Serum MicroRNA Expression Signatures Identified From Genome-wide MicroRNA Profiling Serve As Novel Noninvasive Biomarkers For Diagnosis And Recurrence Of Bladder Cancer

Objective:Recent studies have shown that miRNAs exist stably in human serum and have potential role in the diagnosis and prognosis of various cancers. However, unique serum miRNA signatures for the diagnosis of bladder cancer (BC) have not been determined. The aim of this study was to identify serum miRNA expression signatures in patients with BC and establish new models for BC diagnosis.Methods:1. Serum samples were taken from 250 patients with BC and 240 age-and sex-matched controls. Genome-wide serum miRNA analysis by Miseq sequencing was performed for an initial screen of miRNAs in separately pooled serum samples from 10 non-muscle-invasive BC (NMIBC) patients,10 muscle-invasive BC (MIBC) and 10 controls. In the discovery phase, we screened miRNAs that were significantly differentially expressed revealed by Miseq sequencing.2. In the training phase, the candidate diagnostic miRNAs were first tested with qRT-PCR in an independent cohort from 120 patients with BC and 120 controls to construct the diagnostic miRNA panel based on the logistic regression model for the differentiation between the BC group and the control group.3. In the validation phase, serum samples from another cohort of 110 patients with BC and 110 controls were entered into the discriminatory model to validate the diagnostic accuracy of the constructed algorithm, while urine samples from the same cohort were obtained for traditional urine cytology.4. In the validation phase, the correlation between the diagnostic miRNAs and BC recurrence was further assessed. In NMIBC group and MIBC group, survival curves were respectively estimated with the Kaplan-Meier method and comparisons were made using the log-rank test. The Cox proportional hazards regression model was used to identify the independent prognostic factors.Results:1. Among the 529 serum miRNAs that were scanned by Miseq sequencing (≥1 copy),180,259 and 206 miRNAs were detectable (≥10 copies) in controls, NMIBC patients and MIBC patients, respectively. The expression of a miRNA was considered altered only if at least 50 copies were detected by Miseq sequencing, together with a larger than two-fold change in its expression level between the BC and control groups. Based on these criteria,26 miRNAs were found differentially expressed in BC.Among the 26 miRNAs,18 miRNAs were down-regulated in BC(miR-484, let-7b-5p, let-7a-5p, miR-30a-5p, miR-181b-5p, miR-1228-5p, miR-181a-5p, miR-27a-3p, miR-3135b, miR-3187-3p, miR-185-5p, miR-15a-5p, miR-15b-5p, miR-92b-5p, miR-342-3p, miR-501-3p, miR-148a-3p, and let-7i-5p), and 8 miRNAs were up-regulated in BC (miR-24-3p, let-7c, miR-128, miR-98, miR-148b-3p, miR-30d-5p, miR-1294, and miR-152)2. In the training phase, qRT-PCR analysis revealed six miRNAs (miR-152, miR-148b-3p, miR-3187-3p, miR-15b-5p, miR-27a-3p, and miR-30a-5p) that showed differential expression patterns between BC group and control group (all p <0.001). miR-152 and miR-148b-3p were upregulated in BC. miR-3187-3p, miR-15b-5p, miR-27a-3p, and miR-30a-5p were downregulated in BC. The corresponding AUCs of the six miRNAs were 0.738,0.729,0.814,0.715,0.703 and 0.645, respectively A diagnostic miRNA panel based on the six miRNAs for BC was finally developed by logistic regression model with an area under the receiver operating characteristic curve (AUC) of 0.956 (95% CI,0.922 to 0.978, sensitivity=90.00%, specificity=90.00%) in the training phase.3. In the validation phase, the AUC of the diagnostic miRNA panel was 0.899 (95% CI,0.851 to 0.936, sensitivity=80.00%, specificity=89.09%). Moreover, the corresponding sensitivities of this panel for Ta, T1 and T2-T4 were 90.00%, 84.85%,89.36%, significantly higher than those of urine cytology, which were 13.33%,30.30%,44.68%, respectively (all at p<0.001).4. In the validation phase, Kaplan-Meier analysis showed that NMIBC patients with high miR-152 level and low miR-3187-3p level had worse recurrence-free survival (RFS) (p=0.023, and p=0.043, respectively). In multivariate Cox regression analysis, miR-152 and tumor stage were independently associated with tumor recurrence of NMIBC (p=0.028). However, none of the six deregulated miRNAs influenced patient predicted recurrence of MIBC.Conclusion:1. Our results suggested that a serum miRNA signature (miR-152, miR-148b-3p, miR-3187-3p, miR-15b-5p, miR-27a-3p, and miR-30a-5p) may have considerable clinical value in diagnosing BC.2. Expression level of serum miR-152 could provide information on the recurrence risk of NMIBC.