- 學位論文
大量表現微小核醣核酸miR10-b及miR-20b可促進人類大腸癌COLO320DM細胞的非貼附依存性生長及侵入作用
Overexpression of miR-10b and miR-20b promotes anchorage-independent growth and invasion in human colon cancer COLO320DM cells
摘要
研究背景：本研究的目的在於釐清大腸癌細胞株COLO320DM不同種的細胞特性，貼覆性及漂浮性的細胞其miRNA表現的差異性與腫瘤的特性是否具關連性。 材料與方法：本研究所使用的細胞株為人類大腸癌細胞COLO320DM，在初步的觀察中發現，當細胞的miR-10b及miR-20b大量表現的時候，人類大腸癌細胞COLO320DM會呈現懸浮型以及貼附型兩種形態，因此針對細胞生長、非貼附依存性生長、細胞貼附能力、轉移及侵入能力、化療藥物5-FU的感受性、癌症幹細胞標幟分子、訊息傳導路徑分子表現、細胞內ROS表現、抗氧化酵素表現和抗氧化酵素活性等細胞癌症特性相關實驗釐清COLO320DM細胞不同種細胞型態的差異性。 研究結果：本論文之研究結果發現有大量miR-10b以及miR-20b的表現的人類大腸癌COLO320DM-S細胞，是以懸浮狀態生長在培養液中。而miR-10b及miR-20b表現量少的COLO320DM-A細胞是貼附在培養盤中生長的細胞。此miRNA的差異可能對COLO320DM這株人類大腸癌細胞的癌化特性有著特別的意義。在細胞正常生長的環境中，懸浮性COLO320DM-S細胞之生長速率和貼附性COLO320DM-A細胞沒有統計上的差異，而懸浮性COLO320DM-S細胞之生長速率還是略快於貼附性COLO320DM-A細胞。但是在貼附依存性生長的Soft agar實驗中卻有明顯差異，懸浮性COLO320DM-S細胞生長速率遠大於貼附性COLO320DM-A細胞，且具極顯著性差異。觀察此兩種細胞與細胞外基質分子Collagen、Fibronectin、 Laminin、Vitronectin貼附的情形，意外的發現懸浮性COLO320DM-S細胞與Collagen、Fibronectin、 Laminin、Vitronectin的結合能力大於貼附性COLO320DM-A細胞。且懸浮性COLO320DM-S細胞在細胞轉移或細胞侵入的能力都優於貼附性COLO320DM-A細胞。對於化學治療藥物5-FU的藥物敏感性，懸浮性COLO320DM-S細胞對於5-FU的敏感性也相對低於貼附性COLO320DM-A細胞。在懸浮性COLO320DM-S細胞內具活性的p-AKTS473和p-AKTT308的表現量也高於貼附性COLO320DM-A細胞。但在貼附性生長和懸浮性生長的COLO320DM兩群細胞中大腸癌幹細胞的含量都低於1%，兩者間差異不大。而過氧化氫和氫氧根離子為主的ROS在懸浮性COLO320DM-S細胞內表現量比較多;而抗氧化酵素的表現上Catalase和Glutathione peroxidase1/2在貼附性COLO320DM-A細胞中表現量比較多，且Glutathione peroxidase的活性在貼附性COLO320DM-A細胞也高於懸浮性COLO320DM-S細胞。但貼附性COLO320DM-A細胞中Glutathione的含量低於懸浮性COLO320DM-S細胞。 結論：實驗結果顯示，在大腸癌的細胞中miR-10b及miR-20b是一個具有致癌特性的oncomir。當miR-10b及miR-20b大量表現時，可能會增加大腸癌細胞的生長、轉移、侵入及抗藥性等能力。但miR-10b及miR-20b在大腸癌中確實扮演的角色及它的生物功能，甚至於它下游的標靶基因都需要更多的研究去確認。本論文之研究尚有許多不足之處，如以miR-10b或miR-20b的inhibitor去Knockdown其表現，看其是否會降低細胞之癌化特性，或以動物實驗證明miR-10b及miR-20b表現高的細胞，其腫瘤確實生長的比較快而大。這些都是後續應執行的實驗。
並列摘要
Objective：The objective of this study is to understand the relation of different subtype (adhesion and suspension) in colon cancer cell line, COLO320DM between the miR express that might change cancer property. Methods and Materials：In primary data, we find when miR-10b and miR-20b overexpression COLO320DM will grow in suspension type. Then, we design experiences, including cell growth of COLO320 DM, anchorage-dependent growth of colon cancer cell lines, cell adhesive ability to ECM, the difference of migratory and invasive activity, drug sensitivity of chemotherapy drug 5-FU, expression of cancer stem cell markers, the expressed levels of AKT, GSK3β and ERK, the levels of intracellular ROS, the expressed levels of antioxidative enzymes, antioxidative enzyme activity to prove whether cell malignant property change. Results：The results shows COLO320DM cell grows in suspended type while miR-10b and miR-20b overexpress. And COLO320DM cell grow in adhesive type while miR-10b and miR-20b express low. In normal culture environment, suspended COLO320DM cell growth rate is faster than adhesive COLO320DM cell. In anchorage-independent surrounding, suspended COLO320DM cell growth rate is faster than adhesive COLO320DM cell. And cell adhesive ability of ECM molecules, suspended COLO320DM cell is stronger than adhesive COLO320DM cell. The resistant ability of chemotherapy drug, 5-FU suspended COLO320DM cell is higher than adhesive COLO320DM cell. The expression of p-AKTS473 and p-AKTT308 is suspended COLO320DM cell is higher than adhesive COLO320DM cell. The cancer stem cell containing have on difference between this two cell types. And expression of antioxidant enzyme are that catalase and glutathione peroxidase expression are adhesive COLO320DM cell higher than suspended COLO320DM cell. The activation of glutathione peroxidase is adhesive COLO320DM cell higher than suspended COLO320DM cell. The glutathione containing is suspended COLO320DM cell is higher than adhesive COLO320DM cell. Conclusion：In colon cancer, miR-10b and miR-20b are oncomir. When miR-10b and miR-20b overexpresses, malignancy (cell growth rate, migratory ability, invasive ability, resistant of chemotherapy drug 5-FU) of colon cancer will increase. But the real function and the mechanism of downstream molecules needs more study to solve. And our study is not using inhibitor to knockdown expression of miR-10b and miR-20b. The other is not using animal model to know growth rate in xenograft.
參考文獻
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