余榮熾Yu, Lung-Chih臺灣大學:生化科學研究所潘國偉Pan, Guo-WeiGuo-WeiPan2010-05-042018-07-062010-05-042018-07-062008U0001-1906200816303300http://ntur.lib.ntu.edu.tw//handle/246246/178817MicroRNAs (miRNAs)為由21～23個核醣核酸所組成，不具轉譯意義的小片段單股RNA，能藉由translational repression或mRNA cleavage的方式抑制標的基因的表現；它們被證實在人體內參與許多重要生理現象的調控，包括胚胎細胞的發育、細胞的分化及腫瘤細胞的生成和增生等。過去許多的研究顯示，microRNA-221/222 (miR-221/222)於多種腫瘤細胞中呈現大量表現的現象。因此，本論文即以miR-221/222為目標；我們首先從miRNA資料庫中搜尋miR-221/222可能的標的基因，從其中我們篩選出了HIF-1β基因為可能的標的基因。HIF-1β為組成Hypoxia-inducible factor-1 (HIF-1)轉錄因子的其中一子單元，HIF-1轉錄因子在許多重要的細胞活動扮演重要的角色。證實HIF-1β是否確實受到miR-221/222的調控，我們以消化道系統癌細胞株做為研究的細胞模型；我們於細胞株中轉殖miR-221/222 mimic或其inhibitor，接著以西方點墨法分析HIF-1β蛋白的表現；實驗的結果證實HIF-1β蛋白的表現確可受到miR-221/222的抑制。而後我們建構載有正常或miR-221/222 seed site辨識序列突變的HIF-1β基因3’-untranslated region (3’-UTR)片段的報導基因質體；報導基因活性分析的實驗，證實HIF-1β確為miR-221/222的標的基因。我們也以即時同步定量RT-PCR分析HIF-1β蛋白表現受miR-221/222抑制的細胞，其HIF-1β transcript的表現量，來探討miR-221/222對HIF-1β表現的抑制是藉由translational repression或mRNA cleavage的方式。述一系列的研究，顯示miR-221/222確實可調控HIF-1β的表現；而這些初步的研究成果，也顯現了調控HIF-1轉錄因子活性的一條可能的新路徑。由於miR-221/222在胰臟癌癌化過程中，呈現大量表現的情形；因此我們也進一步以miRNA inhibitor抑制胰臟癌細胞株的內生性miR-221/222，並將之注射至裸鼠，觀察miR-221/222對胰臟癌細胞增生的影響。但在兩種不同胰臟癌細胞株中我們卻觀察到不同的結果，一為明顯的抑制腫瘤細胞的增生，另一卻為促進。此項研究還需要未來更多的實驗進一步探討，但此兩種不同的結果皆已顯示miR-221/222對胰臟癌細胞增生的顯著影響。MicroRNAs (miRNAs) are non-coding, single-stranded RNAs of 21～23 nucleotides in length, which suppress the expressions of target genes through translational repression or mRNA cleavage. MiRNAs involve in the regulations of many important cellular activities, including embryo development, cell differentiation, tumorigenesis, and tumor cell proliferation, etc.. Previous investigations have shown that the expressions of microRNA-221 and microRNA-222 (miR-221/222) significantly elevate in the oncogenetic processes of several cancers; thus we focus on miR-221/222 in the present investigation. First, we searched for the potential target genes of miR-221/222 in miRNA databanks. HIF-1β was selected for further investigation. HIF-1β is one of the two subunits that constitute HIF-1 (hypoxia-inducible factor-1) transcription factor, which plays significant roles in many cellular activities.e employed gastrointestinal cancer cell lines as study models to investigate whether miR-221/222 regulate the expression of HIF-1β. MiR-221/222 mimics and specific inhibitors for miR-221/222 were transfected into the gastrointestinal cancer cells, and western blotting analyses for the expressions of HIF-1β were followed. The results demonstrated the inhibitory function of miR-221/222 on HIF-1β expression. In the successive experiments, the reporter plasmids bearing the 3’-untranslated region (3’-UTR) fragments, wild-type or miR-221/222-seed site mutated, of HIF-1β gene were constructed, and the results obtained from the reporter gene activity assays of the constructs suggested that HIF-1β gene is the target of miR-221/222. In addition, the expressions of HIF-1β transcript in the cell models were quantitatively analyzed through real-time RT-PCR to elucidate which mechanism, translational repression or mRNA cleavage, leading to the suppression of HIF-1β-protein expression by miR-221/222.ur investigations demonstrate that miR-221/222 can inhibit the expression of HIF-1β. This suggests a novel route through which the activity of HIF-1 transcription factor could be regulated.s the significant elevation of miR-221/222 