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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

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Introdução: Ocupação foi identificada como o segundo fator de risco mais importante para o câncer de bexiga depois de fumar sendo responsável por até 20% de todos os cânceres de bexiga em países industrializados. Apesar dos esforços consideráveis para investigar ocupações em relação ao risco de câncer de bexiga, muitas não foram encontrados de forma consistente. Material e Métodos: Foram incluídos 200 pacientes com diagnóstico de câncer de bexiga entre os anos de 2009 e 2013. Foi aplicado um questionário para obter informações sobre a profissão, tempo de exposição e hábitos diários, sintomas, e também dados de doenças incluindo estágio, grau e número e tamanho de lesões. Os pacientes do Grupo 1 foram aqueles sem emprego previamente associados com o risco de câncer de bexiga. Grupo 2 representado pacientes em risco devido a profissões. Resultados: Os pacientes do Grupo 2 apresentaram uma proporção significativamente maior de pT2 CaB (P = 0,037), enquanto que os pacientes do grupo 1 apresentaram significativamente mais pTa (p = 0,002) da doença. Analisando preditores de pT2, a presença de ocupação aumento de alto risco por 2,80 vezes a chance de desenvolver uma doença invasiva. Ao analisar o grau do tumor descobriram que um tempo de exposição de 10 anos ou mais aumenta o risco de tumores de alto grau em 4,28 vezes (p = 0,001). Conclusão: Pacientes com história de exposição a agentes cancerígenos devido à sua atividade profissional podem estar em maior risco de desenvolvimento de tumores invasivos e aqueles que estão expostos a estes agentes para mais de 10 anos podem desenvolver doença de alto grau com mais freqüência. População em risco pode, portanto, beneficiar de rastreamento para o câncer de bexiga / Occupation was identified as the second most important risk factor for bladder cancer after smoking accounting for up to 20% of all bladder cancers in industrialized countries. Despite considerable efforts to investigate occupation against the risk of bladder cancer, many have not been found consistently. We included 200 patients diagnosed with bladder cancer between 2009 and 2013. A questionnaire was applied to obtain information about the profession, exposure time and daily habits, symptoms, and also diseases of data including stage, grade and number and lesion size. Patients in Group 1 were those without jobs previously associated with the risk of bladder cancer. Group 2 represented patients at risk because of professions. Group 2 patients had a significantly higher proportion of pT2 CaB (P = 0.037), whereas patients in group 1 had significantly more pTa (p = 0.002) of the disease. Analyzing predictors of pT2, the presence of high-risk occupation increases by 2.80 times the chance of developing invasive disease. In considering the degree of tumor found that a time of 10 or more years of exposure increases the risk of high-grade tumors in 4.28 times (p = 0.001). Patients with a history of exposure to carcinogens because of their duties may be at greater risk of developing invasive tumors and those who are exposed to these agents for more than 10 years can develop high-grade disease more often. Population at risk can therefore benefit from screening for bladder cancer

Introdução: Um dos principais obstáculos para compreensão dos eventos biológicos envolvidos no câncer é a falta de modelos adequados para o estudo in vitro em especial em relação ao câncer de próstata (CaP) e ao câncer de bexiga (CaB). Há um número limitado de linhagens celulares de CaP e de CaB sendo a maioria proveniente de tumores invasivos e metastáticos. Sabe-se ainda, que existem diferenças étnicas entre as populações quanto ao comportamento de neoplasias. Desta forma, a pesquisa baseada em linhagens de uma população homogênea seria fonte de resultados limitados, não contemplando a diversidade que sabidamente ocorre entre os diferentes grupos. Além desse aspecto, as linhagens celulares comerciais são na sua maioria adquiridas na Coleção Americana de Culturas de Tecido (ATCC, do inglês American Tissue Cell Culture) que apesar de serem bem padronizadas, requerem processos de importação com aumento do custo e demandas burocráticas que dificultam a pesquisa. Portanto, consideramos vital para a compreensão dos fenômenos relacionados à carcinogênese, assim como estudos de resistência a drogas, quimioprevenção e novas estratégias terapêuticas, o desenvolvimento de linhagens tumorais derivadas de tumores primários que acometem a nossa população, peculiarmente miscigenada. No presente trabalho, fragmentos de carcinoma urotelial da bexiga e de adenocarcinoma da próstata foram obtidos durante cirurgia para remoção de tumores primários de pacientes tratados e acompanhados na Divisão de Urologia da Faculdade de Medicina da Universidade de São Paulo (FMUSP) e no Hospital Sírio Libanês. As linhagens estabelecidas a partir destes fragmentos foram caracterizadas através da análise da cinética de crescimento, análises imunocitoquímicas e anormalidades cromossômicas incluindo cariótipo e hibridização in situ por fluorescência (FISH). Além disso, as linhagens obtidas foram submetidas a estudos de quimiossensibilidade com o uso dos compostos curcumin e Prima-1. Avaliamos ainda, a tumorigenicidade de nossas linhagens em camundongos atímicos. Os resultados deste trabalho demonstram o desenvolvimento de três linhagens de CaB e três linhagens de CaP sendo as mesmas não tumorigênicas em camundongos atímicos. Além disso, demonstramos que o curcumin na concentração de 50 M induziu morte celular em todas as linhagens estudadas, sendo seu efeito mais evidente nas linhagens de CaP. Por fim, Prima-1 reduziu a viabilidade celular independente do status de p53 nas linhagens de CaB / Introduction: One of the main obstacles for understanding biological events involved in cancer is the lack of appropriated models for in vitro studies especially for prostate cancer (PC) and bladder cancer (BC). There are a limited number of PC and BC cell lines being the majority originated from metastatic and invasive tumors. Also it is well known that there are ethnic differences between populations concerning the behavior of tumors. In such a way, the research based on cell lines derived from a homogenous population should be source of limited results, not contemplating the diversity known to occur among different groups. In addition the commercial cell lines are generally acquired at American Tissue Cell Culture (ATCC) that although wellestablished requires importation processes with cost increase and bureaucratic demands that difficult the research. Therefore we consider vital to the comprehension of the carcinogenesis phenomena, as well as drug resistance studies, chemoprevention and new therapeutic strategies, the development of tumor lineages derived from primary tumors that assail our miscigenated population. At the present work, fragments of bladder urothelial carcinoma and prostate adenocarcinoma were obtained by surgical resection of primary tumors from patients treated and followed in the Division of Urology of the Clinical Hospital of the São Paulo University (FMUSP) and Syrian Lebanese Hospital. The cell lines established from these fragments were characterized through growth kinetic, immunocytochemistry and chromosome abnormalities including karyotyping and Fluorescence in situ hybridization (FISH). Moreover, the cell lines were submitted to chemosensitivity studies using curcumin and Prima-1 and analyzed regarding their tumorigenicity in athymic mice. The results of this work show the development of three BC and three PC cell lines that were not tumorigenic in athymic mice. Curcumin at 50 M concentration induced cell death in all studied lineages, being more effective in PC cell lines. Finally, PRIMA-1 reduced the cellular viability independent of the p53 status in BC cell lines

