自噬作用是細胞的自我吞噬機制，通過這種機制，使得受損的蛋白質與細胞胞器被聚集到自噬體並與溶酶體融合，特別是在營養缺乏期間進行大量降解和回收。自噬作用的失調與各種疾病有很多的關聯，包括癌症。ATG4B是自噬機制所需的半胱氨酸蛋白酶。最近的報導顯示，ATG4B升高促進腫瘤發生、惡性腫瘤與藥物的抗藥性，這表明ATG4B可調節自噬以促進腫瘤發展。然而，自噬對ATG4B在癌細胞中的作用仍然未知。在這項研究中，我們發現在自噬誘導條件下，包括雷帕黴素和飢餓，腦神經膠質瘤H4細胞和神經母細胞瘤SHSY5Y細胞中ATG4B蛋白水平降低。此外，自噬抑製劑氯喹(Chloroquine)或巴弗洛黴素(Bafilomycin A1)和蛋白體抑製劑(MG132)在細胞中適度恢復自噬降解的ATG4B。沉默ATG7也部分恢復了用雷帕黴素處理的細胞中的ATG4B蛋白水平，而它在飢餓細胞中沒有恢復作用。此外，在雷帕黴素處理的腦神經膠質瘤H4細胞中ATG4B的mRNA水平降低，但在飢餓的細胞並沒有。使用螢光素酶與ATG4B的3'UTR融合作為報告基因測定來評估miRNA在自噬條件下對細胞中ATG4B的作用。用雷帕黴素處理的腦神經膠質瘤H4細胞中螢光素酶活性顯著降低。然而，螢光素酶活性對恢復幾乎沒有影響。相應地，miR-34a在雷帕黴素處理的細胞中增加，而miR-34a在BafA1和CQ藥物處理的細胞中被抑制。總之，雷帕黴素可調節自噬和miR-34a以減少神經膠質瘤細胞中的ATG4B。我們的結果可能揭示雷帕黴素對腫瘤抑制的潛在機制。

Autophagy is a self-eating mechanism in cells through which damaged proteins and organelles are recruited to autophagosomes and fused with lysosome for their bulk degradation and recycling during nutrient deprivation. Dysregulation of autophagy is associated with various diseases, including cancer. ATG4B is a cysteine protease required for autophagy machinery. Recent reports have shown that elevated ATG4B promoted tumorigenesis, malignancy and drug resistance, suggesting ATG4B might modulate autophagy to facilitate tumor progression. However, the role of autophagy on ATG4B in cancer cells remains unknown. In this study, we found ATG4B protein level was decreased in glioma H4 and SHSY5Y cells during autophagy inducing conditions, including rapamycin and starvation. Moreover, autophagy inhibitors chloroquine or BafA1and proteosome inhibitor MG132 modestly recovered autophagy downregulated ATG4B in cells. Silencing ATG7 also partially recovered ATG4B protein level in cell treated with rapamycin, whereas it had no recovery effects in starved cells. Furthermore, mRNA level of ATG4B was decreased in rapamycin-treated H4 cells, but not starved cells. Luciferase fusion with 3’UTR of ATG4B as reporter assay was used to evaluate the effects of miRNA on ATG4B in cells during autophagy conditions. The luciferase activity was significantly decreased in H4 cells treated with rapamycin. However, the luciferase activity had little effects on recovery. The miR-34a was accordingly increased in the rapamycin-treated cells, while miR-34a was inhibited in BafA1 and CQ pretreated cells. Taken together, rapamycin may regulate autophagy and miR-34a to reduce ATG4B in glioma cells. Our results may shed a light on potential mechanisms of rapamycin on tumor suppression.

論文審定書 ⅰ
致謝 ⅱ
摘要 ⅲ
Abstract ⅳ
前 言 1
細胞自噬作用的過程與功能 1
自噬基因(Autophagy gene)參與自噬作用 1
ATG4的介紹與功能 2
ATG4B與疾病的關聯性 3
材料與方法 5
一、細胞培養 5
二、質體的製備 6
三、蛋白質萃取 7
四、西方墨點法(Western blotting) 8
五、RNA萃取 9
六、互補DNA合成 10
七、即時定量聚合酶連鎖反應 10
(Real-time Quantitative Polymerase Chain Reaction,Q-PCR)
八、永久轉染細胞株 (stable transfection) 11
九、RNA干擾技術(RNA interference) 11
結果 13
誘發細胞自噬作用降低ATG4B的蛋白質表達 13
抑制自噬基因表達影響細胞自噬作用 14
誘發細胞自噬作用觀察ATG4B的mRNA表達 14
進一步探討pMIR-REPROT 評估內源性miRNA表達 15
誘發細胞自噬作用觀察不同microRNA的表現 15
討論 18
細胞可能藉由降解ATG4B的蛋白質表達協助細胞自噬作用進行 18
MicroRNA調控自噬核心基因 19
抑制自噬基因影響細胞自噬 20
總結與展望 22
圖表 23
參考文獻 32
補充結果圖表 34
附錄 39

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