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系統識別號 U0026-1907201219073000
論文名稱(中文) 過氧小體醯基輔酶A硫酯酶8在癌症之角色
論文名稱(英文) The role of peroxisomal acyl-coenzyme A thioesterase 8 in cancer
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) Department of Biochemistry and Molecular Biology
學年度 100
學期 2
出版年 101
研究生(中文) 詹宜鑫
研究生(英文) Yi-Shin Chan
學號 s16994085
學位類別 碩士
語文別 中文
論文頁數 75頁
口試委員 指導教授-賴明德
口試委員-張文粲
口試委員-陳昌熙
中文關鍵字 醯基輔酶A硫酯酶8  肝癌 
英文關鍵字 acyl-coenzyme A thioesterase 8  hepatocellular carcinoma 
學科別分類
中文摘要 醯基輔酶A硫酯酶可以將醯基輔酶A水解成游離脂肪酸以及輔酶A。雖然在1952年時就已經知道醯基輔酶A硫酯酶的催化活性,但是對於ACOTs家族在細胞和生物中具有的生理功能並不清楚。目前人類部分已發現ACOTs家族有十個成員,包含ACOT1、ACOT2、ACOT4、ACOT6、ACOT7、ACOT8、ACOT9、ACOT11、ACOT12和ACOT13,其中ACOTs家族中的ACOT8是位在過氧小體這個單膜的胞器之中。過去有很多研究指出當位於過氧小體中的酵素功能出現異常的時候可能會造成許多疾病包含老化甚至是癌症。此外,有研究指出ACOT8的mRNA在卵巢癌的檢體中發現會大量表現。雖然如此,ACOTs在癌症中扮演的角色至今尚未釐清。在此研究中,我們想要去了解位於過氧小體的ACOT8是否在肝癌中扮演重要的角色。一開始我們由GEO資料庫找尋關於肝癌檢體的數據庫並且進行生物晶片數據的分析,結果發現在肝癌病人中的ACOT8的基因拷貝數以及mRNA表現量有異常增加的現象。接下來我們進一步想知道ACOT8在肝癌中所具有的功能,為此我們利用Huh-7肝癌細胞株建立了將內生性ACOT8抑制的轉染細胞株。我們的數據指出當ACOT8表現量減少時會顯著的抑制細胞的生長能力。然後我們更進一步發現當ACOT8表現量減少時會透過活化TGF-beta路徑來增加p15INK4b和p27KIP1的蛋白質表現量,並誘導細胞週期延遲。此外,我們想要知道為何ACOT8表現量下降時可以影響TGF-beta的表現。由過去研究發現活性氧化物 (ROS) 的增加會抑制TGF-beta的表現,我們也發現在ACOT8被抑制後活性氧化物含量高的細胞比例會減少。
英文摘要 Acyl-Coenzyme A thioesterases (ACOTs) hydrolyze acyl-CoA esters to the free fatty acids (FFAs) and coenzyme A (CoASH). Although the catalytic activity of ACOTs was first identified in 1952, the physiological functions of ACOTs are not completely understood. The human ACOTs family consists of ten members, including ACOT1, ACOT2, ACOT4, ACOT6, ACOT8, ACOT9, ACOT11, ACOT12 and ACOT13. Among the Family, ACOT8 are localized in peroxisomes, which are single-membrane lined organelles. Previous studies show that aberrant functions of peroxisomal enzymes may cause many diseases such as aging and cancer. Moreover, some papers indicated that the mRNA expression of ACOT8 is increased in ovarian cancer. Nevertheless, the role of ACOTs in cancer is unclear. In this study, we are interested in studying the role of peroxisomal ACOT8 in liver cancer. At first, we searched for datasets relevant to hepatocellular carcinoma (HCC) specimens from gene expression omnibus (GEO) database and analyzed the microarray data. As a result, the gene copy number and mRNA expression level of ACOT8 was increased in HCC specimens. To investigate what are the functions of ACOT8 in liver tumor progression, we established ACOT8 knockdown clones of Huh-7 cells. Our data show that downregulation of ACOT8 reduces cell proliferation. Then, we further identified that downregulation of ACOT8 may increase the protein expression of p15INK4b and p27KIP1 through TGF-beta pathway and induce the cell cycle delay. Furthermore, we want to know why downregulation of ACOT8 may affect the expression of TGF-beta. Previous studies show that upregulation of reactive oxygen species (ROS) inhibit the expression of TGF-beta. We found that the percentage of high ROS cells was decreased in ACOT8 knockdown clones.
論文目次 目錄
緒論
一、脂質代謝異常與癌症的關聯性 1
二、ACOT在脂質代謝上所扮演的角色 2
三、過氧小體在脂質代謝及肝癌的關聯性 4
四、ACOT與疾病或癌症的關聯性 5
五、研究目標與策略 6

材料與方法
一、細胞培養與藥物處理(Cell culture and treatment) 8
二、質體製備(Plasmid preparation) 13
三、抽取Total RNA(Isolate total RNA) 16
四、反轉錄聚合酶連鎖反應(Semi-quantitative RT-PCR) 17
五、即時聚合酶連鎖反應(Real-time PCR) 19
六、基因殖入轉染(Transfection)與細胞株的篩選(Selection) 21
七、西方點墨法(Western Blotting) 23
八、細胞生長分析(MTT assay) 29
九、細胞群落形成分析(Colony formation assay) 30
十、細胞爬行分析(Boyden chamber migration assay) 32
十一、細胞週期分析(Cell cycle analysis) 34
十二、細胞內活性氧分析(intracellular ROS analysis) 36
十三、微陣列生物晶片分析(microarray analysis) 37

結果
一、肝癌檢體中ACOT8的基因拷貝數和mRNA表現量會增加 39
二、減少ACOT8的表現可以抑制細胞生長能力以及爬行能力 40
三、抑制ACOT8的表現可以抑制腫瘤生長能力 41
四、抑制ACOT8表現導致細胞週期的延遲 41
五、抑制ACOT8表現會增加p15及p27的表現量 42
六、抑制ACOT8會影響TGF-beta的表現 43
七、抑制ACOT8可能增加Smad3的磷酸化 43
八、ACOT8表現量下降抑制細胞內ROS的產生 44

討論 46
結論 51
參考文獻 52
附圖 58
附註 74


圖目錄
附圖
一、ACOT8的基因拷貝數在肝癌檢體中有放大的現象 58
二、肝癌腫瘤的ACOT8表現量高於周圍正常的肝組織 59
三、ACOT8的mRNA表現量以肝癌細胞Huh-7最高 60
四、利用核糖核酸干擾技術成功抑制內生性的ACOT8 61
五、降低人類ACOT8基因表現會抑制細胞的生長速率 62
六、降低人類ACOT8基因表現會抑制細胞貼附性生長 63、64
七、降低人類ACOT8基因表現會抑制細胞的爬行能力 65
八、抑制ACOT8表現能輕微減緩腫瘤生長 66
九、抑制ACOT8造成細胞週期延遲的現象 67
十、抑制ACOT8後分析和細胞週期相關的蛋白質表現 68、69
十一、抑制ACOT8可能影響TGF-beta的表現及下游路徑的活化 70
十二、抑制ACOT8後TGF-beta的mRNA有顯著上升 71
十三、抑制ACOT8後分析Smad3的磷酸化情形 72
十四、抑制ACOT8導致細胞中ROS下降 73
附註
一、本篇研究假說 74
二、利用Metacore平台進行生物晶片分析的結果 75
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