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Detection and identification of miRNA in cell-free urine: potential utility in bladder cancer
Jessica DeLong, MD, Niall Harty, MD, Spencer Kozinn, MD, Kimberly Rieger-Christ, PhD, Kelly Summerhayes, BS, John Libertino, MD, Ian Summerhayes, PhD.
The Lahey Clinic, Burlington, MA, USA.

BACKGROUND: MicroRNAs are small, non-coding RNAs that have been shown to play an important role in tumorigenesis. In this study we isolated RNA from cell-free urine in an attempt to characterize miRNA profiles indicating the presence of urothelial carcinoma and its potential use as a non-invasive assay to identify patients with cancer progression.
METHODS: Urine was collected from patients diagnosed with bladder cancer and control patients with no history of cancer under an IRB-approved protocol. Urine was centrifuged and total RNA was isolated from the supernatants using the mirVana Paris™ kit. Samples were grouped according to grade and stage (healthy controls (18), TaG1 (19), T1G3 (16), ≥T2 (30), carcinoma in situ (CIS; 28) and no evidence of disease following therapy (50). Seven hundred and thirty miRNAs were profiled by qRT-PCR on pooled samples within each group. Validation was performed on individual samples using qRT-PCR.
RESULTS: Cell-free RNA was isolated from urine of 18 healthy controls and 143 patients with bladder cancer. Of the 730 miRNAs tested, 420 were detected in at least one of the pooled samples with 29 of these miRNAs present in all samples. MiRNAs detected across all experimental groups showed a trend toward increased expression throughout disease progression. In contrast, miR-346 and miR-490-3p displayed similar expression levels in all groups, presenting as potential candidates for normalization. Six miRNAs were detected solely in the healthy control group whereas 20 miRNAs were identified only in the cancer groups. Eliminating CIS from the analysis resulted in the detection of an additional 33 miRNAs in the remaining cancer subgroups. When the T1G3, ≥T2 and ≥T2 alone groups were examined 71 and 165 miRNA were identified, respectively. Validation by qRT-PCR on individual samples confirmed the increased expression of numerous miRNA in cell-free urine from patients with T1G3 and ≥T2 disease.
CONCLUSIONS: We have demonstrated successful isolation of cell-free miRNA from the urine of patients. Utilizing a qPCR platform, we then identified a miRNA panel present in cancer patients but consistently absent in healthy controls. Preliminary analysis involving the grouping of miRNAs to form diagnostic panels useful in tumor detection, recurrence, and progression hold the promise for the development of useful clinical tools.


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