CUHK electronic theses & dissertations collection

目的：結直腸癌是世界上第三常見惡性腫瘤，結腸鏡檢查是診斷的金標准。但其創傷性、昂貴的設備以及人力的需求阻礙了廣泛應用。本研究評估了糞便miRNA作為非損傷性分子生物標記物篩查結直腸腺瘤和腫瘤的可行性，並深入探究了致癌miRNA的基因靶點。方法：我們評估了糞便miRNAs的穩定性以及檢測的可重復性。糞便樣本收集自88例結腸直腸癌患者，57例結直腸息肉患者和101名健康對照，用實時定量逆轉錄PCR檢測miRNA水平。所有候選miRNA標記物在配對的癌及癌旁組織中進行驗証。我們共測試了糞便中7種miRNAs，包括前期報道在結直腸癌中上調的miR-21和miR-92a(第一部分)，以及在667個miRNA中在結直腸癌上調最高的5個miRNA(第二部分)。我們研究了它們的水平與腫瘤分期及位置的關系。並隨訪了病人經腫瘤或腺瘤切除術后其糞便miRNA水平，從而証實它們是否與腫瘤相關。我們應用了彗星試驗、細胞活力試驗、集落形成試驗，以及細胞凋亡分析試驗研究了miR-18a在腫瘤的發展過程中的作用(第三部分)。結果：第一部分，我們確定糞便miRNA的穩定性，能被實時定量逆轉錄PCR檢測並顯示高重復性。糞便miR-92a標記物的靈敏度和特異性分別為71.6%和73.3%，miR-21分別為55.7%和73.3%。MiR-92a水平顯示遠端結直腸癌比近端結直腸癌的檢測具有更高靈敏度，晚期腺瘤比小息肉更具靈敏度。腫瘤切除后，miR-21和miR-92a水平顯著下降。第二部分，基於結直腸腫瘤miRNA的表型，我們發現糞便miR-18a, miR-20a, miR-135b和miR-221能作為標記物鑒別結直腸癌，miR-18a (敏感度: 51.1%, 特異性: 90.1%); miR-20a (72.7%, 81.2%) ; miR-135b (81.8%, 68.3%); miR-221 (69.3%, 77.2%)。腫瘤切除后，這四種標記物會顯著下降。MiR-135b和miR-221也能鑒別腺瘤。四種標記物對遠近端結腸癌的檢測無顯著差異。第三部分，通過程序和熒光素酶報告基因活性預測和驗証，我們發現一種重要的DNA修復蛋白---共濟失調毛細血管擴張突變(ATM)是miR-18a的靶蛋白。MiR-18a的異位表達減弱細胞DNA雙鏈損傷修復機制，導致腫瘤發生的易感性。結論：我們發現糞便中miR-21, miR-92a, miR-18a, miR-20a, miR-135b 和miR-221標記物能夠鑒別結直腸癌。MiR-92a, miR-135b 和miR-221能鑒別結直腸腺瘤。MiR-18a抑制共濟失調毛細血管擴張突變基因表達並減弱細胞DNA雙鏈損傷修復機制。糞便miRNA是結直腸癌篩查的有效生物標記物。Objective: Colorectal cancer (CRC) is the third most common cancer worldwide. Colonoscopy is the current gold standard for diagnosing CRC. However, its invasive nature, the cost of equipment and the demand for manpower have hampered the wide application of this procedure. This study evaluated the feasibility of using stool-based miRNA as non-invasive biomarkers for the screening of colorectal adenoma and cancer, and investigated the gene target of a candidate oncogenic miRNA.Methods: The reproducibility of detection and stability of stool-based miRNAs were first evaluated. Stool samples were collected from 88 CRC patients, 57 patients with colorectal polyp and 101 healthy controls MiRNA levels were detected by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). AII candidate miRNA markers were validated in a cohort of paired tumor and adjacent normal tissues. In total, we tested 7 miRNAs in the stool, including miR-21 and miR-92a which were reported to be up-regulated in CRC in previous studies (part one), and 5 miRNAs which were found to be the most up-regulated in colorectal tumor based on the profiling of 667 miRNAs (part two). Their levels with tumor stage and location were evaluated. Their change in level was followed up in a subset of patients after the removal of tumor or adenoma. We investigated miR-18a for its function in cancer development using comet assay, cell viability assay, colony formation assay, and analysis on apoptosis (part three).Results: In part one, we found stool miRNAs stable and detectable with high reproducibility by qRT-PCR. In detecting CRC, stool miR-92a had a sensitivity of 71.6% and a specificity of 73.3%, stool miR-21 had a sensitivity of 55.7% and a specificity of 73.3%. Stool miR-92a level had higher sensitivity for distal CRC than proximal CRC, and a higher sensitivity for advanced