微型核糖核酸是從先導體(precursor)3臂端跟5臂端產出的20到24個核苷酸的短片段非編碼核糖核酸。目前文獻指出，在不同的組織、發育階段跟物種中，成熟微型核糖核酸對於先導微型核糖核酸(precursor miRNA)會有不同的臂端選擇，然而在乳癌中，微型核糖核酸臂端選擇改變(arm switch)的功能跟機制仍然不清楚。本研究，我們利用生物資訊以及實驗的方法去評估乳癌中微型核糖核酸臂端選擇改變(arm switch)的情形，利用癌症基因組圖譜(TCGA)資料庫分析，全面地分析在乳癌中微型核糖核酸臂端選擇的改變，我們找到幾個在乳癌中臂端選擇會發生明顯改變的微型核糖核酸。進一步驗證miR-193a跟miR-324發現5p跟3p的使用率，在乳癌的臨床檢體中明顯發生變動。並且miR-193a-5p/-3p 跟 miR-324-3p的表現量在乳癌檢體中明顯下降，而miR-324-5p的表現量卻是有意義的增加。利用異位表達實驗， miR-193a-5p/-3p跟miR-324-5p/-3p會抑制細胞生長，而miR-193a-3p 跟 miR-324-5p/-3p會抑制爬行以及侵入能力，但miR-193a-5p沒有參與細胞的爬行以及侵入。另外，內生性的miR-193a-5p/-3p跟 miR-324-5p/-3p會因為送進去個別人造目標基因(五個串聯的標的位置)，導致有意義的提高他們的表現量。因此臂端選擇改變的現象可能是癌化的過程中微型核糖核酸的目標基因變多造成，臂端選擇偏好的機制可以用來調控微型核糖核酸的功能，以及增加訊息核糖核酸 (mRNA)網絡的複雜度，總之本研究中，我們發現在乳癌中微型核糖核酸臂端存在選擇改變是非常普遍的，同時提出微型核糖核酸參與乳癌發展的一個新見解。

MicroRNAs (miRNAs) are short noncoding RNAs (20~24 nt) which can be derived from 3' and/or 5' ends of the precursor. Recent literatures suggested that different arms of precursor miRNA can be selected cross different tissues, developmental stages or species. However, the biological function and mechanism of miRNA arm switch remain unclear in breast cancer. In this study, we assessed the roles of miRNA arm switch in breast cancer by bioinformatics and experimental approaches. By comprehensively identifying the miRNA arm switch in the breast cancer by analyzing The Cancer Genome Atlas database (TCGA database), we identified several miRNAs showing significant changes in arm selection in breast cancer compared with adjacent normal tissues. Further examining the expression levels of miR-193a-5p/-3p and miR-324-5p/-3p revealed that the usage of -5p and -3p arm significantly changed in breast cancer compared with adjacent normal tissues. Furthermore, the expression levels of miR-193a-5p/-3p and miR-324-3p significant decreased but miR-324-5p significantly increased in breast cancer samples. Ectopic expression of miR-193a-5p and miR-193a -3p、miR-324-5p and miR-324-3p reduced breast cancer cell growth and migration/invasive ability, whereas miR-193a-5p suppressed breast cancer growth without contributing to breast cancer cell motility. The endogenous levels of miR-193a-5p, -3p, miR-324-5p and -3p significantly increased by transfecting their artificial targets (five tandem target sites) into breast cancer cells. Taken together, the "arm switching" phenomenon may result from the amount of their target genes during breast cancer progression. The arm selection preference of miRNA change may be a way to modulate miRNA function which further complicates the mRNA regulatory network. Our findings suggest that miRNA arm switch is remarkably prevalent in breast cancer, which emerges a new insight in breast cancer progression.

論文審定書 i
誌謝 ii
Abbreviations iii
Abstract in Chinese v
Abstract in English vi-vii
Contents viii-ix
1. Introduction 1
1.1 Breast cancer 1
1.2 MicroRNAs 2
1.3 MicroRNA in Breast cancer 3
1.4 MicroRNA arm switch 3
2. Specific Aims 5
3. Materials and Methods 6
4. Result 13
4.1 Identifly miRNA arm switch using TCGA database 13
4.2 The 5p/3p arm selection of mir-193a and mir-324 in breast cancer cell line
13
4.3 miR 193a-5p/-3p and miR-324-5p/-3p contribute to breast cancer cell growth 15
4.4 Ectopic expression of miR-193a-3p and miR-324-5p/-3p contribute to breast cancer cell motility 16
4.5 Target-mediated miRNA protection (TMMP) involved in miR-#-5p/-3p ratios changes 17
5. Discussion 20
6. References 25
7. Tables 30
8. Figures 37
9. Supplementary data 54

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