expressions in pancreatic cancer has been well documented, we used miRNA inhibitors to neutralize the endogenous miR-221/222 in pancreatic cancer cell lines, and employed human-nude mice xenograft model to examine the effects of miR-221/222 on the tumor formation of pancreatic cancer cells. However, opposite results were observed from the experiments of two different cell lines, with significant inhibition of tumor formation observed in one cell line while promotion in the other. More studies are expected for further exploration on this subject; nonetheless, the two opposite results obtained from the two cell lines both demonstrate the significant role of miR-221/222 in pancreatic cancer.目錄寫表..............................................................1要................................................................3文摘要............................................................4論、 miR-221/222的生成與生理意義.....................................5. miRNA的生成....................................................5. miRNA的功能....................................................6. miR-221/222的生理意義............................................6、 HIF-1的組成與生理意義...........................................7. HIF-1的組成單元.................................................7. HIF-1的調控.....................................................8. HIF-1的功能及生理意義...........................................9、 研究構想與目的..................................................11驗材料與方法、 細胞培養.......................................................12、 細胞蛋白萃取與西方點墨法 (Western blot)分析.......................12、 小片段RNA萃取及miRNA同步定量RT-PCR........................13、 轉殖miRNA mimic及miRNA inhibitor..............................13、構築報導質體與Luciferase-Reporter assays............................14、 RNA萃取及同步定量RT-PCR分析.................................15、腫瘤增生實驗....................................................16果、 miR-221/222可能的標的基因的預測.................................17、分析HIF-1β蛋白質及miR-221在消化道癌細胞株中的表現.............18、 miR-221及miR-222對HIF-1β蛋白表現的影響........................19、 miR-221及miR-222調控HIF-1β基因表現的專一性探討................20、 miR-221/222抑制HIF-1β表現的機制探討............................22、 miR-221/222對胰臟癌細胞增生的影響...............................23論...............................................................25考文獻...........................................................41表目錄1.Primer序列.....................................................292.實驗中所選用的癌細胞株.........................................301. HIF-1β為miR-221/222的可能標的基因.............................312.HIF-1β蛋白及miR-221在消化道癌細胞株中的表現...................323.轉殖miR-221 mimic及miR-222 mimic抑制HIF-1β表現................334.轉殖miR-221 inhibitor及miR-222 inhibitor回復HIF-1β表現.............345. miR-221/222對Luciferase報導基因表現的影響.......................356.以Luciferase活性變化分析預測的seed site對miR-221/222調控標的基因的 重要性........................................................367.分析miR-221/222抑制HIF-1β表現的機制............................378.以miR-221/222 inhibitor抑制內生性miR-221/222......................389. miR-221/222對MIAPaCa-2胰臟癌細胞增生的影響....................3910.miR-221/222對ASPC-1胰臟癌細胞增生的影響......................40圖1.細胞轉殖時間表...............................................50圖2.報導質體pGL3-control Map......................................51application/pdf2276428 bytesapplication/pdfen-US胰臟癌細胞株轉錄因子MicroRNAHIF-1β[SDGs]SDG3http://ntur.lib.ntu.edu.tw/bitstream/246246/178817/1/ntu-97-R95b46030-1.pdf