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MiRNA profiles in bladder carcinoma cells linked to epithelial-mesenchymal transition (EMT) and different biomarker proteins in bladder cancer
Niall J. Harty, MD, Spencer I. Kozinn, MD, Jessica M. DeLong, MD, Christina S. Deliyiannis, BS, Kimberly M. Reiger-Christ, PhD, Jason Gee, MD, John A. Libertino, MD, Ian C. Summerhayes, PhD.
Lahey Clinic, Burlington, MA, USA.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is involved in tumor progression where the underlying cellular changes associated with EMT have been identified in in vitro models and confirmed in a limited number of in vivo studies. Here, we report an extended miRNA profile in 45 bladder carcinoma cell lines and the respective alterations in EMT related adhesion proteins and established bladder biomarkers.
METHODS: 45 bladder carcinoma cell lines represent the panel to be analyzed. Total RNA was isolated from cells using the mirVana Paris™ (Ambion, Austin, TX). Specific quantitative real-time PCR experiments were carried out using Taqman® MicroRNA Assays (ABI, Foster City, CA) for all miRs according to the manufacturer’s instructions. Western blot analysis was performed in a standard format. Bladder cells were transfected with 30nM of selected pre-miRs or a scrambled pre-miR-negative control (Ambion) using siPORT NeoFX transfection reagent (Ambion). Forty-eight hours post-transfection cells were used for standard migration/invasion assays. Seventy-two hours post-transfection cells were harvested for protein analysis.
RESULTS: MiRNA analysis demonstrated that the miR-200 family (miRs-200a, 200b, 200c, 205, 141, 429) as well as miR-30b, miR-31, and miR-99a were altered in EMT. A sub-group of invasive bladder cell lines showed a decreased expression of members of the miR-200 family accompanied by novel expression of ZEB1 and a concomitant reduction in expression of PKP3 as reported. Alternative EMT related adhesion proteins including desmoglein, desmocollin, plakoglobin and N-cadherin displayed alterations in response to restoration of different miRs in transfectants with no change observed in the same cells for E-cadherin and vimentin expression levels. Bladder biomarkers including MUC1, maspin and gelsolin displayed inconsistent expression throughout bladder tumorigenesis and no expression response to the introduction or knockdown of miRs used in transfection experiments.
CONCLUSIONS: Modulation of miRNAs implicated in bladder EMT plays a role in tumorigenesis but are clearly not the sole factor(s) driving the urothelial neoplastic process. This detailed characterization of progression events driven by altered miR expression in a panel of bladder carcinoma cell lines reveals established molecular and cellular routes to transformation along with novel urothelial progression pathways.


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