Abstract 4193: Exposure to arsenic and miRNA deregulation: a potential non-invasive screening tool for urothelial cell carcinoma.
Authors: Michailidi Christina , Hadar Tal , Zenner Kaitlyn , Schoenberg Mark , Netto George , Sidransky David , Hoque Mohammad O. 
Published: August 2014

Abstract
Background: We hypothesize that gene deregulation caused by aberrant miRNA expression may play a role in arsenic-induced transformation. To confirm this hypothesis and to identify the molecular mechanisms involved, we developed a cell culture model that permit us to study the progressive, transforming effects of arsenic in uro-epithelial cell behavior and to assess the impact of arsenic exposure on bladder tumorigenesis. Identified miRNAs may also have potential for non-invasive screening test using urine.
Materials and Methods: We first aimed to determine which fraction of urine is suitable for miRNA analysis. To this end we extracted RNA from 10 cases of urine samples using a) 600 μL of voided urine directly after collection from patients; b) 600 μL of supernatant after centrifuging for 10 min at 1500 rpm and c) from the urine sediment. Our initial observation suggested that RNA from urine supernatant is suitable for miRNA analysis. Furthermore, we aimed to identify a housekeeping miRNA with consistent non-differential expression levels between controls and urine from UCC patients. Total RNA, including miRNA, was extracted from urine samples obtained from non-neoplastic individuals and UCC patients, using the MirVana miRNA Isolation Kit. For quantitative real-time reverse transcriptase PCR (qRT-PCR) of selected miRNA, cDNA was synthesized from 20 ng of total RNA using TaqMan miRNA specific primers (Applied Biosystems) for each candidate and a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). PCR reactions were carried out in 384-well plates in a 7900 sequence detector (Perkin-Elmer Applied Biosystems) and were analyzed by a sequence detector system (SDS 2.2.1; Applied Biosystems).
Results: RNU6b, RNU48, miR-222, miR-191 and miR-16 were tested as potential housekeeping genes in urine samples from controls and cases. Among the above listed candidates, miR-222 was found to be the optimal miRNA for normalization across the samples. As such, we normalized expression of miR-200a, -200b, -200c, and -205 to miR-222 in 51 urine samples from UCC cases and 5 urine samples from controls. Expression of miR-200a, -200b, -200c, and -205 was observed in 15/51(29%), 22/51(43%), 29/51(57%) and 38/49(78%) respectively, in urine of UCC cases. No expression of any of the miRNAs tested was observed in any of the 5 controls by an optimal cut point. When we consider expression of any of the four miRNAs tested, UCC cases were detected with 100% sensitivity and 100% specificity.
Conclusions: Our initial findings support the assumption that miRNAs may be used as potential biomarkers for non-invasive detection of UCC. Further work will focus specifically in urine of UCC patients with a known exposure to arsenic, as well as urine with matched tumor tissue. Appropriate cohort with age and gender matched controls needs to be tested for alterations of a panel of miRNAs expression to determine its clinical utility.
Citation Format: Christina Michailidi, Tal Hadar, Kaitlyn Zenner, Mark Schoenberg, George Netto, David Sidransky, Mohammad O. Hoque. Exposure to arsenic and miRNA deregulation: a potential non-invasive screening tool for urothelial cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4193. doi:10.1158/1538-7445.AM2013-4193