Mir-29 Repression in Bladder Outlet Obstruction Contributes to Matrix Remodeling and Altered Stiffness Mari Ekman 1,3 , Anirban Bhattachariya 1 , Diana Dahan 1 , Bengt Uvelius 2 , Sebastian Albinsson 1 , Karl Swärd 1* 1 Department of Experimental Medical Science, Lund University, Lund, Sweden, 2 Department of Urology, Lund University, Lund, Sweden, 3 Department of Biology, Lund University, Lund, Sweden Abstract Recent work has uncovered a role of the microRNA (miRNA) miR-β9 in remodeling of the extracellular matrix. Partial bladder outlet obstruction is a prevalent condition in older men with prostate enlargement that leads to matrix synthesis in the lower urinary tract and increases bladder stiffness. Here we tested the hypothesis that miR-β9 is repressed in the bladder in outlet obstruction and that this has an impact on protein synthesis and matrix remodeling leading to increased bladder stiffness. c-Myc, NF-κB and SMADγ, all of which repress miR-β9, were activated in the rat detrusor following partial bladder outlet obstruction but at different times. c-Myc and NF-κB activation occurred early after obstruction, and SMADγ phosphorylation increased later, with a significant elevation at 6 weeks. c-Myc, NF-κB and SMADγ activation, respectively, correlated with repression of miR-β9b and miR-β9c at 10 days of obstruction and with repression of miR-β9c at 6 weeks. An mRNA microarray analysis showed that the reduction of miR-β9 following outlet obstruction was associated with increased levels of miR-β9 target mRNAs, including mRNAs for tropoelastin, the matricellular protein Sparc and collagen IV. Outlet obstruction increased protein levels of eight out of eight examined miR-β9 targets, including tropoelastin and Sparc. Transfection of human bladder smooth muscle cells with antimiR-β9c and miR-β9c mimic caused reciprocal changes in target protein levels in vitro. Tamoxifen inducible and smooth muscle-specific deletion of Dicer in mice reduced miR-β9 expression and increased tropoelastin and the thickness of the basal lamina surrounding smooth muscle cells in the bladder. It also increased detrusor stiffness independent of outlet obstruction. Taken together, our study supports a model where the combined repressive influences of c-Myc, NF-κB and SMADγ reduce miR-β9 in bladder outlet obstruction, and where the resulting drop in miR-β9 contributes to matrix remodeling and altered passive mechanical properties of the detrusor. Citation: Ekman M, Bhattachariya A, Dahan D, Uvelius B, Albinsson S, et al. (β01γ) Mir-β9 Repression in Bladder Outlet Obstruction Contributes to Matrix Remodeling and Altered Stiffness. PLoS ONE 8(1β): e8βγ08. doi:10.1γ71/journal.pone.008βγ08 Editor: Partha Mukhopadhyay, National Institutes of Health, United States of America Received September 4, β01γ; Accepted October ββ, β01γ; Published December 10, β01γ Copyright: © β01γ Ekman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was financially supported by The Swedish Research Council, The Greta and Johan Kock’s Foundation, The Crafoord Foundation, The Medical Faculty at Lund University, Gösta Jonsson’s foundation, Olle Engkvist Byggmästare’s foundation and Avtal Läkare Forskning. AB is supported by the Marie Curie Initial Training Network SmArt from the European Union. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. * E-mail: karl.sward@med.lu.se Introduction MicroRNAs (miRNAs) are small single-stranded RNAs that control protein synthesis via messenger RNA (mRNA) degradation and translational repression [1]. Individual miRNAs have hundreds of predicted targets and close to one third of all protein-coding genes and, essentially, all biological pathways are influenced by miRNAs [β]. MiRNA-based therapeutics have progressed considerably in recent years, suggesting that this novel strategy may be brought to clinical application soon [γ]. Considerable work is needed to fully uncover the potential of miRNAs as therapeutic targets, and the exploration of miRNAs in urologic conditions other than cancer is in its infancy. The endonucleases Drosha and Dicer are required for miRNA maturation [4], and in recent work, we and others have taken advantage of conditional and smooth muscle-specific deletion of Dicer to probe the role of miRNAs in detrusor smooth muscle [5,6]. This leads to impairment of cholinergic neurotransmission, to a relative increase in the purinergic component of detrusor activation and to a disturbed micturition pattern. The miR-β9 cluster has gained recognition as a modulator of extracellular matrix production [7-10]. It comprises three miRNAs (miR-β9a, miR-β9b, and miR-β9c) derived from two independent genes [10]. Among the targets are several collagen isoforms, laminin, and elastin (Eln or tropoelastin) [11]. The extracellular matrix molecule elastin is one of the best established targets of miR-β9, and its message has 14 binding sites dispersed over the coding sequence and the γ’UTR [1β]. The matricellular protein Sparc has three miR-β9 binding sites PLOS ONE | www.plosone.org 1 December β01γ | Volume 8 | Issue 1β | e8βγ08