microRNA implication in urinary bladder cancer A. Zaravinos 1, J. Radojicic 3, G.I. Lambrou 2, D. Volanis 1,4, D. Delakas 4, E.N. Stathopoulos 3, D.A. Spandidos 1 1 Laboratory of Virology, Medical School, University of Crete, Heraklion, Crete, Greece. 2 1 st Department of Pediatrics, Choremeio Research Laboratory, University of Athens, Athens, Greece. 3 Department of Surgical Pathology, Medical Faculty, University of Crete, Heraklion, Crete, Greece. 4 Department of Urology, Asklipieio General Hospital, Voula, Athens, Greece. Urinary bladder cancer is the most common malignancy of the urinary tract, responsible for significant mortality and morbidity worldwide. MicroRNAs (miRNAs) are a group of endogenous, small, noncoding RNA molecules of ~22 nucleotides. The interaction of miRNAs and target genes is intricately regulated, in that one miRNA may modulate multiple target genes whereas one target gene may be regulated by various miRNAs. MiRNAs negatively affect the expression level of their target genes through two distinctive mechanisms, depending on the degree of their complementarity to target sequences. In the first mechanism, a perfect or near-perfect match between miRNAs and their binding sequences within the 3’ untranslated regions (UTR) of their target mRNAs induces the RNA-mediated interference pathway. The RNA-induced silencing complex then recognizes the miRNA-mRNA interaction and cleaves the mRNA through an endonuclease activity. In the second mechanism, miRNAs control gene expression at the translational level through imperfect target matching. MiRNAs influence tumorigenesis through their regulation of specific proto-oncogenes and tumor suppressor genes. Figure 1. ROC curve analysis. miR-145, miR-221 and miR p had the best sensitivity and specificity to distinguish urinary bladder cancer from control samples. Seventy-seven urinary BC cases, along with 77 matched tumor-associated normal samples were investigated for the expression of 11 miRNAs using qPCR. The relationship among the expression of miR-10b, miR-19a, miR-19b, miR-21, miR-126, miR-145, miR-205, miR-210, miR-221, miR p and miR-378-1, as well as with the pathologic features of the tumors, was further examined. The influence of miR expression on the overall and cancer-specific survival of the patients, as well as putative target genes of the up- and down-regulated miRNAs were also predicted. As expected, the majority of the microRNAs studied (miR-10b, miR-145, miR-221, miR-296-5p and miR-378) exhibited significant down-regulation in BC vs. the normal tissue (Table 1). A great range in the x-fold expression values of all microRNAs was noticed. miR-145 was found to be the most down-regulated in bladder cancer compared with normal, and miR-205, miR-210 and miR-21 were the most up-regulated in cancer. High expression of miR-21 correlated with worse overall survival (p=0.0099) (Figure 2). 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 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 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) (Table 2). Our goal was to investigate the expression profile of a various oncomiRs and tumor-suppressor miRs in urinary bladder cancer (BC). The data reported here demonstrate that several microRNAs are deregulated in BC through down-regulation. miR-145 was found to be the most down-regulated in bladder cancer compared with normal, and miR-205, miR-210 and miR-21 were the most up-regulated in cancer. miR-21 can be used as independent prognostic factor both for patient survival and metastasis. miR-210 can be used as an independent prognostic factor for overall survival. Table 1. Mean and median fold values, represent the change in the expression levels of the microRNAs, between urinary bladder cancer and normal tissue. *Mann-Whitney U test. microRNAmean foldSDminimummaximumSEMmedian foldp-value* miR-10b miR-19a miR-19b miR miR miR < miR miR miR < miR-296-5p < miR < Figure 2. Kaplan-Meier curve analysis revealed that high miR-21 expression correlated with worse overall survival (p=0.0099). VariableRelative risk95% Confidence IntervalP value Overall SurvivalmiR miR miR RecurrencemiR-19a miR-19b2.1E miR miR MetastasismiR Table 2. Multivariate logistic regression analysis was utilized to assess the potential prognostic factors in overall survival, tumour recurrence and metastasis.