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研究生:張言溥
研究生(外文):Yen-Pu Chang
論文名稱:MAPK傳導路徑相關微型RNA在黑色素瘤細胞中功能之研究
論文名稱(外文):Study of functions of MAPK signaling pathway related microRNAs in melanoma cells
指導教授:馬念涵
指導教授(外文):Nianhan Jia-Lin Ma
學位類別:碩士
校院名稱:國立中央大學
系所名稱:系統生物與生物資訊研究所
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:2014
畢業學年度:102
語文別:英文
論文頁數:56
中文關鍵詞:MAPK 訊息傳導路徑微型RNA黑色素瘤
外文關鍵詞:MAPK signaling pathwaymicroRNAMelanoma
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先前的研究指出,大約有50-70%的黑色素瘤包含BRaf基因的突變,其中80%是V600E的取代突變,這個突變持續性地誘導MAPK/ERK訊息傳導路徑的活化也造成癌症的惡性表現型。很多訊息傳導路徑被發現會被微型RNA調控;然而,微型RNA、黑色素瘤與MAPK路徑之間的關聯性尚不明確。從先前微陣列晶片實驗和GEO資料庫篩選出與MAPK相關的微型RNA。為了更深入的研究,未來將會實施動物實驗。闡明微型RNA在黑色素瘤中的角色可能可以提供其作為癌症生物標記或微型RNA的標靶治療。
It has been reported that approximately 50-70% of melanoma samples contain BRaf mutations, 80% of which is V600E. The mutation constitutively induces high activity in MAPK/ERK signaling pathway and causes malignant phenotypes of cancers. MAPK/ERK signaling may be modulated by microRNA (miRNA); however, correlations between miRNA, melanoma and MAPK/ERK pathway are still unclear. We screen out MAPK-related miRNA from the prior microarray results and GEO data analysis. For further investigation, experiments should be performed in vivo in the future. Elucidating the role of miRNAs in melanoma may provide as biomarkers or a miRNA-targeting therapy for cancers.
Abstract in Chinese i
Abstract in English ii
Abbreviation list iii
Acknowledgements iv
Table of contents v
List of tables viii
List of figures viii
I. INTRODUCTION 1
1. miRNA (microRNA) 1
1.1 Biogenesis of miRNA 1
1.2 Mechanisms of miRNA regulation 2
2. miRNA dysregulation in cancer 3
3. MAPK/ERK signaling pathway and human cancer 4
4. Melanoma 6
5. Genetic defects and target therapy of melanoma 7
6. Significance and purpose of the study 7

II. MATERIALS AND METHODS 9
1. Materials 9
1.1 Cell lines 9
1.2 miRNA (microRNA) mimics 9
1.3 Plasmids 10
1.4 Antibodies 10
2. Methods 11
2.1 miRNA mimic transfection 11
2.2 Cell proliferation assay – Alamar blue 11
2.3 Cell proliferation assay – Colony formation assay 11
2.4 Transwell assay 12
2.5 Wound healing assay 12
2.6 Soft agar assay 13
2.7 Construction of miRNA Stable clones 13
2.8 GEO data analysis 14
2.9 Preparation of protein extracts 14
2.10 Western blot analysis 15
2.11 Data process software and hardware 16

III. RESULTS 17
1. Screen miRNAs which is related to MAPK signaling pathway by microarray and GEO data 17
2. Overexpression of MAPK-related miRNAs reduce MAPK signaling pathway activity 18
3. Overexpression of MAPK-related miRNAs cause down-regulation of MAPK signaling pathway activity 18
4. MAPK-related miRNAs induce cell apoptosis and cause cell cycle arrest 19
5. MAPK-related miRNAs decrease cancer cell proliferation 20
6. MAPK-related miRNAs repress anchorage-independent growth
21
7. MAPK-related miRNAs inhibit cell migratory ability 21
8. Stable-expressed MAPK-related miRNAs inhibit cell migration
22 
IV. DISCUSSION 23
1. miRNA affect cell signaling pathways 23
2. miRNA influence cancer cells progress 23
3. Studies related to our MAPK-related miRNAs 24
4. Future aspect 25

V. REFERENCES 26
Tables 29
Figures 32
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