adenoma than minor polyps. The removal of tumor resulted in reduced stool miR-21 and miR-92a levels.In part two, based on miRNA profiling of CRC tumors, we found that stool-based miR-18a, miR-20a, miR-135b, and miR-221 can discriminate colorectal cancer patients from healthy individuals: miR-18a (sensitivity: 51.1%, specificitiy: 90.1%); miR-20a (72.7%, 81.2%); miR-135b (8 1.8%, 68.3%); miR-221 (69.3%, 77.2%). Levels of these 4 stool-based markers dropped after removal of tumors. Stool-based miR-135b and miR-221 also discriminated patients with adenoma from healthy individuals. MiR-18a, miR-20a, miR-135b and miR-221 showed no desparity in detecting proximal or distal colon cancer.In part three, based on in silico prediction and validation with luciferase reporter activity, we identified Ataxia Telangiectasia Mutated (ATM ), a protein crucial to DNA repair, as a target of miR-18a. Ectopic expression miR-18a attenuates DNA double strand break repair mechanism, creating a genetic predisposition to the development of cancer.Conclusion: Stool-based miR-21, miR-92a, miR-18a, miR-20a, miR-135b and miR-221 can discriminate patients with CRC from healthy individuals. Notably, a subset of these miRNAs (miR-92a, miR-135b, and miR-221) can discriminate patients with colorectal adenoma from healthy individuals MiR-18a suppressed ATM gene expression and attenuated cellular repair mechanism to DNA double strand breaks. Stool-based miRNAs are useful CRC screening biomarkers.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.Wu, Chung Wah.Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.Includes bibliographical references (leaves 80-92).Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.Abstract also in Chinese.Chapter CHAPTER ONE --- INTRODUCTION --- p.1Chapter 1.1 --- Colorectal cancer --- p.1Chapter 1.2 --- Current screening methods --- p.3Chapter 1.2.1 --- Colonoscopy --- p.3Chapter 1.2.2 --- Sigmoidoscopy --- p.4Chapter 1.2.3 --- Stool-based tests --- p.4Chapter 1.2.3.1 --- Fecal occult blood test --- p.5Chapter 1.2.3.2 --- Stool-based DNA test --- p.6Chapter 1.2.3.3 --- Stool-based RNA and protein test --- p.7Chapter 1.3 --- MiRNA and its role in cancer --- p.8Chapter 1.4 --- Aims of study --- p.9Chapter CHAPTER TWO --- METHODOLOGY --- p.10Chapter 2.1 --- Subjects and sample collection --- p.10Chapter 2.2 --- MiRNA extraction in tissue and stool samples --- p.13Chapter 2.3 --- MiRNA quantitation by quantitative reverse transcription quantitative reverse transcription polymerase chain reaction --- p.14Chapter 2.4 --- Determining the stability of miRNA in stool samples --- p.15Chapter 2.5 --- Determining the reproducibility of miRNA quantitation in stool samples --- p.16Chapter 2.6 --- Reverse transcription for miRNA array --- p.16Chapter 2.7 --- Quantitative polymerase chain reaction for miRNA array --- p.16Chapter 2.8 --- Cell culture, miRNA precursors and transfection --- p.17Chapter 2.9 --- Dual-luciferase reporter assay --- p.17Chapter 2.10 --- Quantitative reverse transcription polymerase chain reaction for