運用miRNA微陣列手段，我們篩選到一批在結直腸腫瘤內高表達和低表達的miRNA。其中miR-133a是在腫瘤中最顯著降低的miRNA之一， 但其在腫瘤的發生發展過程中的作用尚未可知，因此我們選擇miR-133a最為研究目標，論證其的生物學功能，分子機理，及其在結直腸癌中的靶分子。 / 我們首先在較大規模樣本中驗證miR-133a的低表達。定量PCR結果顯示，對比正常的癌旁組織，miR-133a在94例結直腸癌組織中顯著低表達（p < 0.001）。我們進一步分析miR-133a在9種常用的腫瘤細胞系內的表達情況。對比正常的直腸組織，miR-133a在9種常用的結直腸細胞系內的表達均明顯下降。在腫瘤發生發展過程中，癌細胞趨向於降低具有抑癌功能的基因的表達。因此我們推測miR-133a是一個潛在的腫瘤抑制miRNA。 / 為了論證我們的假設，我們首先選取miR-133a低表達的結直腸癌細胞系HCT116和LoVo最為研究模型，將miR-133a在這兩種細胞系內過表達。升高的miR-133a可以抑制腫瘤細胞生長（p < 0.01和p < 0.05），抑制腫瘤克隆集落形成（p < 0.01）。我們進一步發現過表達miR-133a可以抑制裸鼠體內腫瘤的生長（p < 0.05）。細胞週期分析顯示miR-133a抑制細胞生長的能力表現為誘導腫瘤細胞週期阻滯於G0/G1期。細胞週期特異性CDK抑制蛋白p21和p27對於細胞週期調節十分關鍵。基於此項觀察，我們進一步分析了p21和p27的表達。Western-blot實驗證實過表達miR-133a可以促進HCT116和LoVo細胞內p21和p27的蛋白上調。但miR-133a在p53突變型的HT29過表達後並沒有引起p21的變化。通過對比p53野生型和突變性細胞系，我們發現p53對於miR-133a誘導p21是十分關鍵的。為了證明miR-133a可以引起p53的活性，我們通過啟動子螢光報告實驗證實，miR-133a不僅可以促進p53結合DNA 的能力，而且可以引起p21啟動子的活性。其次，我們發現在p53野生型細胞株HCT116中，轉染si-p53可以拮抗miR-133a誘導p21。我們通過蛋白降解實驗發現，miR-133a可以延長p53蛋白的半衰期。 / 基於以上實驗事實，我們推測miR-133a誘導p21是通過穩定p53蛋白來實現的。通過數個miRNA靶基因預測軟體，我們判斷RFFL可能是miR-133a發揮效力的直接靶基因。RFFL是E3連接酶，負責非磷酸化p53和磷酸化p53的降解。在RFFL的3側非翻譯區有一個進化保守的miR-133a識別序列。我們克隆此段序列到螢光素酶基因的3側非翻譯區，並進行雙螢光素酶報告基因檢測。測試發現miR-133a可以直接結合到RFFL的此段序列上，並抑制前段基因的翻譯。體外實驗證實，過表達miR-133a可以減少HCT116和HT29細胞內的RFFL蛋白，但並不影響其mRNA。過表達RFFL可以降低p53的表達，反之降低RFFL的蛋白表達，可以提高p53和p21蛋白。上述實驗均有助於證實miR-133a是通過抑制RFFL來提高p21的表達，進而抑制細胞週期。 / 我們也發現miR-133a具有增敏抗癌藥物的效力。單獨轉染miR-133a並不能顯著引發細胞凋亡，而聯合使用miR-133a及抗癌藥物doxorubicin，或者Oxaliplatin都可以顯著增強細胞的早期凋亡。 / 基於上述實驗結果，我們證實miR-133a是一個抑制腫瘤生長的miRNA，其機制可能是通過抑制RFFL蛋白的表達，並啟動p53/p21信號通路引起的。我們的實驗說明miR-133a可以對抗腫瘤藥物起到增敏的作用。 / We found that miR-133a was significantly down-regulated in colorectal cancer (CRC) tissues using miRNA array. However, the role of miR-133a in CRC is largely unknown. We sought to clarify its biologic function, molecular basis, and target gene in CRC. The down-regulation of miR-133a was verified in 94 primary CRC tumours compared with the matched adjacent normal tissues (p < 0.001) and in 9 human colon cancer cell lines. Ectopic expression of miR-133a in colon cancer cell lines (HCT116 and LoVo) significantly suppressed cell growth as evidenced by cell viability assay (p < 0.01 and p < 0.05), and colony formation assay (p < 0.01). Cell cycle analysis revealed that miR-133a caused an increased proportion of cells arrested at G0/G1 phase in HCT116 and LoVo, concomitant with the up-regulation of key G1 phase regulators CDK inhibitors p21 and p27. Promoter-luciferase activity assays indicated that miR-133a markedly increased p53 binding activity and induced p21 transcription. We further revealed that miR-133a decelerated p53 degradation, suggesting that miR-133a was an important positive modulator of the p53/p21 pathway. In silico search showed that the 3’UTR of ring finger and FYVE-like domain containing E3 ubiquitin protein ligase (RFFL), an E3 ubiquitin protein ligase targeting p53 for degradation, contains an evolutionarily conserved miR-133a binding site. Co-transfection with miR-133a repressed RFFL-3’UTR reporter activity for up to 53% (p < 0.01) and remarkably reduced