Chan Wing Yan Michael. / "August 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 168-200). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

膀胱尿路上皮腫瘤發病率在泌尿道腫瘤中排第二位，它具有高複發性的特點。目前，有創性尿道膀胱鏡檢查是診斷的金標準。儘管先後有很多血液或尿液中的分子被先後用於診斷膀胱癌的診斷研究，但到目前為止尚未有任何一種方法可以取代膀胱鏡檢查。有證據表明在膀胱上皮腫瘤組織中有很多異常表達的microRNA，但是內在機制的有關研究相對缺乏。在本研究中，我們利用在尿液上清中異常表達的microRNA來評估它們在膀胱癌診斷中的價值。而且，我們揭示了其潛在的調控機理。通過microRNA基因芯片，我們結合并對比來自膀胱腫瘤病人和正常對照患者的9個尿液上清樣本，以及4對腫瘤組織及臨近正常黏膜上皮中microRNA的表達，初步篩選出10個異常的microRNA。然後我們使用定量RT-PCR的方法在另外獨立的18對腫瘤組織和正常黏膜中進一步驗證芯片結果。最後我們就6個被帥選出來的microRNA在71例的膀胱癌患者和正常對照組的尿液上清中進行檢測並評估其診斷效能。我們發現，miR-125b和miR-99a的表達在膀胱癌患者的尿液上清中明顯下調。另外，它們下調程度與腫瘤的病理分級相關。結合miR-125b和miR-99b兩者作為診斷膀胱癌的指標，靈敏度達86.7%，特異度達81.1%，同時有陽性預測值達91.8%。當作為腫瘤分級指標，miR-125b具有81.4%的敏感度，87.0%的特異度，陽性預測值達93.4%。膀胱腫瘤切除之後，和術前比較，兩個microRNA的表達水平再度上升。我們將miR-99轉染到三個膀胱腫瘤細胞株中（T24，UMUC3和J82）。我們發現miR-99a對UMUC3細胞具有輕微的抗增殖功能。同時，miR-99a在3個細胞株中顯示均顯示具有抗遷移和抗侵襲能力。為尋找miR-99a的目標mRNA，我們結合數據庫算法預測，在Western blot中驗證到miR-99a能顯著下調VLDLR蛋白。隨後我們將帶有VLDLR的3'UTR質粒轉染進入細胞中并證實VLDLR mRNA是miR-99a直接作用的目標。另外，當VLDLR siRNA被轉入3個細胞株之後，我們觀察到相似的抗遷移和抗侵襲的現象。最後我們發現N-cadherin是該通路中的下游抑制遷移和侵襲的分子。本項研究證實研究尿液上清中的microRNA是可行的。MiR-125b和miR-99a是膀胱腫瘤的診斷和分級的有效指標。此外，miR-99a能夠通過和VLDLR mRNA直接結合從而抑制膀胱腫瘤遷移和侵襲功能。 / Urothelial carcinoma of the bladder (UCB) is the second most common malignancy in the urological system with high recurrence rate. Current gold standard examination for diagnosis is urethrocystoscopy, which is an invasive procedure. Although numerous molecular markers in blood or urine have been proposed as diagnostic biomarkers for bladder cancer, none of them could replace urethrocystoscopy in clinical practice. There are accumulating evidences suggesting microRNA dysregulation might be related to the pathogenesis of UCB. However, the exact functions of these microRNAs in UCB remain unknown. In this thesis, the role of selected microRNAs in urine supernatant was investigated in the diagnosis of UCB and also the carcinogenesis of UCB. / In brief, a high-throughput microarray was carried out on nine supernatants of urine from UCB and normal subjects, and also four pairs of tissue from UCB and normal mucosa. Ten microRNA candidates were then identified. Quantitative RT-PCR was used to validate these microRNAs on a set of 18 pairs of tumor tissue and normal mucosa. Eventually, six potential candidate microRNAs were selected and then validated as diagnostic tools on the samples of urine supernatants from 71 patients (50 of known UCB and 21 of normal subjects). The expression levels of these selected microRNAs were further evaluated in the urine supernatants of 20 patients after tumors resections. MiR-125b and miR-99a were the two most significantly down-regulated microRNAs in the urine supernatants of patients with UCB. Moreover, the degree of down-regulation was associated with the pathological grade of the tumor. A combined index of miR-125b and miR-99a in urine supernatant had a sensitivity of 86.7%, specificity of 81.1%, and a positive predicted value of 91.8% for diagnosing UCB. When used to discriminate high-grade from low-grade UCB, miR-125b alone had a sensitivity of 81.4%, specificity of 87.0% and PPV of 93.4%. After transurethral resections, the expression levels of both microRNAs were significantly