Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20122
標題: 視神經萎縮蛋白1在肺癌之表現與其生物意義
The expression and biological significance of optic atrophy 1 in lung cancer.
作者: 王俞婷
Wang, Yu-Ting
關鍵字: optic atrophy 1;視神經萎縮蛋白1
出版社: 生物醫學研究所
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摘要: 
Opa1, a dynamin related protein, participates in mitochondrial fusion and plays a role in mitochondria-dependent apoptosis. Although Opa1 has been studied in ADOA, correlation between Opa1 and lung cancer has not been well studied. First, we show that Opa1 mRNA is expressed in every lung cancer cell lines. To further study the role of Opa1 in lung cancer, we generated monoclonal antibodies to recognize Opa1 protein and analyze its expression in lung cancer. The results showed that Opa1 protein is expressed in lung, breast and cervical cancer cell lines. Opa1 is highly expressed in tumor sections of lung cancer patients, especially in the early stage, demonstrating that Opa1 might play a role in lung carcinogenesis, particularly in the early stage of lung cancer. Results of immunostain showed that Opa1 was expressed in tumor nest of lung cancer specimens and in the cytoplasm of lung cancer cell lines. Subcellular fractionations showed that Opa1 was indeed located in mitochondria. Immunoblotting showed that Opa1 recognized low molecular weight proteins in mouse organs, indicating the tissue specificity or the degradation of Opa1 protein. Low serum induced apoptosis and influenced Opa1 protein level. By double thymidine block and release, levels of Opa1 protein increase during S phase and decrease at M phase, suggesting that Opa1 expression is correlated with cell cycle progression. Unexpectedly, isoelectric point in a two dimensional gel electrophoresis implicated that Opa1 might bind to acidic proteins or became phosphorylated. Opa1 protein levels decreased by treatment with Calphostin C, a PKC inhibitor, demonstrating that Opa1 was phosphorylated by Protein kinase C. The survival rate decreases in Opa1 knockdown lung cancer cells after Cisplatin treatment. Opa1 may induce drug resistance in cancer cells. These data show that Opa1 may be a potential target for lung cancer therapy in the future. Studying the molecular mechanism and physiological significance of Opa1 may help us realize the correlation in carcinogenesis.

Opa1蛋白為Dynamin家族成員之一,是參與粒線體融合的重要蛋白;此外也與粒線體依賴的細胞凋亡機制有關,目前廣為研究Opa1基因的缺陷會造成體染色體顯性遺傳疾病,但並沒有研究探討其蛋白與肺癌之間是否有相關性。首先我們偵測Opa1於mRNA層面上與肺癌細胞株及肺癌患者檢體之表現,結果顯示Opa1 mRNA於各肺癌細胞株都有表現。之後自製Opa1單株抗體並檢測肺癌、乳癌與子宮頸癌細胞株,發現Opa1蛋白的確有高度表現的情形;於肺癌患者組織檢體也觀察到有六成以上的Opa1蛋白有表現;而於各肺癌分期的患者檢體中更能發現Opa1蛋白於Ia與Ib的表現量高達八成以上,顯示Opa1蛋白可能於肺癌早期之生成有關連。而使用組織與細胞化學免疫染色法發現Opa1於肺癌患者組織檢體都表現於癌化組織中,而於各肺癌細胞也都有表現的情況,更發現其分布位置主要位於細胞質;使用亞細胞分離方式觀察於細胞內分布情況,結果顯示Opa1的確分布於粒線體。而於各小鼠器官組織表現會發現有辨認到低分子量的情況,推測或許有組織特異性或降解可能性。隨著血清飢餓時間增加,Opa1低分子量蛋白表現有累積提升的現象,反之高分子量蛋白則減少表現,顯示細胞飢餓導致凋亡而造成Opa1蛋白表現之差異;於雙胸腺嘧啶阻斷實驗更發現Opa1蛋白與細胞週期進程有關,於S期表現量為最高,M期有下降的趨勢。使用二維電泳分析Opa1等電點發現其酸鹼性與軟體預測不同,推測可能與較酸蛋白結合或有磷酸化修飾而造成其等電點下降;最後使用各種絲胺酸、蘇胺酸抑制劑發現Protein kinase C抑制劑能有效抑制Opa1蛋白表現,顯示Opa1蛋白與磷酸化有關且受PKC嚴格調控。使用抗癌藥物Cisplatin作用發現Opa1蛋白受抑制的癌細胞存活率明顯下降,顯示Opa1之高度表現有提升癌細胞抗藥性之作用。由以上結果顯示,Opa1有潛力成為治療肺癌的標靶,更深入研究其分子機制與生理意義有助於了解其與細胞癌化間的關係。
URI: http://hdl.handle.net/11455/20122
其他識別: U0005-0102201015225300
Appears in Collections:生物醫學研究所

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