RFFL protein level, indicating that miR-133a directly bound to RFFL mRNA and inhibited RFFL translation. Moreover, miR-133a sensitized colon cancer cells to chemotherapeutic agents (doxorubicin and oxaliplatin) by enhancing apoptosis (p < 0.01) and inhibiting cell proliferation (p < 0.001), adding further weight to the potential significance of miR-133a as a tumour suppressor in inhibiting CRC. In conclusion, miR-133a serves as a functional tumour suppressor in CRC through direct inhibition of the oncogenic RFFL and induction of the p53/p21 pathway. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Dong, Yujuan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 139-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / ABSTRACT --- p.I / ACKNOWLEDGMENTS --- p.VI / LIST OF FIGURES --- p.VII / LIST OF TABLES --- p.IX / ABBREVIATIONS --- p.X / TABLES OF CONTENT --- p.XIII / Chapter CHAPTER ONE --- INTRODUCTION --- p.1 / Chapter 1.1 --- Colorectal cancer --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.1 / Chapter 1.1.2 --- Etiology --- p.4 / Chapter 1.1.3 --- CRC prevention, screening and therapy --- p.7 / Chapter 1.2 --- microRNA (miRNA) --- p.13 / Chapter 1.2.1 --- Biogenesis of miRNA --- p.13 / Chapter 1.2.2 --- Diagnostic value of miRNAs in CRC --- p.16 / Chapter 1.2.3 --- Prognostic value of miRNAs in CRC --- p.25 / Chapter 1.2.4 --- Predictive value of miRNAs for treatment response in CRC --- p.28 / Chapter 1.2.5 --- miRNA-related single-nucleotide polymorphisms and CRC --- p.29 / Chapter 1.2.6 --- miRNAs and their function in CRC genesis --- p.33 / Chapter 1.2.7 --- Future perspective of miRNAs in CRC --- p.41 / Chapter 1.3 --- Hypothesis and objectives --- p.41 / Chapter CHAPER TWO --- METHODOLGY --- p.43 / Chapter 2.1 --- Cell cultures --- p.43 / Chapter 2.2 --- Patients and clinical specimens --- p.43 / Chapter 2.3 --- miRNA extraction from tissue and cell --- p.46 / Chapter 2.4 --- Micro-dissection and RNA extraction from paraffin sections --- p.47 / Chapter 2.5 --- Real-time quantitative PCR for miRNA microarray --- p.48 / Chapter 2.6 --- miRNA expression analysis --- p.48 / Chapter 2.7 --- mRNA expression analysis --- p.49 / Chapter 2.8 --- RNA interference --- p.52 / Chapter 2.9 --- Colony formation assay --- p.52 / Chapter 2.10 --- MTT cell viability assay --- p.53 / Chapter 2.11 --- Flow cytometry for cell cycle analysis --- p.53 / Chapter 2.12 --- Flow cytometry for cell apoptosis analysis --- p.54 / Chapter 2.13 --- Protein degradation assay --- p.54 / Chapter 2.14 --- Western blot analysis --- p.55 / Chapter 2.15 --- Immunofluorescence staining --- p.56 / Chapter 2.16 --- Plasmids construction --- p.58 / Chapter 2.16.1 --- pRFFL plasmid --- p.58 / Chapter 2.16.2 --- pMIR-RFFL-3’UTR and pMIR-RFFLmut-3’UTR --- p.58 / Chapter 2.17 --- Construction of stable cell lines --- p.59 / Chapter 2.18 --- Dual-luciferase reporter assay for p53 signaling pathway --- p.61 / Chapter 2.19 --- Dual-luciferase reporter assay for RFFL 3’UTR and miR-133a binding activity --- p.62 / Chapter 2.20 --- 5-Aza-2'-deoxycytidine (5-Aza-dC) treatment --- p.62 / Chapter 2.21 --- Tumour xenografts in nude mice model (miRNA intratumoural injection model) --- p.63 / Chapter 2.22 --- Tumour xenografts in nude mice model (miRNA stable cell line subcutaneous injection) --- p.64 / Chapter 2.23 --- Statistical analysis --- p.64 / Chapter CHAPTER THREE --- RESULTS --- p.66 / Chapter 3.1 --- Identification of differentially expressed miRNAs in CRC --- p.66 / Chapter 3.2 --- miR-133a is down-regulated in primary human CRC and colon cancer cell lines --- p.69 / Chapter 3.3 --- Ectopic expression of miR-133a inhibits tumourigenic properties of CRC cells --- p.75 / Chapter 3.3.1 --- miR-133a suppresses cell viability and colony formation --- p.75 / Chapter 3.3.2 --- miR-133a inhibits tumour growth in nude mice --- p.78 / Chapter 3.3.3 --- miR-133a suppresses cell cycle progression --- p.81 / Chapter 3.4 --- G0/G1 phase arrest by miR-133a is mediated through up-regulation of CDKN1A and CDKN1B --- p.83 / Chapter 3.5 --- miR-133a activates p53/p21 pathway through stabilization of p53 protein --- p.88 / Chapter 3.5.1 --- miR-133a induces p21 expression in a p53 wild-type cells --- p.88 / Chapter 3.5.2 --- miR-133a induces p21 promoter transcription activity and p53 binding activity --- p.90 / Chapter 3.5.3 --- Silence of p53 abolished miR-133a induced p21 --- p.92 / Chapter 3.5.4 --- miR-133a increases p53 activity through increase of p53 protein stability --- p.95 / Chapter 3.5.5 --- miR-133a has no effect on c-Myc level --- p.98 / Chapter 3.6 --- miR-133a increases p53 protein level by directly down-regulating RFFL --- p.100 / Chapter 3.7 --- Knock-down of RFFL inhibits cancer cell growth --- p.105 / Chapter 3.8 --- miR-133a sensitized CRC cells to chemotherapeutic drugs treatment --- p.110 / Chapter 3.9 --- Pharmacological demethylation restores miR-133a expression in CRC cells --- p.117 / Chapter 3.10 --- Association between miR-133a expression in tumour and clinicopathological characteristics of CRC patients --- p.119 / Chapter 3.11 --- Validation of other dysregulated miRNAs in CRC --- p.123 / Chapter CHAPTER FOUR --- DISCUSSION --- p.128 / Chapter 4.1 --- Biological role of miR-133a as a tumour suppressor --- p.128 / Chapter 4.2 --- p53/p21 pathway is a critical mediator of miR-133a in CRC --- p.129 / Chapter 4.3 --- Functional significance of RFFL in miR-133a induced p53/p21 signaling --- p.131 / Chapter 4.4 --- Clinical potential of miR-133a in CRC --- p.133 / Chapter 4.5 --- Other dysregulated miRNA --- p.136 / Chapter 4.6 --- Limitations and improvements of the study --- p.136 / Chapter 4.7 --- Conclusions --- p.137 / REFERENCE --- p.139 / PUBLICATIONS --- p.153
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328257 |
| Date | January 2012 |
| Contributors | Dong, Yujuan., Chinese University of Hong Kong Graduate School. Division of Surgery. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xv, 154 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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