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研究生：	陳煥文 Chen, Huan-Wen,
論文名稱：	研究microRNA生成機制對大腸癌抗藥性之影響 Study of MicroRNA Processing Machinery on Drug-resistant Colon Cancer
指導教授：	賴明德 Lai, Ming-Derg
學位類別：	碩士 Master
系所名稱：	醫學院 - 生物化學暨分子生物學研究所 Department of Biochemistry and Molecular Biology
論文出版年：	2018
畢業學年度：	106
語文別：	英文
論文頁數：	91
中文關鍵詞：	DROSHA 、MAF1 、大腸癌、抗藥性、微RNA
外文關鍵詞：	DROSHA, MAF1, colon cancer, drug resistance, micro RNA
相關次數：	點閱：50 下載：0
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大腸癌是診斷比率第三高的癌症，其晚期病人五年存活率極差。目前大腸癌第一線治療方為手術和化療。然而大腸癌容易因為抗藥性，使治療效果受限，而其抗藥機制仍未清楚。miRNA修飾因子(miRNAs processing factors)可能在抗藥機制中扮演重要角色。miRNA修飾因子使miRNA成熟並具有功能性，而其失調（如DROSHA）也與許多癌症不良預後相關。DROSHA的調節會受到其他蛋白質交互作用的影響，RNA聚合酶III（RNAP III）的阻遏蛋白MAF1，可能會提供我們一些線索。在這項研究中，我們發現了下調DROSHA會使HCT-116大腸癌細胞株生長受到抑制，並對化療藥物降低敏感性。而在抗奧沙利鉑(oxaliplatin)的HCT-116細胞中也發現內源性DROSHA的表達量下降，且受到化療藥物刺激下，DROSHA表達量比起一般HCT-116更快速地降低。而在下調MAF1的HCT-116細胞中觀察到DROSHA會高表現的特性，並且通過免疫沉澱法及共聚焦顯微鏡觀察了DROSHA及MAF1其潛在的交互作用。最後利用NGS分析了下游miRNA群體的變異。綜上所述，在大腸癌中DROSHA的失調對抗藥性有重要影響，而MAF1可能是其調節因子之一。最後也提供了一些功能尚未明確的miRNAs作為研究大腸癌抗藥性的潛在標的。

Colon cancer is the third most commonly diagnosed cancer with poor 5-year survival rate of patients in an advanced stage. The main treatment option of colon cancer are surgery and chemotherapy. However, the therapeutic efficacy with colon cancer is limited by drug resistance and the mechanism of resistance remains largely unclear. We proposed that micro RNAs (miRNAs) processing factors, which carry out the maturation of miRNAs, might play an important role in drug resistance. Dysregulation of these miRNA processing factors, such as DROSHA, may correlate to worse clinical outcomes. The regulation of DROSHA could be conducted by the interacting proteins. MAF1, the repressor of RNA polymerase III (RNAP III), may also give a clue to understanding drug resistance. In this study, we demonstrated the proliferation inhibition and chemo-drug insensitivity in DROSHA-downregulated HCT-116 colon cancer cells. Lower endogenous DROSHA expression was found in oxaliplatin-resistant HCT-116 cells. In addition, DROSHA expression was reduced during drug treatment. DROSHA upregulation were observed in the MAF1-downregulated colon cancer cells, and the potential DROSHA/MAF1 interaction were examined by immunoprecipitation and confocal microscopy. The effects of DROSHA and MAF1 on miRNAs were also analyzed by NGS. In conclusion, the dysregulation of DROSHA plays an important role in drug resistance. MAF1 might be a regulator of DROSHA in colon cancer cells and contributes to drug resistance. Finally, we identified certain un-explored miRNAs which may function potential therapy targets for drug resistance in colon cancer.

1. Chinese Abstract
 
2. Abstract
 
3. Table of Content.……………………………………………….I
 
4. List of Table...…………………………………………………III
 
5. List of Figure……………………………………………….…IV
 
6. Introduction....……………………………………………….…1
I. Colon cancer and chemotherapy resistance
II. The regulation of miRNAs in colon cancer
III. DROSHA in the cancer progressions
IV. MAF1 may affect miRNAs biogenesis through the microprocessor complex
 
7. Material and Method..…………………………………………5
I. Cell culture
II. Plasmid preparation
III. Cell transient transfection establishment
IV. Lentivirus-mediated gene knockdown
V. Colony formation assay
VI. Soft agar assay
VII. In vitro drug treatment and cell viability assay
VIII. Western blot
IX. Immunoprecipitation
X. Reverse transcription polymerase chain reaction (RT-PCR)
XI. Real-time PCR (qPCR)
XII. Fluorescent plasmids construction
XIII. Fluorescence resonance energy transfer (FRET)
XIV. Next Generation Sequencing (NGS)
XV. Bioinformatic analysis
XVI. Statistical analysis
 
 
8. Result.…………………………………………………………39
I. DROSHA expression affects colon cancer tumorigenesis
II. Down-regulated DROSHA desensitized the colon cancer to chemotherapy
III. Identification of the resistance relating miRNAs and multiple DROSHA associated anti-chemotherapy miRNAs in HCT-116
IV. MAF1 affects drug sensitivity and miRNAs alterations via upregulates miRNAs processing factors in HCT-116
V. MAF1 regulates DROSHA in nucleus by indirect interactions
 
9. Discussion.………………………………………………….…44
I. The emerging roles of DROSHA in tumorigenicity
II. The stress signals may deliver to DROSHA via MAF1 lactations changing
III. Mutations and PTMs that decide DROSHA functions
IV. The mediators that involved in DROSHA and MAF1 interaction
 
10. Conclusion.……………………………………………………48
 
11. Reference.……………………………………………………..49
 
12. Table…………………………………………………………..55
 
13. Figure…………………………………………………………66
 
14. Addendum……………………………………………………83
Table S1. qPCR primer
Table S2. In-Fusion cloning PCR primer
Table S3. Short hairpin RNA ID and sequence
Table S4. DROSHA co-expression genes in Staub Colon & Vilar Colon
Figure S1. Plasmid maps that used in this study
Figure S2. DROSHA mutations and PTMs sites
Figure S3. Overview of the results
 
                                    

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校內：2023-07-01公開
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