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A Role for Micro RNA-200c in the Invasive Bladder Tumor Phenotype
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A Role for Micro RNA-200c in the Invasive Bladder Tumor Phenotype
Matthew F. Wszolek, MD, Justin J. Gould, MD, Patrick A. Kenney, MD, Kimberly M. Rieger-Christ, Ph.D., Brasil S. Neto, MD, John A. Libertino, MD, Ian C. Summerhayes, MD.
Lahey Clinic, Burlington, MA, USA.
Background: MicroRNA (miRNA) has been shown to play a role in different malignancies regulating the expression of genes involved in tumor suppression and oncogenesis. Several studies have reported differential expression of miRNA between normal and tumor tissue and recent investigations have identified miRNA protein targets involved in tumorgenesis. In this study we have focused on the potential involvement of miR-200c in the invasive process in bladder cancer.
Methods: RNA was extracted from cell lines and paraffin embedded tissue using the mirVanaTM miRNA Isolation Kit and RecoverAllTM Total Nucleic Acid Isolation, respectively. From a panel of known miRNAs implicated in bladder carcinoma invasion, differential miRNA expression was confirmed using RT-PCR in 14 bladder carcinoma cell lines and 50 paraffin embedded patient samples using TaqMan miRNA quantification kits. SiRNA knockdown experiments were performed on RT112 and UM-UC-3 via transfection with pre-miR-200c, anti-mir200c or a pre-miR negative control using siPORT NeoFX transfection reagent (Ambion). The ZEB1 gene and ZEB1 knockdown constructs (Origene, Rockville, MD) were introduced into cells using lipofectamine (Gibco BRL, Gaithersburg, MD). Modulation of protein expression was confirmed in western blot analysis and invasive/migration behavior was assessed using standard assays.
Results: A panel of twelve miRNAs has been identified with potential involvement in the invasive phenotype of bladder carcinoma cells. QRT-PCR using 14 bladder carcinoma cell lines and 50 paraffin embedded patient samples validated differential expression between non-invasive and invasive lesions in nine of the original twelve miRNAs panel. Initial studies have focused on the role of miR-200c in the invasion process where miR-200c was found significantly down-regulated in invasive bladder carcinoma cell lines that displayed an epithelial to mesenchymal transition (EMT) phenotype. Furthermore, miR-200c has been linked to ZEB1, a transcriptional regulator implicated in EMT. Constitutive expression of ZEB1 in bladder carcinoma cell lines revealed an inverse correlation between miR-200c and ZEB1 expression. To validate the relationship between ZEB1 expression and miR-200c levels, we performed knock-in and knock-down of miR-200c in bladder cell lines with low (UM-UC-3) or high (RT112) miR-200c expression. Modulation of expression of miR-200c resulted in changes in ZEB1, N-cadherin and E-cadherin expression. In addition, ZEB1 knockdowns exhibited decreased invasive potential on invasion/migration assays. Novel expression of ZEB1 was recorded in bladder tumors in immunohistochemistry where an inverse correlation was recorded between ZEB1 and miR-200c expression.
Conclusions: In this study, we have identified differential expression of miR-200c between non-invasive and invasive bladder lesions linked to the expression of the transcriptional regulator ZEB1. Expression of ZEB1 in bladder carcinoma cell lines was correlated with miR-200c expression with novel expression of ZEB1 recorded in bladder tumor tissue. Both miR-200c and ZEB1 are linked to the invasive phenotype in bladder carcinoma cells likely contributing toward EMT.
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