臺灣博碩士論文加值系統

Oct4 被視為保持幹細胞多潛能性的重要分子。在許多的人類腫瘤生成的過程中，Oct-4也扮演了重要的角色。在肺癌細胞中，Oct-4的持續表現使得癌細胞維持幹細胞的特性，使得癌細胞更不容易被殺死。Egr-1 (early growth response gene-1)是一種鋅指結構(zinc-finger)的轉錄分子，已知能夠調控許多基因的表現，包括發炎反應、細胞分化、生長及發育。雖然Egr-1在從前被認為是一種抑癌基因，但是近年來的研究發現，Egr-1其實也具有致癌基因的特性，所以可以說是一種具有雙面功能的基因。Egr-1已知參與上皮-間葉細胞轉換(epithelial-mesenchymal transition，EMT)與血管新生(angiogenesis)，並且會活化有關腫瘤轉移的基因，例如osteopontin (OPN)。根據微陣列分析結果，將老鼠胚胎幹細胞的Oct4抑制後，會讓Egr-1 mRNA的表現量下降，在我們的實驗結果也發現轉移程度越高的肺癌細胞株，Egr-1的表現量也越高。Oct4與Egr-1可能在肺癌細胞致病機制與轉移上扮演重要角色，但是二者之間的關係與重要性仍然未知。因此本研究利用轉染方式將Oct4 cDNA送入H1299肺癌細胞，並利用RT-PCR與西方點墨法分析結果，發現Egr-1 RNA與蛋白質隨著Oct4 cDNA的表現量升高而增加。同樣的分析以反轉錄病毒感染而持續表現Oct4的A549細胞株，也可以發現Egr-1 基因的持續表現。接著利用螢光報導基因分析發現，在A549以及H1299肺癌細胞中，Oct4能夠活化Egr-1的啟動子活性，並利用將啟動子分段切除方式與染色質免疫沉澱分析發現Egr-1啟動子上有Oct4的結合位置。利用Boyden chamber分析在Oct4過量表現的A549細胞與H1299細胞，發現在抑制Egr-1的表現後，細胞移動的能力明顯下降。另外，OPN的表現量也在抑制Egr-1的表現後有明顯的下降。在動物實驗中，以靜脈注射的方式打入NOD/SCID小鼠不同處理的A549細胞，觀察癌細胞轉移到肺的情況，結果發現過量表現Oct4並抑制Egr-1的A549細胞，轉移的現象較少。由以上的結果顯示，Oct4參與肺癌發展的過程中，會藉由增加Egr-1的表現而影響其轉移的機制。

Oct4 is regarded as one of the key regulators of stem cell pluripotency. It has been detected in several human cancers, suggesting a potentially critical role in tumorigenesis. Oct4 expression also maintains cancer stem-like properties in lung cancer. Early growth response gene-1 (Egr-1), a zinc-finger transcription factor, has been implicated in the regulation of many genes involved in inflammation, differentiation, growth, and development. Egr-1 is generally thought to play a tumor suppressor function. However, recent studies have shown that Egr-1 is a Janus-faced protein in cancer because it may be involved in epithelial-mesenchymal transition (EMT) and angiogenesis. Egr-1 also upregulates the expression of osteopontin (OPN) that is involved in modulating cell functions and increasing lung cancer cell migration. Previous microarray data revealed that knockdown of Oct4 in embryonic stem cell decreases Egr-1 mRNA expression. Our study also showed that protein levels of Egr-1 were increased in metastatic lung cancer cell lines. Oct4 and Egr-1 may play important roles in the pathogenesis of lung cancer. However, little is known about their relationship and significance in metastatic lung cancer. Therefore, I transfected H1299 human lung cancer cell line H1299 with Oct4 cDNA and examined the expression of Egr-1 by RT-PCR and immunoblot analysis. Expression of Egr-1 was increased in cells stably transduced with Oct4. Same results were also obtained from A549 cells stably overexpressing Oct4 via retrovirus-mediated gene transfer. In addition, luciferase reporter and chromatin immunoprecipitation assays also revealed that Oct4 upregulated Egr-1 expression through direct binding to its promoter. Knockdown of Egr-1 decreased the invasive potential of Oct4-overexpressing A549 and H1299 cells by Boyden chamber analysis. Expression of OPN was also decreased after knockdown of Egr-1 in Oct4-overexpressing A549 cells. Animal studies also showed that knockdown of Egr-1 reduced tumor nodules in the lung after intravenous injection of Oct4-overexpressing A549 cells to NOD/SCID mice. In conclusion, these results suggest that Oct4 may be involved in lung cancer progression and metastasis through upregulation of Egr-1 expression.

