Abstract
The microRNAs (miRNAs) miR-132 and miR-212 have been found to regulate synaptic plasticity and cholinergic signaling and recent work has demonstrated roles outside of the CNS, including in smooth muscle. Here, we examined if miR-132 and miR-212 are induced in the urinary bladder following outlet obstruction and whether this correlates with effects on gene expression and cell growth. Three to seven-fold induction of miR-132/212 was found at 10 days of obstruction and this was selective for the detrusor layer. We cross-referenced putative binding sites in the miR-132/212 promoter with transcription factors that were predicted to be active in the obstruction model. This suggested involvement of Creb and Ahr in miR-132/212 induction. Creb phosphorylation (S-133) was not increased, but the number of Ahr positive nuclei increased. Moreover, we found that serum stimulation and protein kinase C activation induced miR-132/212 in human detrusor cells. To identify miR-132/212 targets, we correlated the mRNA levels of validated targets with the miRNA levels. Significant correlations between miR-132/212 and MeCP2, Ep300, Pnkd and Jarid1a were observed, and the protein levels of MeCP2, Pnkd and Ache were reduced after obstruction. Reduction of Ache however closely matched a 90% reduction of synapse density arguing that its repression was unrelated to miR-132/212 induction. Importantly, transfection of antimirs and mimics in cultured detrusor cells increased and decreased, respectively, the number of cells and led to changes in MeCP2 expression. In all, these findings show that obstruction of the urethra increases miR-132 and miR-212 in the detrusor and suggests that this influences gene expression and limits cell growth.
Highlights
MicroRNAs are evolutionarily conserved small RNA molecules (19–24 nucleotides) that hybridize with mRNAs leading to degradation or translational repression [1]
In view of the cholinergic neuroeffector transmission defect that we have reported for the bladder following smooth musclespecific deletion of Dicer [3], we tested if miR-132/212 regulates cholinergic activation of the bladder via effects on acetylcholine esterase (Ache)
We found that the detrusor:mucosa ratios of both miR-132 and miR-212 were reduced in Dicer KO bladders (Fig. 2C)
Summary
MicroRNAs (miRNAs) are evolutionarily conserved small RNA molecules (19–24 nucleotides) that hybridize with mRNAs leading to degradation or translational repression [1]. Bladder Outlet Obstruction and miR-132/212 ribonuclease activities of Dicer and Drosha are required for processing of miRNA precursors [2]. In recent work, using conditional and smooth muscle-specific deletion of Dicer, we established roles of miRNAs in contractility, matrix deposition and cholinergic neuro-effector transmission in the urinary bladder [3]. In subsequent work we discovered that miR-29b and miR-29c key roles in extracellular matrix homeostasis, and, using microarrays, we found over 50 miRNAs that were differentially expressed following outlet obstruction [4]. These findings support the notion that miRNAs play important roles in urological pathologies beyond cancer
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