Validation of a microRNA profile in urine liquid biopsy with diagnostic and stratification value for bladder cancer classification, available through the open app BladdermiRaCan
Júlia Oto, Raquel Herranz, Emma Plana, Javier Pérez‐Ardavín, David Hervás, Fernando Cana, P Verger, David Ramos-Soler, Manuel Martínez-Sarmiento, C.D. Vera Donoso, Pilar Medina
Abstract
We aimed to identify a profile of urine microRNAs (miRNAs) with diagnostic and stratification potential in the whole range of bladder cancer (BC) categories, to avoid current invasive, harmful and expensive procedures. We collected a first morning urine sample from the screening (35 BC patients and 15 age- and gender-matched controls) and validation cohorts (172 BC and 94 controls). In the screening stage we analyzed the expression level of 179 miRNAs by real-time reverse transcription quantitative PCR in urine supernatants. miRNA levels in each sample were normalized by the levels of the previously identified and stably expressed miR-29c-3p. We performed an ordinal regression for each miRNA with False Discovery Rate (FDR) adjustment to identify dysregulated miRNAs, and an ordinal elastic net logistic regression model to identify a miRNA profile for BC diagnosis and stratification with the software R (v3.5.1). Next, we validated the most dysregulated miRNAs, and empirically identified the real miRNA targets in BC cells by miR-eCLIP immunoprecipitation and sequencing. We identified 70 dysregulated miRNAs in BC patients (p < 0.05 FDR-adjusted). With the expression level of 7 miRNAs in urine (miR-221-3p, miR-93-5p, miR-362-3p, miR-191-5p, miR-200c-3p, miR-192-5p, miR-21-5p) we could stratify BC patients and control subjects. To enable the global use of our model, we developed the free BladdermiRaCan online tool. Furthermore, we identified miR-21-5p, miR-425-5p and miR-99a-5p as follow-up markers for BC relapse, and miR-21-5p and miR-221-3p as markers for metastasis. These miRNAs were also dysregulated in BC tissue sections from a subgroup of patients from which urine samples were studied. In conclusion, we have validated and patented a 7-miRNAs urine profile able to diagnose and stratify BC patients; BladdermiRaCan will enable the global use of our model. The experimentally verified target proteins identified for these miRNAs may unravel novel therapeutic targets.