increased compared to pre-operative levels. / In further studies on the role of microRNAs on the development of UCB, miR-99a was selected for further studies. The precursor of miR-99a was temporally transfected into 3 bladder cancer cell lines: T24, UMUC3 and J82. The proliferation ability was noticed to be suppressed mildly in UMUC3, but not the other. Meanwhile, migration and invasion abilities were inhibited by miR-99a in the all 3 cell lines. Potential targets of miR-99a were predicted from several prediction databases. Subsequently, in Western Blot study, the protein level of very low density lipoprotein receptor (VLDLR) was showed to be down-regulated by miR-99a. Thereafter, a plasmid constructed with 3’UTR of VLDLR was transfected into cytoplasm, which confirmed VLDLR mRNA was a direct target of miR-99a. All 3 cells lines showed the same effect on suppression of migration and invasion after knockdown of VLDLR. N-cadherin was identified as a down-stream molecule responsible for the migration and invasion suppression in this pathway. / This study confirmed microRNA expression in urine supernatants was a feasible approach for the assessment of biomarkers, and miR-125b and miR-99a showed promising results in the diagnosis and grading of UCB. Furthermore, we showed that miR-99a suppressed tumor migration and invasion by directly targeting VLDLR. / Detailed summary in vernacular field only. / Zhang, Dingzuan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 107-131). / Abstract and appendix also in Chinese. / Abstract --- p.I / 摘要 --- p.III / Acknowledgments --- p.V / Abbreviations --- p.VII / List of figures --- p.IX / List of Tables --- p.XI / Content --- p.XII / Chapter Chapter I: --- General Introduction / Chapter 1.1 --- Bladder cancer --- p.1 / Chapter 1.1.1 --- The incidence of bladder cancer / Chapter 1.1.2 --- The burden of bladder cancer to the health care system / Chapter 1.1.3 --- Risk factors for bladder cancer / Chapter 1.1.4 --- Pathology grading system in bladder cancer / Chapter 1.1.5 --- Current diagnostic methods and treatment for bladder cancer / Chapter 1.2 --- Biomarkers for bladder cancer --- p.7 / Chapter 1.2.1 --- The advantages of biomarkers in blood and urine for the diagnosis of bladder cancer / Chapter 1.2.2 --- Biomarkers in blood for bladder cancer / Chapter 1.2.3 --- Biomarkers in the urine for bladder cancer / Chapter 1.2.4 --- Current concerning problems with biomarkers / Chapter 1.3 --- MicroRNAs and bladder cancer --- p.11 / Chapter 1.3.1 --- Post-trancriptional function of microRNAs / Chapter 1.3.2 --- The function of microRNAs in tumor / Chapter 1.3.3 --- Prospects of detecting microRNA in cell-free fluid in tumor / Chapter 1.4 --- MicroRNA target identification --- p.15 / Chapter 1.4.1 --- Prediction of microRNA target / Chapter 1.4.2 --- Validation of microRNA target / Chapter 1.4.3 --- Validation of direct interaction between microRNA and target RNA / Chapter 1.4.4 --- Validation of direct binding of microRNA and mRNA in vivo / Chapter 1.5 --- Migration and invasion of bladder cancer --- p.19 / Chapter 1.5.1 --- The biological process of migration in bladder cancer / Chapter 1.5.2 --- Epithelial to mesenchymal transition in