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標題: 小眼畸形相關轉錄因子(MITF)抑制肺癌細胞侵襲及腫瘤形成能力之探討
Expression of Microphthalamia-associated Transcription Factor (MITF) Suppresses Lung Cancer Cell Invasion and Tumorigenicity
作者: 邱素琴
Chiu, Su-Chin
關鍵字: MITF;小眼畸形相關轉錄因子;siRNA;lung cancer;metastasis;invasion;microarray;Real-time RT-PCR;In vivo tumorigenicity;RNA干擾;肺癌;癌轉移;侵襲能力;微陣列晶片;即時定量RT-PCR;In vivo腫瘤生成能力
出版社: 生命科學院碩士在職專班
近二十年來,肺癌是全世界癌症死亡率增加最快的疾病。在台灣,肺癌的平均死亡人數也直逼肝癌,在女性人口則為第一位,而且死亡率持續竄升。癌細胞轉移(metastasis)是癌症病患的主要死因,已被廣泛的研究多年。在過去幾十年,有許多參與腫瘤細胞侵入及轉移作用的基因或蛋白質被驗証及定性。然而,這些分子並非獨一且專一的存在於癌轉移細胞中,在大部分的例子,也存在於正常細胞中。而微陣列(microarray)為近年發展可大量平行分析基因表現的有力工具,亦已應用於許多差異表現基因的搜尋及臨床疾病的分類研究上。先前實驗中,利用cDNA微陣列研究具不同侵入能力(invasion)的肺癌模式細胞株(侵入能力:CL1-0 本論文著重於進一步探討MITF在肺癌生成及轉移過程中可能扮演的角色,首先藉由即時定量RT-PCR(Real-time RT-PCR)分析62個非小細胞肺癌組織檢體中MITF表現量與其臨床診斷結果的相關性。結果發現,MITF表現高的病人有較高的存活率(p值為0.008),且肺癌組織中MITF表現量較高的病患亦較慢復發(即治療後未發現腫瘤的時間,或稱disease free,p值為0.0244)。利用RNA干擾(RNA interference)技術抑制侵襲能力低的CL1-0細胞中MITF的表現,結果發現MITF表現量少的CL1-0,會促使細胞增加侵襲及轉移能力;此外將MITF表現受抑制的轉染細胞株進行In vivo腫瘤生成能力(In vivo tumorigenicity)實驗,結果發現免疫不全鼠(SCID mice)體內腫瘤體積相較對照組癌細胞(scramble 對照組)為大。為探討MITF在肺癌轉移及侵入過程中可能調控的機制,將MITF基因表現持續受抑制的CL1-0細胞株進行Affymetrix基因晶片(Affymetrix HG-U133 Plus 2)分析,並針對實驗結果以即時定量RT-PCR確認。
藉由晶片分析結果顯示,MITF可能與血管新生(angiogenesis)、細胞訊號傳遞(signal transduction)、細胞遷移(cell migration)及轉移相關之基因調控有關,此研究結果顯示降低MITF的表現可增加癌細胞侵襲能力及腫瘤生成的作用。

Lung cancer has increased the most in mortality among all cancers worldwide in recent twenty years. In Taiwan, the average death of lung cancer every year is catching up those of liver cancer, and lung cancer takes the first place of death in female population and keeps on aggravating. Metastasis is the leading cause of death among patients with cancers and has been studied extensively for years. Many genes or proteins involved in invasion and metastasis have been identified and characterized in past decades; however, most of the molecules don't exist in metastatic cancer cells exclusively, in most cases, they also exist in normal cells. Microarray is a powerful tool to analyze gene expressions parallel and in large quantity, and has been utilized in search the differences of gene expressions as well as classification of diseases.
In earlier studies, studying a series of lung adenocarcinoma cell lines with varying degrees of invasiveness (invasiveness: CL 1-0 In this thesis, we stress on exploring the possible role of MITF in tumorigenesis and metastasis further. We measured MITF mRNA expression by real time PCR in 62 non-small cell lung cancer surgical specimens, and correlated with the patient's clinical outcome. The result showed that the expression of MITF negatively correlated with survival and disease free of lung cancer patients (p=0.008 and 0.024, respectively). We investigated the effect of MITF on the invasion and migration of the lung adenocarcinoma cell line (CL1-0) with less invasive capacity by RNA silencing technology, which revealed that the reduction of MITF promoted tumor cell migration and invasion. In addition, the tumorigenesis analysis in SCID mice showed that the tumor volume derived from the transfectants of MITF-specific siRNA was larger than the scramble controls. To further elucidate the potential mechanisms by which MITF contributes to tumor invasion and tumorigenesis, Affymetrix genechips were used to profile the alterations of gene expression after silencing MITF. Quantitative real-time RT-PCR confirmed the expressions of certain of the targeted genes. The microarray data demonstrated that MITF regulated genes involving in angiogenesis, cell proliferation, signal transduction cell migration and metastasis. Our studies herein provided a new insight into how MITF may contribute to tumor invasion and tumorigenesis.
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