臺灣博碩士論文加值系統

背景：發現早期偵測的生物指標，對於口腔癌之診斷和預防是很重要的。微型RNA（miRNA）是非編碼的短RNA，其在腫瘤進程中扮演著重要的角色。
方法：本研究檢測了在Nitroquinoline 1-Oxide (4NQO) 誘導的小鼠舌癌模式中，組織、唾液和血漿中致癌miRNA的表現，藉以探討其潛在的診斷或治療方面之應用性，並以口腔癌細胞探究miR-31影響代謝之致病機制。
結果：組織學染色的結果發現，口腔上皮長時間的暴露於致癌物4NQO的接觸，可以觀察到表皮層病灶一步一步持續性的發展過程，從過度角化到上皮異常增生，最後形成鱗狀上皮腫瘤。在舌頭上皮的染色結果發現miR-31表現與體液中之miR-31，與上皮的發病進程，有相同上升的趨勢。而miR-31之標靶SIRT3則隨著病態進行而逐漸降低，SIRT3扮演著抑癌基因角色，人類口腔癌中SIRT3表現下降，在口腔癌中miR-31-SIRT3共同調控了粒線體膜電位語與粒線體結構，進而將口腔癌細胞之代謝途徑從有氧代謝切換為無氧代謝。
結論：本研究發現在小鼠舌頭腫瘤發生的過程中，致癌miRNA的異常表現與人類的表現是相似的。miR-31-SIRT3促使口腔癌細胞中的粒線體功能障礙和代謝狀態的轉移，導致腫瘤進展之惡化。由於miR-31在口腔癌中為致癌的角色，所以阻斷miR-31表達對於預防口腔癌發展將是有價值的。

Background: Oral squamous cell carcinoma (OSCC) is one of the leading causes of cancer death in Taiwan, and it occurs more frequently in male population due to the abuse of tobacco and betel quid. To develop biomarkers for early cancer detection may bestow substantial benefits in diagnosis and the prevention of tumor progression. miRNAs are short, non-coding RNAs, which play important roles in oncogenesis. This study investigated the disruptions in miR-31, which could be associated with metabolic change and tumor progression.
Methods: Mice were fed with 4-Nitroquinoline 1-Oxide (4NQO) in drinking water for 12 weeks and were kept for extended periods for tumor induction. The seahorse machine was used to detect the metabolic states of OSCC cell lines. The SIRT3 gene as potential miR-31 target to affect metabolism and tumor progression was also explored.
Results: The histological and miR-31 level in body fluid evaluation showed the consecutive increase in the severity of epithelial pathogenesis from hyperkeratosis, hyperplasia to epithelial dysplasia and squamous tumors following the increase of 4NQO exposure. The increase of miR-31 staining parallels the severity of epithelial pathogenesis in tongue tissue. In addition, the expression of miR-31 target, SIRT3, was declined. SIRT3 expression is down-regulated in human OSCC. SIRT3 drives suppressor activity. miR-31-SIRT3 regulates the mitochondrial membrane potential and mitochondrial structure. miR-31 switched OSCC cell lines from aerobic metabolism to anaerobic metabolism.
Conclusion: The disruption in the expression of oncogenic miRNAs in thus mouse model is similar to the disruption being identified in human oral cancer counterparts. miR-31-SIRT3 regulated the mitochondrial dysfunction and shift of the metabolism status in OSCC cell lines and worsened the tumor progression. As miR-31 was functionally oncogenic, the blockage of miR-31 expression would be valuable for the prevention of OSCC development.

目錄
目錄 i
Abstract v
中文摘要 vi
壹、緒論 1
一、口腔癌 1
二、 miRNA 2
1. miRNA之生物合成 2
2. miRNA與口腔癌之關係 2
3 .體液中的miRNA表現 3
三、SIRT3 5
1. SIRT3家族 5
2. SIRT3在癌症中扮演的角色 5
3 . SIRT3與代謝的相關性 6
四、粒線體 7
1. 粒線體 7
2. 粒線體與氧化壓力 7
3 . 粒線體與癌症 8
五、4NQO誘導鱗狀上皮癌致癌模式 9
貳、研究目標 10
一、研究動機 10
二、研究目標 10
参、材料與方法 12
一、 口腔鱗狀上皮細胞培養 12
二、細胞處理4NQO、AG1478與Triciribine 13
三、4NQO誘導小鼠鱗狀上皮癌致癌模式 13
四、小鼠唾液與血液採集 13
五、舌頭組織處理 14
六、原位雜交法 14
七、免疫組織染色 15
八、微型RNA之反轉錄即時定量聚合酶連鎖反應 15
九、即時聚合酶連鎖反應 (Real-time RT-PCR) 15
十、細胞能量運用測定 16
十一、細胞氧化壓力 16
十二、SIRT3過表現與shSIRT3之細胞株建立 17
十三、病毒製備與感染 17
十四、西方點墨法 18
十五、微型RNA模擬物（miR-31 mimic）、抑制物（miR-31 inhibitor）與報導者質體之轉染法 19
十六、報導者分析法（Reporter assay） 19
十七、細胞增生實驗 20
十八、細胞刮痕移行試驗 20
十九、細胞侵襲孔洞試驗 20
二十、粒線體形狀結構觀察 21
二十一、粒線體膜電位membrane potential change (Δψ)分析 21
二十二、統計 21
肆、結果 23
一、基本生理指標與癌症惡病質現象 23
二、miRNA隨著舌癌的進展而增加 23
三、致癌基因隨著舌癌的進展而增加 23
四、唾液與血漿將中miRNA隨著舌癌的進展而增加 24
五、唾液與血漿將中miRNA相關性 25
六、miRNA具有分辨疾病與健康狀態的能力 25
七、4NQO誘導EGFR和AKT的活化藉由miRNA的調控 25
八、miR-31與SIRT3在4NQO的舌癌病變動物模式中之表現 26
九、miR-31與SIRT3在口腔癌病人檢體中之表現 26
十、SIRT3為miR-31調控的標靶基因 26
十一、SIRT3對口腔癌細胞增生能力之調控 27
十二、SIRT3對口腔癌細胞侵襲能力之調控 27
十二、miR-31對口腔癌細胞氧化壓力之調控 27
十三、SIRT3對口腔癌細胞爬行能力之調控 27
十四、miR-31與SIRT3對於粒線體複合體之調控 27
十五、miR-31與SIRT3對於粒線體結構之調控 28
十六、miR-31與SIRT3對於粒線體膜電位差(Δψ)分析 28
十七、miR-31對口腔癌細胞粒線體功能性之影響 28
十八、miR-31與SIRT3對口腔癌細胞代謝之調控 28
十九、miR-31與SIRT3對口腔癌細胞氧化壓力之調控 29
伍、討論 30
陸、總論 37
柒、參考文獻 56
捌、圖 68
圖一 68
圖二 69
圖三 70
圖四 71
圖五 72
圖六 73
圖七 74
圖八 75
圖九 76
圖十 77
圖十一 78
圖十二 79
圖十三 80
圖十四 81
圖十五 82
圖十六 83
圖十七 84
圖十八 85
圖十九 86
圖二十 87
圖二十一 88
圖二十二 89
圖二十三 90
圖二十四 91
圖二十五 92

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