1. MiR-21 can be used as independent prognostic factor for survival and metastasis in urinary bladder cancer. Apostolos Zaravinos 1, Jelena Radojicic 3, George I. Lambrou 2, Dimitrios Volanis 1,4, Ioannis Boulalas 1,4, Dimitris Delakas 4, Demetrios A. Spandidos 1. 1, Laboratory of Virology, Medical School, University of Crete, 71110 Heraklion, Crete, Greece. 2, 1 st Department of Pediatrics, Choremeio Research Laboratory, University of Athens, 11527 Athens, Greece. 3, Department of Surgical Pathology, Medical Faculty, University of Crete, 71110 Heraklion, Crete, Greece. 4, Department of Urology, Asklipieio General Hospital, 16673 Voula, Athens, Greece. Gene expression were acquired by qPCR, from 77 BC specimens. Correlation of the gene expression with survival, recurrence and metastasis was employed by SPSS 16.0. Our goal was to correlate the expression of 12 micro-RNAs (miR-10b, miR19a, miR19b, miR-21, miR-122a, miR-145_1, miR-205_1, miR-210, miR-221, miR-222, miR-378-1 and miR-296-5p) with the corresponding expression of FGF2, OPN and VEGFA. Gene expression was correlated with the overall and cancer-specific survival of patients suffering from urinary bladder cancer (BC), as well as with recurrence and metastasis. High expression of miR-21 correlated with worse overall survival (p=0.0099). Univariate analysis showed that miR-21 and miR-210 can be used as independent prognostic factors for overall survival (p=0.015 and p=0.049, respectively). Moreover, univariate analysis revealed that miR-21 can be used as independent prognostic factor for metastasis (p=0.049). Multivariate analysis revealed that miR-21, miR-210 and miR-378_1 can be used as independent prognostic factors for overall survival (p=0.005, p=0.033 and p=0.012, respectively); miR-21 and miR-378_1 can be used as independent prognostic factors for recurrence (p=0.030 and p=0.031, respectively); and miR-21 can be used as independent prognostic factors for metastasis (p=0.049). FGF2 was positively correlated with the majority of the miRs both in BC and normal tissue (p<0.001). OPN was positively correlated with miR-145_1 (p=0.015) in BC, and with miR-296-5p (p=0.017) in normal tissue. VEGFA was positively correlated with miR-21 in BC (p=0.043), and with miR-205_1 (p=0.045), FGF2 (p=0.004) and OPN (p<0.001) in normal tissue. FGF2 expression was positively correlated with miR-10b, miR19a, miR19b, miR-21, miR-122a, miR- 145_1, miR-210, miR-221, miR-222, miR-378-1 and miR-296-5p MiR-21 can be used as independent prognostic factor both for overall patient survival and metastasis of BC. MiR-210 can be used as an independent prognostic factor for overall survival for patients suffering from urinary bladder cancer. Urinary bladder cancer is the most common malignancy of the urinary tract, responsible for significant mortality and morbidity worldwide. MicroRNAs (miRs) are short non-coding RNA molecules that post- transcriptionally modulate protein expression. VariableRelative risk95% Confidence IntervalP value Overall SurvivalmiR-10b0.7360.265-2.0480.558 miR-19a0.6000.213-1.6890.333 miR-19b0.6000.213-1.6890.333 miR-210.2420.077-0.7580.015 miR-126_10.6000.213-1.6890.333 miR-145_10.4520.157-1.2990.140 miR-205_10.7360.265-2.0480.558 miR-2100.3350.113-0.9960.049 miR-2210.4210.146-1.2110.108 miR-296-5p0.6000.213-1.6890.333 miR-378-11.2660.455-3.5200.651 FGF20.4210.146-1.2110.108 OPN1.1800.424-3.2810.751 VEGFA1.4570.524-4.0560.471 RecurrencemiR-10b0.7940.310-2.0370.632 miR-19a0.7500.291-1.9310.551 miR-19b1.1900.465-3.0500.716 miR-210.6300.244-1.6240.339 miR-126_10.9450.369-2.4240.907 miR-145_11.7330.671-4.4760.256 miR-205_11.0000.391-2.5591.000 miR-2100.8670.338-2.2210.766 miR-2210.9170.358-2.3460.856 miR-296-5p0.6880.267-1.7710.438 miR-378-12.0090.772-5.2290.153 FGF20.5440.209-1.4120.211 OPN1.0580.413-2.7120.907 VEGFA0.5930.229-1.5400.283 MetastasismiR-10b0.9660.349-2.6740.946 miR-19a1.0360.374-2.8690.946 miR-19b1.0360.374-2.8690.946 miR-210.3350.113-0.9960.049 miR-126_11.0360.374-2.8690.946 miR-145_10.6000.213-1.6890.333 miR-205_10.7360.265-2.0480.558 miR-2100.7900.284-2.1960.651 miR-2210.7360.265-2.0480.558 miR-296-5p0.6000.213-1.6890.333 miR-378-10.9660.349-2.6740.946 FGF20.5590.199-1.5750.271 OPN0.9000.325-2.4940.839 VEGFA0.6440.229-1.8110.404 VariableRelative risk95% Confidence IntervalP value Overall SurvivalmiR-210.1190.027-0.5270.005 miR-2100.2300.060-0.8880.033 miR-378_17.3161.544-34.6610.012 RecurrencemiR-19a0.0000.000-1.0000.999 miR-19b2.1E+090.000-1.0000.999 miR-210.2050.049-0.8560.030 miR-378_15.9841.176-30.4470.031 MetastasismiR-210.3350.113-0.9960.049 Table 1. Univariate logistic regression was utilized to assess the potential prognostic factors in overall survival, tumour recurrence and metastasis. Table 2. Multivariate logistic regression was utilized to assess the potential prognostic factors in overall survival, tumour recurrence and metastasis. Figure 1. Kaplan-Meier curves depicting overall and cancer-specific survival (%) of urinary bladder cancer patients, regarding to the expression of 11 miRNAs, FGF2, VEGFA and OPN genes (77 patients). Survival differences were assessed using Log-rank (Mantel Cox). Statistical significance was set at the 95% level (p<0.05). Normal Tissue miR-10bmiR-19amiR-19bmiR-21miR-126_1miR-145_1miR-205_1miR-210miR-221miR-296-5pmiR-378-1FGF2OPNVEGFA Normal Tissue Bladder Cancer miR-10b<0.001 0.008<0.001 0.0680.363 miR-19a<0.001 0.0140.3790.774 miR-19b<0.001 0.0030.2940.582 miR-21<0.001 0.0740.4720.265 miR-126_1<0.001 0.0090.1420.215 miR-145_1<0.001 0.001<0.001 0.5970.2980.603 miR-205_1<0.001 0.015<0.001 0.1470.4860.045 miR-210<0.001 0.0180.3300.553 miR-221<0.001 0.2670.5950.789 miR-296-5p<0.001 0.004<0.001 0.0170.490 miR-378-1<0.001 0.0020.1170.914 FGF2<0.001 0.3810.020<0.001 0.004 OPN0.8060.5020.3410.1330.2150.0150.1420.6790.0850.1510.1240.403<0.001 VEGFA0.4610.6990.7980.0430.8130.1630.1920.3930.1030.4960.5010.2970.548 Bladder Cancer Table 1. Table 2. Table 2. Spearman’s rank correlation coefficient among microRNA, FGF2, OPN and VEGFA expression levels in urinary bladder cancer and adjacent normal tissue samples.