MicroRNAs can have large-scale effects by regulating expression of a variety of genes during mammalian development and carcinogenesis. As a result, understanding the mechanisms and function of individual miRNAs has generated great interest. Despite the accumulating evidence regarding the role of various miRNAs in cancer, very limited information is available about the function of miRNAs in bladder cancer, and only few miRNA targets have been identified.
Here we report that miR-23b to be down-regulated in bladder cancer tissues compared to normal adjacent tissues and this was also observed in bladder cancer and non-malignant cell lines. Our data suggests a potential diagnostic/prognostic role for miR-23b in predicting overall survival and discriminating malignant from normal tissues and indicates that miR-23b is a tumor suppressor in bladder cancer.
To determine the biological relevance of miR-23b in bladder cancer, we performed functional assays. Ectopic expression of miR-23b resulted in significant inhibition of cell proliferation, colony formation, migration/invasion and induction of cell cycle arrest and apoptosis in bladder cancer cells. Expression of miR-23b in cancer is somewhat controversial because it has been found to be either up-regulated and oncogenic in kidney cancer where it caused translational repression of tumor suppressor PTEN gene 
or down-regulated and a tumor suppressor in prostate cancer where it directly targets Src kinase and Akt oncogenes 
, while our study indicates it is a tumor suppressor in bladder cancer. Previous studies have shown that microRNAs are highly tissue specific and they can act as tumor suppressor or oncogenes 
. MicroRNAs possess several features that make them attractive candidates as new prognostic biomarkers and powerful tools for the early diagnosis of cancer 
. In this study, we found that miR-23b was predictive of overall survival such that patients with higher miR-23b expression had longer overall survival compared to patients with low miR-23b expression. MicroRNA-23b expression was also able to distinguish malignant from normal tissues indicating the diagnostic significance of miR-23b in bladder cancer although additional studies with a larger cohort of tissue samples are required.
A significant obstacle to understanding miRNA function has been the relative paucity of experimentally validated targets. To determine the effectors of miR-23b, in-silico algorithms and functional analyses identified Zeb1 as its target. We demonstrated that miR-23b directly targets the 3′UTR of Zeb1, as its over-expression was associated with suppression of luciferase activity. In addition, a significant down-regulation in the level of Zeb1 protein was observed after miR-23b over-expression, indicating post-transcriptional regulation of Zeb1 via targeting its 3′UTR. Functional assays performed after Zeb1 depletion by siRNA transfection mimicked the results obtained with miR-23b overexpression. These results indicate that effects of miR-23b in bladder cancer are partly by directly targeting Zeb1, though other targets may also be involved since microRNAs can target thousands of genes. Zeb1 is one of the crucial regulators of epithelial-to-mesenchymal transition (EMT) 
and has been shown to play a major role in invasion and metastasis of epithelial tumors 
. The relevance of ZEB proteins to tumor progression has been studied in several human cancers. Expression of ZEB1 correlated with an aggressive phenotype in various histological types of endometrial carcinoma and was detected in the sarcomatous compartment of endometrial carcinosarcoma 
. In colon cancer, ZEB1 was expressed at the invasive front of tumors, in association with the transient loss of basement membranes 
. Reciprocal expression of ZEB1 and E-cadherin has also been observed in non-small cell lung carcinoma 
. A direct correlation between ZEB1 immunoreactivity and Gleason grade has been reported in human prostate tumors 
and in bladder cancer, ZEB1 has been reported to be over-expressed and responsible for enhanced motility 
. In the present study, we found that over-expression of miR-23b resulted in suppression of oncogene Zeb1 in the bladder cancer cells suggesting that miR-23b can mediate EMT, thereby representing a possible mechanism through which it affects bladder cancer migration and invasion.
In conclusion, this study shows that miR-23b has diagnostic/prognostic significance and directly targets oncogenic Zeb1 in bladder cancer. miR-23b over-expression resulted in suppression of the bladder cancer cell proliferation and invasion, inducing apoptosis, and cell cycle arrest. Finally, this study indicates that miR-23b over-expression may be a therapeutically useful strategy for the treatment of bladder cancer.