臺灣博碩士論文加值系統

　　口腔鱗狀細胞癌為現代常見的癌症之一。Numb蛋白為一高度保守的蛋白，並於過去的文獻中被發現具有多樣且複雜的功能。而在近期的研究發現Numb在癌症中扮演著抑制癌因子的角色，並且具有抑制細胞增生、遷移及幹細胞化…等功能。在本篇研究中發現在口腔上皮鱗狀細胞癌之病患其腫癌組織的Numb mRNA表現相較於癌旁組織大多有下降的趨勢。為了探討Numb在口腔癌中所扮演的角色，以慢病毒感染的方式建立了Numb 1及Numb 4穩定大量表現之細胞株，並運用CRISPR/Cas 9基因編輯系統建立Numb剔除之細胞。實驗結果中發現Numb剔除的細胞其非貼附性生長的能力有上升的情形且在Numb 1、Numb 4大量表現的細胞則有受到抑制的現象。此外，Numb剔除的細胞相較於正常表現Numb之細胞其胞外乳酸的濃度有上升的情形，並且於ECAR/OCR的數值上也呈現較高的比值。而在進一步的結果中發現，Numb剔除的細胞其乳酸通道蛋白MCT1、MCT4的表現有上升的趨勢。本研究亦將釐清口腔癌當中Numb是否會經由調控MCT1/4表現進而影響癌細胞非貼附性生長的能力。

Oral squamous cell carcinoma (OSCC) is one of the common cancers worldwide. Numb is an highly conserved protein and it displays a complex pattern of functions. Recent studies have shown Numb as an oncogenic suppressor by inhibiting proliferation, migration, stemness properties, and others. We found in this study that Numb mRNA expression was down-regulated in a large fraction of OSCC tumors. We established Numb 1 and Numb 4 expression lentiviral constructs, and the gene edition Numb-knockout (KO) OSCC cell subclones using CRISPR Cas9 system. The analysis showed that the anchorage independent growth of OSCC cells was elevated after deletion of Numb, while such ability was repressed after the rescue of the Numb expression by viral infection. In addition, the level of lactate production and the ratio of ECAR to OCR in the Numb-KO cells was higher than parental cells. Reports showed that monocarboxylate transporter (MCT) family members were responsible for transporting lactate out of cells. This study further found that the deletion of Numb was able to up-regulate MCT1 and MCT4 expression to increase the lactate production. Advance studies are undergoing to clarify the interplay between anchorage independent growth potential and MCT1/4 expression modulated by Numb in OSCC.

誌謝...............................i
中文摘要............................ii
英文摘要...........................iii
目次................................iv
壹、緒論 (Introduction).........................1
一、口腔鱗狀細胞癌(OSCC, Oral Squamous Cell Carcinoma)....1
二、Numb的結構及其功能..........................1
三、乳酸與癌症................................3
四、CRISPR/Cas9 基因剪輯技術..............................4
貳、研究動機及目標 (Purpose).................6
一、研究動機.................................6
二、研究目標..............................7
參、實驗材料與方法 (Material and Method)........8
一、細胞培養(Cell Culture)...............8
二、載體構築 (Construction)...................9
三、轉染 (Transfection).....................9
四、慢病毒感染(lentivirus infection)..........9
五、西方墨點法 (Western blot)............10
六、細胞非錨定生長試驗 (Cell anchorage-independent growth assay)......................................11
七、CRISPR/Cas9剔除基因模式(CRISPR/Cas9 knock-out model).12
八、培養液乳酸測定(Lactate analysis)...........12
九、細胞粒線體壓力測定(Mitochondrial stress test).....13
十、統計分析 (Statistical analysis)...........14
肆、實驗結果 (Results)....................15
一、Numb基因剔除之細胞株的建立...............15
二、大量表現Numb異構體之細胞株的建立............16
三、Numb基因缺失對於細胞增生之影響 ..............16
四、Numb基因缺失對於細胞遷移能力之影響.........17
五、Numb表現影響細胞非貼附性生長能力........17
六、Numb表現影響細胞乳酸排放能力.............18
七、Numb表現調控細胞乳酸運輸蛋白的表現..........19
八、抑制MCT1/4影響乳酸排放之能力...........20
九、抑制MCT1/4影響細胞非貼附性生長能力........21
伍、討論 (Discussion)......................22
陸、結果圖表 (Figures)...................26
圖一、Numb基因剔除之細胞株其Numb蛋白表現情形.......26
圖二、Numb基因剔除細胞株其基因變化 ............27
圖三、Numb過度表達之口腔癌細胞株的建製...........28
圖四、Numb基因剔除對於細胞生長之影響.........29
圖五、Numb基因剔除對細胞遷移能力之影響.........31
圖六、Numb基因剔除對細胞非貼附性生長能力之影響......33
圖七、Numb表現對於細胞非貼附性生長能力之影響.....34
圖八、Numb基因剔除影響細胞乳酸排放情形.........36
圖九、Numb基因剔除後不影響粒線體之功能.........37
圖十、Numb基因剔除影響後造成ECAR/OCR的比值上升......38
圖十一、Numb的缺失造成MCT family表現上升.......39
圖十二、抑制Numb對細胞膜上葡萄糖及乳酸運輸蛋白表現之影響....40
圖十三、以siRNA抑制Numb造成胞外乳酸濃度上升......41
圖十四、透過siRNA抑制MCT1及MCT4蛋白表現......42
圖十五、抑制MCT1和MCT4影響胞外乳酸濃度..............43
圖十六、抑制MCT1及MCT4對於細胞非貼附性生長能力之影響.......44
柒、附圖 (Addedums).........................45
附圖一、以預測軟體TargetScan對Numb序列預測miRNA靶定位置...45
附圖二、miR-31、miR-96、miR-182抑制口腔癌中Numb蛋白的表現..46
附圖三、miRNAs標定Numb 3’UTR報導基因分析結果....47
附圖四、17例口腔癌臨床檢體Numb之mRNA表現量之初步分析.......48
附圖五、以CRSPR/Cas9建製Numb基因剔除之細胞流程............49
附圖六、CRISPR/Cas9基因編輯系統之作用原理.................50
附圖七、pSurrogate reporter之設計..........51
附圖八、Numb異構體之結構............................52
捌、附表 (Tables).......................................53
Table 1：本研究使用試劑列表..............................53
Table 2：抗體列表(上：初級抗體；下：次級抗體).............54
Table 3：引子列表 (Numb 過度表現及Numb亞型鑑定PCR).......55
Table 4：引子列表 (MCT family).........................56
Table 5：引子列表 (構築CRISPR/Cas9相關載體構築)..........57
Table 6：引子列表 (Numb基因刪除細胞株序列確認)............58
玖、參考文獻 (References)...............................59

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