微小 RNA16 抑制肺癌細胞增生之研究 = Micro RNA16 inhibit lung cancer cell proliferation
石文昭、蔡孟
E-mail: [email protected]
摘 要
microRNAs是一群小片段的RNA，它的長度約18~24個核?˙艦B不會被轉譯為蛋白質。生物體的運作伴隨而來的是一連串 的調控作用，而microRNA更是這其中的要角，像是胚胎的發育、細胞分化、細胞周期的調控等等都可以發現microRNA參 與其中。腫瘤的形成就是因為細胞在生物體中並沒有受到正常的調控，所以才會無限制的增生或是轉移；而近年來更陸續 的發現乳癌、直腸癌、前列腺癌及神經膠母細胞瘤中都可以發現到microRNA的異常表現，但是在肺癌與microRNA之間的 研究文獻則較少。因此本研究要分析與肺癌相關之microRNA，其中包含了miR-16、miR-24、miR-494等等不同
的microRNA；進一步分析不同肺癌細胞株的結果顯示，若與正常支氣管上皮細胞相比，miR-16在肺癌細胞中的表現量往 往都較正常細胞來的低。我們分別在CL1-5肺腺癌細胞中建立了兩個miR-16表現系統：pSilencerTM3.1-miR16
與pcDNATM6.2-miR16。透過已建立完成的表現系統分析，我們發現miR-16大量表現的細胞株pSilencer3.1-miR16-A7
、A19與pcDNA6.2-miR16-G16、G17細胞的生長速率會受到抑制達50%；但是對肺腺癌細胞的移動(migration)以及侵 入(invasion)能力則不會有明顯的改變。接著透過二維電泳(2-D electrophoresis)的分析以及電腦軟體的預測，我們想找出在肺 癌細胞中受到miR-16調控的基因；目前為止我們利用上述兩種方法找到了兩個可能的目標基因，分別是NYGGF4
和ALDOA 。研究microRNA與癌症將有助於我們瞭解癌症的發生機制及癌症的治療，甚至是作為一個分子標記與預防癌症 的再復發。
關鍵詞 : 微小RNA ; 微小RNA16 ; 肺癌 ; 癌細胞侵入 ; 癌轉移 目錄
目錄 封面內頁 簽名頁 授權書...…iii 中文摘要………
………...iv 英文摘要………v 誌謝…
…...vi 目錄………
….viii 圖目錄………...xi 表目錄………
………xii 1. 前言………...1 1.1 癌症………
……….. …....1 1.2肺癌之分類與臨床症狀……….…...2 1.3 致癌基因與腫瘤抑制基因………
…………..…...3 1.4 小片段RNA之起源……….4 1.5 MicroRNA及其作用機制………
…………...6 1.6 MicroRNA與癌症之間的探討………...7 2. 研究動機………
………....10 3. 材料方法………....11 3.1 細胞株………
………...11 3.2 細胞培養………..…....11 3.2.1 培養液的準備………
…………....11 3.2.2 細胞培養之生長條件………....12 3.3 RNA之萃取………
……...12 3.4 cDNA的製備……….……13 3.5 即時定量PCR………
……...14 3.6 小量質體萃取………...14 3.7 hsa-miR-16表現載體之構築………
………...15 3.7.1 hsa-miR-16的片段取得………..15 3.7.2質體與miR-16片段之酵素切與黏合…………....16 3.7.3 限制酵素切處理後DNA之純化………...18 3.7.4 大量生產表現載體………..19 3.8 CL1-5細 胞轉染miR-16表現載體………..19 3.9 在CL1-5細胞中建立hsa-miR-16穩定表現系統……….20 3.10細胞生長速 率測試……….21 3.11細胞群落形成分析……….21 3.12細胞基質侵 襲力分析……….22 3.13傷口癒合分析……….22 3.14 GIEMSA染 色……….23 3.15二維電泳之蛋白質分析………..23 3.15.1蛋白質樣 品的備製……….23 3.15.2等電點電泳分離……….24 3.15.3聚丙烯胺膠體分析…
……….25 3.15.4銀染……….26 4. 結果與討論………
……….28 4.1肺癌相關microRNA之篩選…….………....28 4.2 在CL1-5肺腺癌細胞株 中miR-16大量表現系統的建立與 細胞功能之探討...30 4.3 miR-16目標基因之鑑定……
………...35 5. 結論………...38 參考文獻………
………57 附錄……….….61
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