中文摘要..........II
Abstract.........IV
誌謝........VI
目錄..........VIII
圖目錄..........X
縮寫..........XII
第一章 緒論..........1
A、肺癌(Lung cancer)..........1
B、癌轉移(tumor metastasis)..........2
C、Oct4..........3
1.概論..........3
2.Oct4與腫瘤..........3
3.Oct4 與癌轉移..........4
4. Oct4 的下游基因..........4
5. Oct4的調控..........5
D、Egr-1..........6
1.概論..........6
2. Egr-1與腫瘤..........6
3.Egr-1與癌轉移..........7
4. Egr-1的調控..........8
5. Oct4與Egr-1..........8
6.Egr-1與Osteopontin(OPN)..........9
E、Osteopontin(OPN)..........10
1.概論..........10
2. OPN與癌轉移..........10
第二章 研究目的..........12
第三章 材料與方法..........13
材料..........13
1.質體..........13
2.細胞株..........13
3.慢病毒..........14
4.實驗動物..........15
5.寡去氧核?酸(Oligodeoxynucleotides,ODN)..........15
方法..........15
1. 細胞株與細胞培養..........15
2. 慢病毒的生產與感染細胞..........16
3. Stable clone的選殖..........17
4. 西方點墨法分析..........18
5. 反轉錄?-聚合?連鎖反應 (Reverse transcriptase-polymerase chain reaction, RT-PCR)..........20
6. 啟動子活性分析..........21
7. Egr-1 啟動子片段切除的建構..........21
8. 細胞移動分析(migration assay)..........22
9. 動物實驗..........23
10. 免疫組織染色(Immunohistochemistry)..........23
11. Enzyme-linked immunosorbent assay(ELISA)..........24
12. 統計學..........25
第四章 結果..........26
一、在肺癌細胞中過量表現Oct4會增加Egr-1蛋白質以及mRNA的表現..........26
二、在肺癌細胞中過量表現Oct4能夠活化Egr-1的啟動子，且發現Egr-1 啟動子上有Oct4的結合位置..........27
三、在肺癌細胞中利用shRNA 抑制Egr-1基因的表現，發現Oct4 mRNA表現量不受影響..........28
四、在動物模式中證明肺癌細胞Oct4過量表現之下Egr-1也會在相同位置的表現..........28
五、Egr-1蛋白質的表現量在轉移程度愈強的肺癌細胞中表現量愈高..........29
六、Egr-1基因剔除的小鼠胚胎纖維母細胞(mouse embryonic fibroblast, MEF) 細胞移動能力明顯低於野生型小鼠MEF..........29
七、過量表現Oct4的肺癌細胞，細胞移動能力會上升，若進一步抑制Egr-1則會降低細胞的移動能力。並在動物模式中證明過量表現Oct4的肺癌細胞會經由Egr-1促進細胞轉移的能力..........30
八、在肺癌細胞中利用shRNA抑制Oct4的表現，Egr-1與Osteopontin (OPN)的mRNA與蛋白質表現也會被抑制。在過量表現Oct4的肺癌細胞A549中進一步抑制Egr-1的基因表現，則會抑制OPN的蛋白質表現。..........31
第五章 總結..........33
第六章 討論..........34
參考文獻..........40
圖..........45
自述..........57

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