Investigation into the role of MicroRNA in bladder cancer

Background: Urothelial bladder cancer is a common disease that arises by at least two different molecular pathways. The biology of bladder cancer is incompletely understood, making the management of patients with this disease difficult. Recent evidence implicates a regulatory role for microRNA in cancer, suggesting the need to study these in more depth. In this thesis I will focus upon these RNAs to better understand their role in bladder cancer. Firstly, I hypothesise that altered microRNA expression contributes to bladder cancer carcinogenesis. Secondly, I will focus upon the use of microRNA in the diagnosis of bladder cancer. Urinary biomarkers are needed to improve the care and reduce the cost of managing bladder cancer. Current biomarkers struggle to identify both high and low-grade cancers due to differing molecular pathways. Therefore, my second hypothesis is that urinary microRNAs reflecting low and high-grade pathways could detect a broad spectrum of bladder cancers to overcome differences in genetic events seen within the disease. Thirdly, I will manipulate the expression of microRNA-1224-3p in urothelial cells and assess the phenotypical outcomes. Current chemotherapeutic regimes in bladder cancer are limited with no gene-based agents in the near horizon. My third hypothesis is that the manipulation of microRNA will alter urothelial cells in an anti-tumourigenic manner. Materials and Methods: To test the first hypothesis, I examined the expression of 322 microRNAs in 78 normal and malignant urothelial samples using real-time rtPCR. To test the second hypothesis, I investigated urinary samples from patients with bladder cancer (n=68) and age-matched controls (n=53) which included patients with a diagnosis of urosepsis (n=6). Fifteen microRNAs were quantified using real-time PCR from these urine samples. To test the third hypothesis, I investigated the under- and over-expression of microRNA-1224-3p on urothelial cells and investigated the phenotypical responses of these cells to microRNA manipulation. Results: Altered microRNA expression is common in bladder cancer, and changes occurs early in tumourogenesis. In normal urothelium from patients with bladder cancer, 11% of microRNA’s have altered expression when compared to disease-free controls. In bladder cancer microRNA alterations occur in a tumor phenotype-specific manner and can predict disease progression. High-grade UCC were characterized by microRNA up-regulation, including microRNA-21 that suppresses p53 function. In low-grade UCC there was down-regulation of many microRNA molecules including miR-99a/100 that regulate FGFR3 and FOXA1. MicroRNA expression is stable within urinary samples despite adverse handling. MicroRNA had the ability to be detected in urine after they were stored at room temperature for 48 hours. We detected differential expression of 10 microRNAs from patients with cancer and controls (miRs-15a/15b/24-1/27b/100/135b/203/212/328/1224-3p, ANOVA p<0.05). Individually, miR-1224-3p had the best individual performance with specificity, positive and negative predictive values and concordance of 83%, 83%, 75% and 77%, respectively. The combination of miRs-135b/15b/1224-3p detected bladder cancer with a high sensitivity (94.1%), sufficient specificity (51%) and was correct in 86% of patients (concordance). Over-expression of microRNA-1224-3p on urothelial cells was shown to be of a reproducible manner. Over-expression of microRNA-1224-3p resulted in a significant decrease of urothelial cell viability. Conclusion: This was one of the first studies to demonstrate that distinct microRNA alterations characterise bladder cancer in a grade-specific manner. These data reveal new insights into disease biology and have implications regarding tumor diagnosis, prognosis and therapy. The use of the panel of urinary microRNAs-135b/15b/1224-3p in patients with haematuria would have identified 94% of bladder cancers, whilst reducing cystoscopy rates by 26%. However, 2 invasive cancers (3%) would have been missed. Over-expression of microRNA-1224-3p leads to the inhibition of urothelial cell growth and viability. With further studies, this finding could potentially be developed to create a chemotherapeutic agent for patients with bladder cancer.