Identification of novel microRNA targets based on microRNA signatures in bladder cancer

Abstract

MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate protein-coding genes. To identify miRNAs that have a tumor suppressive function in bladder cancer (BC), 156 miRNAs were screened in 14 BCs, 5 normal bladder epithelium (NBE) samples and 3 BC cell lines. We identified a subset of 7 miRNAs (miR-145, miR-30a-3p, miR-133a, miR-133b, miR-195, miR-125b and miR-199a*) that were significantly downregulated in BCs. To confirm these results, 104 BCs and 31 NBEs were subjected to real-time RT-PCR-based experiments, and the expression levels of each miRNA were significantly downregulated in BCs (p < 0.0001 in all). Receiver-operating characteristic curve analysis revealed that the expression levels of these miRNAs had good sensitivity (>70%) and specificity (>75%) to distinguish BC from NBE. Our target search algorithm and gene-expression profiling in BCs (Kawakami et al., Oncol Rep 2006;16:521-31) revealed that Keratin7 (KRT7) mRNA was a common target of the downregulated miRNAs, and the mRNA expression levels of KRT7 were significantly higher in BCs than in NBEs (p = 0.0004). Spearman rank correlation analysis revealed significant inverse correlations between KRT7 mRNA expression and each downregulated miRNA (p < 0.0001 in all). Gain-of-function analysis revealed that KRT7 mRNA was significantly reduced by transfection of 3 miRNAs (miR-30-3p, miR-133a and miR-199a*) in the BC cell line (KK47). In addition, significant decreases in cell growth were observed after transfection of 3 miRNAs and si-KRT7 in KK47, suggesting that miR-30-3p, miR-133a and miR-199a* may have a tumor suppressive function through the mechanism underlying transcriptional repression of KRT7.