Guang Fu Chen. / "May 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 194-221). / 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.

Objectives: To establish a mouse orthotopic bladder cancer model with consistent tumor-take rate. This orthotopic model was subsequently used to evaluate small animal imaging techniques and investigate new therapeutic agents for bladder cancer treatment. / Materials and Methods: Different orthotopic implantation techniques have been tested. MBT-2 cells and syngeneic C3H/He mice were used in all experiments. Chemical bladder pre-treatment with different agents (saline, hydrochloric acid, trypsin and poly-L-lysine) and different concentration of instilled tumor cells (1 x 10⁶ or 2 x 10⁶) were investigated. In the second part of the experiment, trans-abdominal micro-ultrasound imaging (MUI) technique was investigated and validated. Bladder tumor growths were monitored with longitudinal measurement. Mice were killed at every MUI session. Bladder tumor volumes were measured and correlated with gross stereomicroscopy. Using the optimized orthotopic bladder cancer model, targeted contrast enhanced micro-ultrasound imaging has been investigated. VEGFR2 targeted contrast agent was prepared and injected intravenously before imaging sessions. The intra-tumoral perfusion, VEGFR2 expression and blood volume in real time were quantified. Contrast enhanced MUI was performed on Days 14 and 21. The feasibility of targeted contrast enhanced micro-ultrasound imaging was confirmed. After the establishment of orthotopic model and in vivo molecular imaging techniques, this robust platform was used for investigating new treatment agent in localized bladder cancer. Tumor-bearing mice were randomized into control and sunitinibtreated (40 mg/kg) groups. Tumor volume, intra-tumoral perfusion, and in vivo VEGFR2 expression were measured using a targeted contrast-enhanced micro-ultrasound imaging system. The effects of sunitinib malate on angiogenesis and cellular proliferation were measured by CD31 and Ki-67 immunohistochemistry. The clinical outcomes including total bladder weight, tumor stage, and survival were evaluated. / Results: A consistent tumor take-rate of over 90% was achieved by using poly-L-lysine pretreatment with 2 x 10⁶ MBT-2 cells in all of the experiments. MUI identified all tumors that were present on final histology. Measurements of tumor size by MUI and gross microscopy had a high correlation coefficient (r = 0.97). Measurements of intra-tumoral perfusion and in vivo VEGFR2 expression were also proved to be feasible. After the technical refinement and modification, complete measurements could be performed in all mice (n = 10) at 2 consecutive imaging sessions. No adverse effects occurred due to anesthesia or the ultrasound contrast agent. This is the first report of applying targeted contrast enhanced MUI in orthotopic bladder cancer model. Finally, sunitinib was found to have significant tumor growth inhibition in both in vitro and in vivo experiments. In the orthotopic model, tumors in sunitinib-treated mice had reduced tumor volume and stage, lower proliferation index and micro-vessel density. Sunitinib prolonged survival in tumor-bearing mice as compared to control group. / Conclusions: The development of reliable orthotopic animal models assists in the discovery of novel therapeutic agents. The establishment in the methods of implantation with improved tumor-take rate and the advances in imaging technology form the important foundation of basic research in bladder cancer. Trans-abdominal MUI is proven to be a valuable tool for translational studies involving orthotopic mouse bladder cancer models. Furthermore, the first report of the application of targeted contrast enhanced MUI in deep-seated tumor in bladder has been published. It enables investigators to monitor tumor angiogenesis and vascular changes after treatment. It will be useful for direct, noninvasive, in vivo evaluation of anti-angiogenesis therapeutic agents. The preclinical study has demonstrated the activities of a new class of targeted therapy against localized bladder cancer in an orthotopic mouse model. Sunitinib inhibits tumor growth and thus decreases the tumor burden and prolongs survival compared with placebo. These results provide a rationale for future clinical trials using VEGFR-targeted treatments of localized bladder cancer in the neo-adjuvant and adjuvant settings. / Chan, Shu Yin Eddie. / Thesis (M.D) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 189-212).