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MicroRNA expression linked to gemcitabine resistance in an in vitro bladder carcinoma model
Spencer I. Kozinn, MD, Niall J. Harty, MD, Jessica M. DeLong, MD, Kimberly M. Rieger-Christ, PhD, John A. Libertino, MD, Ian C. Summerhayes, PhD.
Lahey Clinic Medical Center, Burlington, MA, USA.

BACKGROUND: MicroRNAs (miRNA) are small, non-coding RNAs found to have an important regulatory role in development, differentiation, cell proliferation and cell death. Recent evidence implicates a role for miRNA in tumorigenesis, including a primary role in establishing drug sensitivity. Our goal in this study was to identify specific miRNAs that confer resistance to gemcitabine in bladder cancer.
METHODS: Gemcitabine resistant cells were established from CUBIII, RT112, RT4, J82, TCCSUP, and UM-UC-3 bladder carcinoma cell lines following exposure to escalating concentrations of the drug. Stably resistant cells were established by passaging cells in the presence of gemcitabine over a 2-3 month period. RNA was extracted from cell lines using the mirVana™ miRNA Isolation Kit (Ambion). Differential miRNA expression was identified in a microarray format comparing untreated controls with resistant cell lines representing the maximum tolerated concentration. Results were validated via qRT-PCR across the full concentration range. The involvement of specific miRNAs in chemoresistance was confirmed with transfection experiments, followed by clonogenic assays.
RESULTS: Gemcitabine resistant bladder carcinoma cell lines were generated from RT112 (450nM), UM-UC-3 (450nM), CUBIII (200nM), J82 (150nM), RT4 (150nM) and TCCSUP (100nM). Microarray analysis comparing miRNA expression between gemcitabine resistant cells and parental cell lines identified differential expression across 63 miRNAs. Confirmation of differential expression was recorded in 17/30 miRNAs tested. Within this group let-7b and let-7i exhibited decreased expression with increasing concentrations of gemcitabine, whilst miR-1290 and miR-138 displayed increased expression levels in gemcitabine resistant cells Transfection of pre-miR-138 and pre-miR-1290 into parental cell lines attenuated cell death after exposure to gemcitabine, while transfection of pre-miR-let7b and pre-miR-let-7i into the resistant lines augmented cell death.
CONCLUSIONS: miRNAs 1290, 138, let-7i, and let-7b play a role in conferring gemcitabine resistance in bladder carcinoma cell lines. The potential for both diagnostic and therapeutic utility in the clinical arena is considerable.


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