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研究生:吳柏翰
研究生(外文):WU, Pohan
論文名稱:p53蛋白在表皮細胞中做為一個轉錄因子調控miR-203
論文名稱(外文):P53 acts as a transcription factor to regulate miR-203 in epidermal cell
指導教授:陳正繹
指導教授(外文):Chen, Jang yi
口試委員:陳正繹趙壯飛周成功
口試委員(外文):Chen, Jang yiChao, Chung-FayeChou, Chen-Kung
口試日期:2012-05-28
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物及解剖學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:42
中文關鍵詞:微核醣核酸-203
外文關鍵詞:miR-203
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  • 下載下載:8
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p63蛋白已知做為表皮分化的主要調節蛋白,但在表皮分化中各因子之間的影響仍未清楚。p63蛋白在表皮分化的過程之中,表現量會明顯的下降,因此以p63蛋白做為表皮幹細胞的生物標幟。microRNA-203 (miR-203)為其中一種主要表現在表皮細胞的microRNA,miR-203在表皮分化過程中,有被報導出表現量有上升的現象,或是在某些皮膚的疾病也可發現有表現量異常情形。miR-203會影響p63蛋白在細胞中的表現量;藉由與p63 mRNA 3’UTR結合,使p63 mRNA不穩定而降低p63蛋白在細胞中的表現量,進而影響表皮的分化。在我們實驗室先前的研究指出,與p63蛋白同家族成員的p53蛋白在表皮分化過程中會影響角質蛋白14的表現,對角質蛋白14造成負調控的影響。而p53蛋白被報導指出在表皮細胞中會正向調控miR-203的生成。以此我們推測,在表皮分化過程中,p53蛋白活化下游基因之一的miR-203,而生成的miR-203造成p63蛋白表現的下降。為了進一步了解p53蛋白對miR-203的調控,我們藉由建構不同長度的miR-203啟動子,以報導基因分析初步判斷p53蛋白對啟動子影響的區域;我們發現在此段啟動子較上游的序列會受到p53蛋白的影響,而有較高的冷光活性表現,另外我們也找出在這段區域中,有p53可能的結合位置。因此我們針對這段可能的p53全結合位,以專一位置突變法改變這段p53全結合位,並以報導基因分析法來探討p53對此段序列的影響,最後以合成方式製成p53全結合位序列做為探針,利用DNA親和性沉澱分析法證實p53蛋白會與此段序列直接結合。因此,這些結果證明了,p53蛋白可做為轉錄因子直接結合在miR-203啟動子上的p53全結合位,正向調控miR-203的表現。
p63 acts as a master regulator of epidermal differentiation is well known; although the signaling cascades required for this process is still largely unknown. p63 expression decreases gradually during epidermal differentiation process, so p63 is considered as a biomarker of epidermal stem cells.
MicroRNA-203 (miR-203) is the one of the micro-RNAs expresses abundantly in the terminally differentiated supra-basal epidermal cells. In the previous studied, MiR-203 was found with perfect binding to the target site on 3’untranslated terminal region (UTR) of p63 mRNA to affect the p63 expression during epidermal differentiation. Our previous studies have found that p53 acts as co-repressor to down-regulate the keratin 14 gene expression during epidermal differentiation, and demonstrated that p53 participated in this process. p53 have been mentioned could up-regulate mir-203 in previous reports. To evaluate the phenomenon of p53 regulated miR-203 may further inference epidermal differentiation, we constructed the different large fragment deletion mutants of miR-203 promoter and analyzed their activity under p53 regulation with report assay. We found more upstream promoter region deletion highly inferences the p53 function and we also found a p53 putative binding site within this region through the sequence analysis. Further we used site-direct mutagenesis and reporter assay to evaluate the putative binding region of miR-203 promoter would be affected by p53. Finally, we used DNA affinity purification assay (DAPA) to confirm the interrelationship between p53 and miR-203 promoter Therefore, we conclude that p53 acts as transcription factor, regulate miR-203 through direct binding the miR-203 promoter.

目錄

目錄 …………………………………………………………………… I-II
中文摘要……………………………………………………………… 1-2
英文摘要……………………………………………………………… 3
本文
壹、 緒論………………………………………………………… 4-8
貳、 實驗材料 ………………………………………………… 9-15
1. 腫瘤細胞株………………………………………………… 9
2. 實驗藥品與試劑…………………………………………… 9-11
3. 引子合成…………………………………………………… 11-12
4. 重要儀器…………………………………………………… 12-15
參、 實驗方法…………………………………………………… 16-22
1. 腫瘤細胞繼代培養………………………………………… 16
2. 專一位置突變法…………………………………………… 16-17
3. 轉形作用…………………………………………………… 17
4. 抽取質體DNA ……………………………………………… 17
5. 蟲光酶分析………………………………………………… 17-18
6. 細胞轉染…………………………………………………… 18
7. 製備RNA反轉錄成cDNA ……………………………………18-19
8. 定量PCR…………………………………………………… 19
9. DNA親和性沉澱分析……………………………………… 19-21
10. 西方墨點法………………………………………………… 21-22
11. 統計方法…………………………………………………… 22
肆、 結果………………………………………………………… 23-25
1. p53蛋白可誘導H1299細胞株產生miR-203……………… 23
2. p53蛋白可影響miR-203啟動子的活性…………………… 23
3. 初步篩選miR-203啟動子可能被p53影響的區域………… 23-24
4. p53藉由miR-203啟動子上-1033~-1010區域影響下游基因… 24-25
5. p53可以直接結合在miR-203啟動子-1033~-1010的區域…… 25
伍、 討論 ………………………………………………………… 26-28
陸、 實驗圖表 …………………………………………………… 29-36
圖1.轉殖p53基因進入H1299細胞,影響pri-miR-203的表現…… 29
圖2. p53影響miR-203啟動子的轉錄活性…………………………… 30
圖3. 初步篩選miR-203啟動子上p53可能影響的位置…………… 31-32
圖4. 探討p53對miR-203啟動子上-1033~-1010的影響………… 33-34
圖5. p53與miR-203啟動子上-1033~-1010區域序列直接結合… 35
柒、 參考文獻 …………………………………………………… 36-39

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