MicroRNAs （miRNAs）是一22個核苷酸、不會編碼出蛋白質的單股RNA，具有負向調控基因表現功能。miRNAs調控基因表現的機制與RNAi類似，成熟的miRNAs為一小片段的單股RNA，藉由結合至RISC複合體可以去辨認與其序列互補的標的mRNA，依據與標的mRNA完全互補與否，而有將標的mRNA降解或阻擋蛋白質轉譯的二種機制。近期很多報導指出miRNAs參與了腫瘤形成的過程，由於miRNAs調控的基因包含了細胞生長、爬行能力、入侵能力等，因此，當miRNAs表現失衡時將會大大地影響細胞的行為，甚至導致腫瘤形成的發生。很多研究報告指出特定的miRNAs在特定的腫瘤細胞中扮演重要角色，例如：miR-221與miR-222會負向調控p27Kip1而促進PC3前列腺癌細胞增生，因此，如果將miR-221與miR-222的表現抑制掉或是降低，就可以造成PC3細胞的增生減緩。基於這個想法，如果能夠將miRNAs生合成抑制，就可以影響受miRNAs高度調控的腫瘤細胞，有機會能夠作為一種腫瘤治療的方式，並且抑制miRNAs生合成是非專一性地影響miRNAs熟成，將有助於應用於多種類的腫瘤細胞。首先，建立持續沈默Drosha表現的HeLa細胞株用以觀察影響HeLa細胞中miRNAs生合成路徑對其造成的影響，實驗結果發現在HeLa細胞中持續降低其miRNAs生合成並不會造成其顯著改變，包含了細胞生長、細胞爬行能力、侵襲能力以及F-actin形成。之後選擇建立持續沈默Drosha表現的PC3前列腺癌細胞株，實驗結果顯示持續降低PC3細胞的miRNAs生合成活性會導致細胞生長減緩、細胞爬行及侵入能力顯著降低。隨後設計miRNAs生合成中的另外二個關鍵酵素的shRNA，為shDGCR8以及shXPO5。將miRNAs生合成過程中三個關鍵酵素的shRNA串連後轉染HeLa細胞，實驗結果顯示同時抑制這三個miRNAs生合成關鍵酵素不會造成HeLa細胞的死亡或巨大影響。在另一部份的研究主題中，冷光基因融合載體的分析方式可以很快速地且大量地篩選持續沈默特定基因的細胞株，在篩選持續沈默Drosha表現的PC3細胞、持續沈默HDM2的PC3細胞以及持續沈默IDH3A的HeLa細胞的實驗中，都印證了利用冷光基因的表現情形即可挑選出持續沈默特定基因的細胞株，經由西方墨點法分析也都確認了挑選出來的細胞株有持續沈默特定基因。這個實驗結果將有助於快速地篩選大量的細胞株，兼具省時以及省錢的優點。

MicroRNAs (miRNAs) are an abundant class of small non-protein-coding RNAs that function as negative gene regulators. MiRNAs negatively regulate their target mRNA in one of two ways depending on the degree of complementarity between the miRNAs and their targets. The researchers are just beginning to understand how this novel class of gene regulators is involved in cancer-related-process in humans. Cancer is caused by uncontrolled proliferation and the inappropriate survival of damaged cells, which results in tumor formation. Out of hundreds of miRNAs that have recently been identified, only a small number of human genomes have been characterized. They have been shown to control cell growth, differentiation and apoptosis; consequently, impaired miRNAs expression has been implicated in tumorigenesis. Recently evidence indicates that patterns of misexpression of miRNAs in cancer suggest key functions of miRNAs in tumorigenesis. For example, deregulated expression of miR-221&222 promotes cancerous growth by inhibiting the expression of p27Kip1. If the expression of miR-221&222 were repressed, the cancerous growth should been decreased. Based on the idea, repressing the expression of miRNAs might be an effective approach to block tumour progression. In my study, I established stable Drosha knock-down HeLa cells to study the impact of Drosha knockdown on the cell growth, migration, invasive activity. Unfortunately, stable-knockdown HeLa cells have not revealed the difference with wild type HeLa cells between in cell growth, cell migration, invasive activity and F-actin expression. Afterwards I established stable Drosha knock-down PC3 cells to analyze the above experiments, observing that stable-knockdown Drosha in PC3 cells leaded to repress cell growth, cell migration and invasive activity. Furthermore, I constructed the multiple shRNAs expression vectors for simultaneous inhibition of Drosha, DGCR8 and XPO5. HeLa cells which transfected with the multiple shRNAs expression vectors didn’t cause cell death or serious effect. On the other hand, I developed a method to rapidly screen stable-knockdown cells based on the luciferase activity. This method will be helpful to providing fast and massive cells screening.

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