Identification of miR-29c-3p as a Robust Normalizer for Urine microRNA Studies in Bladder Cancer

Abstract

Bladder cancer (BC) is among the most frequent malignancies worldwide, being the most expensive cancer to treat and monitor and the most lethal urological cancer. Urine microRNAs (miRNAs) have been proposed as novel non-invasive biomarkers to early diagnose and monitor BC patients in order to avoid the performance of current aggressive diagnostic techniques. However, huge discrepancies arise among studies mainly due to the lack of standardization in the normalization, a crucial step in all miRNA studies. Our aim was to identify the best miRNA normalizer for miRNA studies in urine of BC patients. We evaluated the performance of 110 candidate miRNAs in urine of 35 BC patients and 15 healthy controls by Real Time quantitative Polymerase Chain Reaction (RT-qPCR) followed by a stability analysis with RefFinder. In this screening stage, miR-29c-3p arose as the most stably expressed miRNA in BC and controls, with a good expression level. Stability of miR-29c-3p expression was validated in an independent cohort of 153 BC patients and 57 controls. Finally, we evaluated the robustness of miR-29c-3p as normalizer in the expression study of miR-200c-3p, a potential diagnostic marker for BC. We propose miR-29c-3p as a normalizer for miRNA studies in BC urine. This is the first study that characterizes a reliable normalizer that may allow the comparison of future urine miRNA studies as non-invasive biomarkers for BC diagnosis and monitoring.

Keywords

• bladder cancer
• urine
• miRNA
• reference miRNA
• normalizer
• RT-qPCR