探討黑色素腫瘤中p53調控miR-524-5p及miR-596表現之機制

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姓名

曹崴(Wei Tsao) 查詢紙本館藏

畢業系所

系統生物與生物資訊研究所

論文名稱

探討黑色素腫瘤中p53調控miR-524-5p及miR-596表現之機制
(Study of the mechanism of p53 regulation in miR-524-5p and miR-596 expression in melanoma)

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摘要(中)

miRNA在許多癌症基因調控，都參與並且扮演著重要的角色，先前有研究指出，在mitogen-activated protein kinases pathway (MAPK pathway)過度活化的惡性黑色素腫瘤當中， miR-524-5p與miR-596這兩個miRNA的表現量異常的降低，且當重新增強這兩個miRNA的表現量之後，會抑制黑色素腫瘤的增值、爬行和侵襲等諸多細胞功能。是非常具有效力的腫瘤抑制miRNA。但是至目前為止，此二種miRNA在黑色素腫瘤細胞當中所受到的下調機制依舊是未知，找出在黑色素腫瘤當中此二種miRNA之調控機制便是此研究所要探討之主軸
p53是一個週知的腫瘤抑制蛋白，通過轉錄的調控，活化或抑制多種與癌症相關的基因轉錄，也有諸多研究顯示p53也會調控許多癌症相關miRNA的轉錄。在此研究當中，我們假設p53可能為調控miR-524-5p與miR-596這兩個miRNA的轉錄因子。在研究中我們發現，當p53的表現量在黑色素腫瘤細胞中上升時，miR-524-5p與miR-596的表現量便有隨之上升之趨勢，而p53表現受到抑制時，miR-524-5p與miR-596的表現量也有下調的趨勢，p53與這兩個miRNA之表現，是有著正相關的關聯性，且在miR-524-5p當中最為明顯，所以，為了驗證p53是否會直接調控其轉錄，我們也預測了miR-524-5p的啟動子序列，進一步發現此序列上可能有潛在p53連接位點。最後通過建立miR-524-5p的啟動子序列之冷光報導基因，試圖證明與了解p53是否直接的與miR-524-5p的啟動子序列進行連接並調控其轉錄。

摘要(英)

miRNAs are involved in an important role in the regulation of many cancer-related genes. It has been found that the expression levels of miRNA miR-524-5p and miR-596 were significantly low in melanoma with high activated mitogen-activated protein kinases pathway （MAPK pathway）. When the expression levels of the two miRNAs are overexpressed, cell functions such as proliferation, migration, and invasion of melanoma tumors are repressed. However, the down-regulation mechanism of these two miRNAs in melanoma is still unclear. Therefore, the aim of this study is to identify how these two miRNAs are regulated in melanoma.
p53 is a well-known tumor suppressor protein that activates or inhibits the transcription of many cancer-associated genes through transcriptional regulation. Many studies have also shown that p53 as well regulates the transcription of many cancer-associated miRNAs. In this study, we hypothesized that p53 may be a transcription factor that regulates two miRNAs, miR-524-5p and miR-596. The increase of MAPK pathway in melanoma may cause the inhibition of p53 activity, which may result in decrease of miR-524-5p and miR-596 in melanoma.
Our results indicated that the expression levels of p53 can affect the expressions of miR-524-5p and miR-596. The expressions of p53 and the two miRNAs were positively correlated. In order to verify whether p53 is directly regulator for miR-524-5p transcription, we predicted the promoter sequence of miR-524-5p according data base or web and further cloned the potential p53 binding sites on luciferase reporter plasmid. Finally, we try to test whether or not p53 is directly binding to the promoter sequence of miR-524-5p and regulates its transcription.

關鍵字(中)

★ 黑色素癌
★ 黑色素腫瘤
★ 微型核醣核酸
★ 癌症
★ 腫瘤蛋白53
★ miR-524-5p
★ miR-596

關鍵字(英)

★ melanoma
★ micro RNA
★ miRNA
★ p53
★ TP53
★ miR-524-5p
★ miR-596
★ cancer

論文目次

目錄

中文摘要 vi
Abstract vii
致謝 ix
緒論 1
1. 黑色素腫瘤（Melanoma） 1
1-1. 黑色素細胞 1
1-2. 黑色素腫瘤發生與分類 1
1-3. 黑色素腫瘤之治療及預後 2
1-4. 黑色素腫瘤之基因表現 2
2. 微型RNA （miRNA） 4
2-1. miRNA的生成 4
2-2. miRNA的作用機制 6
2-3. 癌症中miRNA扮演之角色 7
2-4. miR-524-5p和miR-596與黑色素腫瘤的關係 7
3. 腫瘤抑制蛋白53 （p53） 8
3-1. p53介紹 8
3-2. p53的活化 9
3-3. p53與癌症的關係 10
3-4. p53和miRNA在癌症中所扮演的角色 11
實驗材料與方法 13
1. 實驗材料（Materials）： 13
1-1. 細胞株（Cell lines） 13
1-2. 藥物（Drug） 13
1-2-1. Nutlin-3 13
1-2-2. Doxycycline 13
1-3. 小分子干擾RNA （Small interfering RNA） 14
1-3-3. siRNA-p53 14
1-4. 質體DNA （Plasmid DNA） 14
1-4-4. miR-524-5p 啟動子冷光報導基因 14
1-4-5. pEF tet-off p53 16
1-4-6. pSV40 17
1-5. 抗體（Antibodies） 17
2. 實驗方法（Methods） 17
2-1. DNA 和 RNA 轉染（DNA and RNA transfection） 17
2-2. 蛋白質萃取的準備（Preparation of protein extraction） 17
2-3. 西方墨點法（Western blot） 18
2-4. 細胞增殖測定實驗（Cell proliferation assay） 18
2-5. 細胞群落形成測定實驗（Colony formation assay） 19
2-6. RT-qPCR 19
2-6-7. RNA 萃取（RNA extraction） 19
2-6-8. 反轉錄PCR（Reverse Transcription PCR） 19
2-6-9. cDNA preamp 20
2-6-10. Realtime-PCR 20
2-7. 冷光報導基因偵測 20
實驗結果 21
3. 在黑色素腫瘤中miR-524-5p 和miR-596的表現通過p53的穩定所調控 21
4. 在黑色素腫瘤中miR-524-5p 和miR-596的表現通過過度表現p53所調控 22
5. 在黑色素腫瘤中miR-524-5p和miR-596的表現受到RNA干擾p53的表達所調控 24
6. p53表現量之變化與其所造成之miRNA表現之影響對黑色素細胞腫瘤之生長調控 26
7. p53 調控MIR-524轉錄之預測 27
8. 利用冷光報導基因偵測p53調控MIR-524轉錄之結果 30
結論與討論 33
1. p53在黑色素腫瘤當中調控miR-524-5p的表現 33
2. p53是否直接調控miR-524-5p之轉錄？ 33
3. 其他可能調控miR-524-5p之轉錄因子 34
4. 未來展望 35
參考資料 37
Plasmid information 54
qPCR raw data 64

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指導教授

馬念涵(NianHan Ma)

審核日期

2018-11-6

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