bladder cancer / Chapter 1.6 --- Objectives of this study --- p.21 / Chapter Chapter II --- MicroRNAs in urine supernatant: potential useful markers for bladder cancer screening / Chapter 2.1 --- Introduction --- p.23 / Chapter 2.2 --- Materials and methods --- p.26 / Chapter 2.2.1 --- Ethics Statement / Chapter 2.2.2 --- Patients and samples / Chapter 2.2.3 --- RNA extraction / Chapter 2.2.4 --- MicroRNA microarray / Chapter 2.2.5 --- Quantitative real-time polymerase chain reaction (RT-PCR) / Chapter 2.2.6 --- Statistical methods / Chapter 2.3 --- Results --- p.31 / Chapter 2.3.1 --- MicroRNA screening by microRNA microarray / Chapter 2.3.2 --- Independent validation of the ten selected microRNAs by qRT-PCR on tissue / Chapter 2.3.3 --- Verification of the six validated microRNAs in urine supernatants as tumor markers / Chapter 2.3.4 --- MiR-125b and miR-99a in urine supernatants were useful for the diagnosis of bladder cancer / Chapter 2. --- 3.5 MiR-125b and miR-99a were two highly correlated microRNAs / Chapter 2.3.6 --- Expression levels of miR-125b and miR-99a increased after tumor resection / Chapter 2.4 --- Discussion --- p.47 / Chapter Chapter III: --- MiR-99a suppresses migration and invasion in bladder cancer by targeting VLDLR / Chapter 3.1 --- Introduction --- p.53 / Chapter 3.2 --- Materials and methods --- p.56 / Chapter 3.2.1 --- Human tissue samples and bladder cancer cell lines / Chapter 3.2.2 --- RNA extraction and Polymerase Chain Reaction / Chapter 3.2.3 --- MicroRNA and plasmid transfection / Chapter 3.2.4 --- Western Immunoblotting / Chapter 3.2.5 --- Agarose gel electrophoresis / Chapter 3.2.6 --- Luciferase assay / Chapter 3.2.7 --- MTT proliferation assay / Chapter 3.2.8 --- Apoptosis assay / Chapter 3.2.9 --- Cell cycle analysis / Chapter 3.2.10 --- Cell migration Assay / Chapter 3.1.11 --- Cell invasion assay: / Chapter 3.2.12 --- Statistical methods: / Chapter 3.3 --- Results --- p.67 / Chapter 3.3.1 --- MiR-99a was significantly down-regulated in bladder cancer / Chapter 3.3.2 --- Precursor microRNA was successfully transfected into bladder cancer cell lines / Chapter 3.3.3 --- MiR-99a had little effect on cell proliferation / Chapter 3.3.4 --- MiR-99a had little effect on cell apoptosis and cell cycle / Chapter 3.3.5 --- Over-expression of miR-99a suppressed cell migration in bladder cancer / Chapter 3.3.6 --- Over-expression of miR-99a also suppressed invasion ability in bladder cancer / Chapter 3.3.7 --- Target prediction for miR-99a using 8 target prediction databases / Chapter 3.3.8 --- Protein level of VLDLR was down-regulated by miR-99a in bladder cancer / Chapter 3.3.9 --- VLDLR was a direct target of miR-99a / Chapter 3.3.10 --- VLDLR mRNA was not down-regulated correspondingly by miR-99a / Chapter 3.3.11 --- MiR-99a suppressed down-stream protein of VLDLR in Reelin pathway / Chapter 3.3.12 --- Knockdown of VLDLR also suppressed cell migration and invasion / Chapter 3.3.13 --- N-cadherin was the down-stream protein responsible for the suppression of migration and invasion in miR-99a/VLDLR pathway / Chapter 3.4 --- Discussion --- p.93 / Chapter Chapter IV: --- Conclusion and prospective --- p.101 / Appendix --- p.105 / Reference --- p.107