Abstract 2108: miR-1 is a putative tumor suppressor gene for urothelial bladder cancer

Abstract

Introduction and Objective High-grade, muscle invasive urothelial bladder cancer is common in the United States and worldwide. microRNAs (miRNA) are highly conserved, endogenously-encoded small RNAs that modulate gene expression and are involved in a variety of physiological processes and disease states. A large body of literature has proven that alterations in miRNA expression and function contribute to cancer development. We and others have reported altered expression of miRNAs in human urothelial cancers. In a previous microarray experiment, we demonstrated that miR-1 levels were reduced in high-grade, muscle invasive bladder cancer by 8 fold (p value= 0.000019). The goal of the present study was to evaluate the role of miR-1 as a tumor suppressor gene for bladder cancer using cell line models. Methods Human bladder cancer cell lines were maintained under standard growth conditions. Tumor and matched normal tissue were obtained with Internal Review Board approval from patients with a confirmed pathologic diagnosis of high-grade, muscle invasive urothelial (bladder) cancer undergoing radical cystectomy. miRNA mimics were transfected according to manufacturers protocol. Cell growth was assessed using cell counting and MTS assays. For cell cycle analysis, cell nuclei were stained with propidium iodide and analyzed by flow cytometry. miR-1 levels were measured using real-time PCR and northern blotting. Western blotting was performed using standard methods. Data represent at least 3 independent experiments. Results miR-1 expression was confirmed as reduced or absent in an additional set of high- grade, muscle-invasive tumors compared to adjacent normal tissue. Restoring expression of miR-1 using transfection with miR-1 mimics resulted in >50% reduction in the growth of UMUC3 cells compared to a control miRNA sequence. Cell cycle analysis revealed a 5-10% reduction in the percentage of miR-1 expressing cells in G1 phase, while a 2-5% increase in apoptotic cell death was observed. Target prediction algorithms identified a conserved miR-1 binding site in the 3′UTR of ERK MAPK, suggesting that loss of miR-1 in bladder cancer may result in increased levels of MAPK and thus contribute to driving proliferation. Indeed, miR-1 expressing cells had reduced protein levels of ERK MAPK in UMUC3 cells compared to a control miRNA. Conclusions Expression profiling identified miR-1 as significantly downregulated in advanced urothelial carcinomas. We confirmed this finding in additional patients. Our data demonstrate that restoring expression on miR-1 in a human bladder cancer cell line resulted in a significant growth reduction that is likely due to slowing down of proliferation rate, rather than being characterized by arrest in a specific phase of the cell cycle. We identified ERK MAPK as a potential target of miR-1. miR-1 induced regulation of ERK MAPK protein levels in bladder cancer may contribute to its role as a tumor suppressor gene. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2108.