title: MiR-133 modulates TGF-β1-induced bladder smooth muscle cell hypertrophic and fibrotic response: implication for a role of microRNA in bladder wall remodeling caused by bladder outlet obstruction.
abstract: Bladder outlet obstruction (BOO) evokes urinary bladder wall remodeling significantly, including the phenotype shift of bladder smooth muscle cells (BSMCs) where transforming growth factor-beta1 (TGF-β1) plays a pivotal role given the emerging function of modulating cellular phenotype. miR-133 plays a role in cardiac and muscle remodeling, however, little is known about its roles in TGF-β1-induced BSMC hypertrophic and fibrotic response. Here, we verified BOO induced bladder wall remodeling and TGF-β1 expression mainly located in bladder endothelium. Furthermore, we uncovered miR-133a/b expression profile in BOO rats, and then explored its regulated effects on BSMCs' phenotypic shift. Our study found that miR-133 became down-regulated during rat bladder remodeling. Next, we sought to examine whether the expression of miR-133 was down-regulated in primary BSMCs in response to TGF-β1 stimulation and whether forced overexpression of miR-133 could regulate profibrotic TGF-β signaling. We found that stimulation of BSMCs with exogenous TGF-β1 of increasing concentrations resulted in a dose-dependent decrease of miR-133a/b levels and transfection with miR-133 mimics attenuated TGF-β1-induced α-smooth muscle actin, extracellular matrix subtypes and fibrotic growth factor expression, whereas it upregulated high molecular weight caldesmon expression compared with the negative control. Also, downregulation of p-Smad3, not p-Smad2 by miR-133 was detected. Additionally, miR-133 overexpression suppressed TGF-β1-induced BSMC hypertrophy and proliferation through influencing cell cycle distribution. Bioinformatics analyses predicted that connective tissue growth factor (CTGF) was the potential target of miR-133, and then binding to the 3'-untranslated region of CTGF was validated by luciferase reporter assay. These results reveal a novel regulator for miR-133 to modulate TGF-β1-induced BSMC phenotypic changes by targeting CTGF through the TGF-β-Smad3 signaling pathway. A novel antifibrotic functional role for miR-133 is presented which may represent a potential target for diagnostic and therapeutic strategies in bladder fibrosis.
authors.affiliation: Department of Urology, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 1665 Kong Jiang Road, Shanghai 200092, China.
authors.forename: Liu Jian
authors.initials: LJ
authors.lastname: Duan
authors.affiliation: Department of Urology, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 1665 Kong Jiang Road, Shanghai 200092, China. Electronic address: jasonqi@sh163.net.
authors.forename: Jun
authors.initials: J
authors.lastname: Qi
authors.affiliation: Department of Urology, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 1665 Kong Jiang Road, Shanghai 200092, China.
authors.forename: Xiang Jie
authors.initials: XJ
authors.lastname: Kong
authors.affiliation: Department of Urology, An Hui Provincial Hospital, 17 Lu Jiang Road, Hefei, Anhui 230001, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Huang
authors.affiliation: Department of Urology, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 1665 Kong Jiang Road, Shanghai 200092, China.
authors.forename: Xiao Qiang
authors.initials: XQ
authors.lastname: Qian
authors.affiliation: Department of Urology, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 1665 Kong Jiang Road, Shanghai 200092, China.
authors.forename: Ding
authors.initials: D
authors.lastname: Xu
authors.affiliation: Department of Urology, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 1665 Kong Jiang Road, Shanghai 200092, China.
authors.forename: Jun Hao
authors.initials: JH
authors.lastname: Liang
authors.affiliation: Department of Urology, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 1665 Kong Jiang Road, Shanghai 200092, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Kang
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001483:Base Sequence; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor / Q000737:chemistry / Q000378:metabolism; D004195:Disease Models, Animal; D015536:Down-Regulation / Q000187:drug effects; D006801:Humans; D006984:Hypertrophy / Q000473:pathology; D008297:Male; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016415:Sequence Alignment; D015398:Signal Transduction / Q000187:drug effects; D051900:Smad3 Protein / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects; D001743:Urinary Bladder / Q000378:metabolism; D001748:Urinary Bladder Neck Obstruction / Q000378:metabolism / Q000473:pathology*
chemical_list: D000199:Actins; D002148:Calmodulin-Binding Proteins; D055513:Connective Tissue Growth Factor; D035683:MicroRNAs; D051900:Smad3 Protein; D053773:Transforming Growth Factor beta1; C526316:CCN2 protein, rat; C549446:MIRN133 microRNA, rat; C498862:Acta2 protein, rat
keywords: Bladder fibrosis; Bladder outlet obstruction; Bladder smooth muscle cells; Extracellular matrix; TGF-β1; miR-133