動蛋白絲重組以及細胞生長的影響。首先,將帶有GSN全長cDNA 的質體DNA -pcDNA6-GSN轉染進入MDA-MB-231中,利用西方轉漬法和即時定量聚合酶連鎖反應檢測細胞內GSN的表現量;並挑選出穩定過量表現GSN的細胞株。觀察GSN過量表現細胞株的細胞型態,再利用細胞計數的方式計算細胞的細胞倍增生長時間 (PDT),以及螢光顯微鏡術觀察細胞內肌動蛋白絲的分子結構;發現:GSN過量表現會使細胞型態變的較寬較長,生長不容易聚集,並且會減緩細胞生長的速度,還會使胞內的肌動蛋白絲進行去聚合反應。進一步探討GSN過表現抑制細胞生長的原因;實驗中使用螢光光譜儀檢測fura-2並計算胞內鈣離子濃度、使用q-PCR檢測脂質相關基因:脂肪酸生成酶 (FAS) 和過氧化體增生因子 (PPAR-γ 2) 的表現量、使用原子力顯微鏡(AFM)偵測細胞表面的黏著力,最後使用流式細胞儀檢測細胞週期。實驗結果發現:GSN過量表現細胞株,胞內鈣離子濃度提高了56%,FAS的表現量增加,但是降低了PPAR-γ2的表現量,細胞表面的黏附力提升了24%,並且增加細胞處於G1時期的比例,這些現象可能都與肌動蛋白絲分子結構發生改變有關。另外,GSN過量表現細胞株會增加Tm1的表現;利用siRNA 靜默GSN 過量表現細胞株中Tm1,並且使用trypsin蛋白酶 處理檢測細胞貼附力的實驗中,發現降低Tm1 的表現會增加細胞對胞外的貼附力,並且可能與GSN有加成的作用。綜合以上結果:GSN過量表現影響細胞生長可能是經由 (1) 肌動蛋白絲進行去聚合反應直接或間接的使胞內鈣離子濃度提高,影響細胞內鈣離子訊息。(2)改變脂質相關基因表現,影響脂質代謝,部分干擾細胞生長。(3) 肌動蛋白絲t進行去聚合反應,會經由β-catenin/cyclin D1 的訊息路徑,使細胞停滯於G1 階段,來影響細胞週期。另外,(4) GSN過量表現會增加 Tm1 的表現量;藉由增加兩者基因的表現量,共同影響細胞對胞外環境的貼附力,進而抑制細胞生長。
Gelsolin (GSN) is one of actin binding proteins (ABPs) that regulate dynamic actin filament organization by severing and capping. In recent years, many studies have devoted to establish the role of dynamic actin filament formation by ABPs in controlling the proliferation of malignant tumors. These studies showed that GSN might act as a tumor activator or as a tumor suppressor, and that is related to tumor stage and its ability of metastasis. Apparently, the effect of GSN on tumor cell proliferation is still not clear yet. In this study, GSN-overexpressed MDA-MB-231 human breast cancer cell line was utilized as a model to examine the effect of GSN overexpression on actin filament reorganization and cell proliferation in breast cancer cells. Firstly, a pcDNA6-GSN that contains full-length GSN cDNA was transfected into MDA-MB-231, and then the stable GSN overexpression cell lines of MDA-MB-231 confirmed by real time q-PCR and western bolt were cloned subsequently. Trypan blue exclusion assay was used to estimate the population doubling time (PDT) for cell proliferation of GSN-overexpressed MDA-MB-231, and then the cell morphology and intracellular actin filament formation were observed by the phase microscopy and the epi-fluorescent microscopy, respectively. The results showed, GSN overexpression would inhibit cell proliferation and make cell morphology wider as well as longer, and reduce cell aggregation, and promote actin filament depolymerization. A futher study was performed to investigate how GSN-overexpression could inhibit cell proliferation. The methods and results were as follows: (1) Measuring fura-2 F340/F380 fluorescence ratio in the cells indicated 1.56 folds of increases in Ca2+ levels for GSN-overexpressed cells. (2) Detecting lipid-related genes by q-PCR showed GSN-overexpression facilitates the fatty acid synthase (FAS) but attenuates the peroxisome proliferator-activated receptor-γ2 (PPAR-γ2). (3) Detecting by atomic force microscopy (AFM) indicated GSN-overexpression increased cell surface adhesion force. (4) Detecting cell cycle by cytometry showed GSN-overexpression cell increased percentage of the cell population at G1 phase. In addition, GSN-overexpressed cells will cause Tropomyosin-1 (Tm1) gene up-regulation. Using siRNA to silence Tm1 and detecting cell adhesion force showed that silenced Tm1 gene increased the cell adhesion to the extracellular matrix. Taken together, results obtained in this study suggested that GSN overexpression might affect cell proliferation through (1) actin filament depolymerization, directly or indirectly increased intracellular calcium concentration, and then affecting intracellular calcium signal; (2) changing the lipid-related gene expression to affect lipid metabolism; (3) modulating actin filament polymerization/ depolymerization to arrest cells in G1 phase by inhibiting the β-catenin/cyclin D1 signal pathway; (4) upregulating Tm1 gene that might affect cell adhesion to the extracellular force, inhibited cell growth.
其他識別: U0005-1108201114485800
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