mRNA --- p.19Chapter 2.11 --- Western blot analysis --- p.19Chapter 2.12 --- Comet assay --- p.19Chapter 2.13 --- Colony formation and cell viability assay --- p.20Chapter 2.14 --- Annexin V apoptosis assay --- p.21Chapter 2.15 --- Statistics --- p.21Chapter CHAPTER THREE --- RESULTS --- p.23Chapter 3.1 --- PART 1 --- p.23Chapter 3.1.1 --- Stability of miRNA detection in stool samples --- p.23Chapter 3.1.2 --- Reproducibility of miRNA quantitation in stool samples --- p.23Chapter 3.1.3 --- Detection and normalization of miRNA levels --- p.25Chapter 3.1.4 --- Expression of miR-21 and miR-92a in CRC tissue samples --- p.28Chapter 3.1.5 --- Levels of stool-based miR-21 and miR-92a in CRC and polyp patients --- p.30Chapter 3.1.6 --- Sensitivity of stool-based miR-21 and miR-92a towards colorectal cancer and polyps --- p.32Chapter 3.1.7 --- Association of stool-based miR-21 and miR-92a with clinicopathological features --- p.34Chapter 3.1.8 --- Follow-up on stool miR-21 and miR-92a levels after removal of lesion --- p.37Chapter 3.2 --- Part 2 --- p.39Chapter 3.2.1 --- MiRNA profiling in colorectal tumors --- p.39Chapter 3.2.2 --- Validation of miRNA profiling results --- p.41Chapter 3.2.3 --- Candidate miRNA levels in stool samples of CRC and adenoma patients --- p.44Chapter 3.2.4 --- Sensitivities and specificities of miRNA candidates for adenoma and CRC --- p.47Chapter 3.2.5 --- Sensitivties of miRNA candidates based on tumor location --- p.49Chapter 3.2.6 --- Follow-up on stool miRNA levels after removal of lesion --- p.51Chapter 3.2.7 --- Association of stool-based miRNAs with nodal involvement in CRC --- p.53Chapter 3.2.8 --- Establishing the miRNA marker panel --- p.55Chapter 3.3 --- Part 3 --- p.57Chapter 3.3.1 --- In Silico prediction of miR-18a target and validation by luciferase assay --- p.57Chapter 3.3.2 --- Expression and correlation between miR-18a and ATM in paired colorectal tumor tissue, cell lines and normal colon biopsies --- p.60Chapter 3.3.3 --- Regulation of double strand DNA damaga recovery by miR-18a --- p.62Chapter 3.3.4 --- Cell sensitization to genotoxin by miR-18a --- p.64Chapter 3.3.5 --- Effect of miR-18a on genotoxin induced apoptosis --- p.66Chapter CHAPTER FOUR --- DISCUSSION --- p.68Chapter 4.1 --- Stability and detection reproducibility of stool-based miRNA --- p.68Chapter 4.2 --- Stool-based miRNAs for screening colorectal cancer and polyps/adenomas --- p.69Chapter 4.2.1 --- MiR-21 --- p.69Chapter 4.2.2 --- MiR-18a, miR-20a and miR-92a --- p.70Chapter 4.2.3 --- MiR -135b and miR -221 --- p.71Chapter 4.2.4 --- MiR -31 --- p.72Chapter 4.3 --- Discriminating proximal and distal CRC --- p.73Chapter 4.4 --- Evaluation of stool-based miRNA level after removal of lesions --- p.74Chapter 4.5 --- MiRNA marker panel --- p.74Chapter 4.6 --- Advantages of stool-based miRNA tests --- p.75Chapter 4.7 --- Ataxia telangiectasia mutated as the direct target of miR -18a --- p.75Chapter 4.8 --- Future directions for study --- p.78Chapter 4.9 --- Conclusion --- p.78REFERENCES --- p.80PUBLICATIONS --- p.9