Abstract
The mammalian bladder urothelium classified as basal, intermediate, and terminally differentiated umbrella cells offers one of the most effective permeability barrier functions known to exist in nature because of the formation of apical uroplakin plaques and tight junctions. To improve our understanding of urothelial differentiation, we analyzed the microRNA (miRNA) expression profiles of mouse urinary tissues and by TaqMan miRNA analysis of microdissected urothelial layers and in situ miRNA-specific hybridization to determine the dependence of these miRNAs on the differentiation stage. Our in situ hybridization studies revealed that miR-205 was enriched in the undifferentiated basal and intermediate cell layers. We then used a quantitative proteomics approach to identify miR-205 target genes in primary cultured urothelial cells subjected to antagomir-mediated knockdown of specific miRNAs. Twenty-four genes were reproducibly regulated by miR-205; eleven of them were annotated as cell junction- and tight junction-related molecules. Western blot analysis demonstrated that antagomir-induced silencing of miR-205 in primary cultured urothelial cells elevated the expression levels of Tjp1, Cgnl1, and Cdc42. Ectopic expression of miR-205 in MDCK cells inhibited the expression of tight junction proteins and the formation of tight junctions. miR-205- knockdown urothelial cells showed alterations in keratin synthesis and increases of uroplakin Ia and Ib, which are the urothelial differentiation products. These results suggest that miR-205 may contribute a role in regulation of urothelial differentiation by modulating the expression of tight junction-related molecules.
Highlights
From the ‡Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan; §Medical Research and Development, Chang Gung Memorial Hospital, Taoyuan 33375, Taiwan; ¶Department of Urology, Chang Gung Memorial Hospital, Taoyuan 33375, Taiwan; ʈDepartment of Microbiology and Immunology, Chung Shan Medical University, Taichung 40201, Taiwan; **Department of Information Management, Chang Gung University, Taoyuan 33302, Taiwan; and ‡‡Graduate Institute of Biomedical Science, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
The urothelium contributes to forming an extensible barrier that accommodates the large changes in urine volume and offers highly specialized umbrella cells that have distinct apical and basolateral membrane domains demarcated by tight junctions [7,8,9]
Identification of miRNAs that are Associated with Differentiation of the Urothelium—To explore which miRNAs may have potent roles in regulating urothelial differentiation, we first performed the exploratory microarray analyses of miRNA expression levels in RNA samples isolated from different urinary tissues including the mouse ureter, kidney, and the urothelial as well as muscularis layers of the bladder
Summary
We constructed the cumulative distribution plots of the log2-transformed protein expression foldchanges for those genes containing miR-205 target sites and all other expressed genes in response to antagomir-mediated knockdown of endogenous miR-205 in primary cultured urothelial cells. MiR-205 Down-regulates Cgnl1 at the Protein and mRNA Levels in the Urothelium—In addition to quantitative proteomic profiling, we conducted a transcriptomic analysis of antagomir-treated urothelial cells and revealed a total of 21,198 gene expression, which included 2154 genes (10.16%) containing the binding sites of miR-205 from more than two prediction programs.
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