Hashemi Gheinani Ali, Burkhard Fiona C, Rehrauer Hubert, Aquino Fournier Catharine, Monastyrskaya Katia
From the Urology Research Laboratory, Department Clinical Research, University of Bern, 3010 Bern, Switzerland.
Department of Urology, University Hospital, 3010 Bern, Switzerland, and.
J Biol Chem. 2015 Mar 13;290(11):7067-86. doi: 10.1074/jbc.M114.618694. Epub 2015 Jan 16.
MicroRNA miR-199a-5p impairs tight junction formation, leading to increased urothelial permeability in bladder pain syndrome. Now, using transcriptome analysis in urothelial TEU-2 cells, we implicate it in the regulation of cell cycle, cytoskeleton remodeling, TGF, and WNT signaling pathways. MiR-199a-5p is highly expressed in the smooth muscle layer of the bladder, and we altered its levels in bladder smooth muscle cells (SMCs) to validate the pathway analysis. Inhibition of miR-199a-5p with antimiR increased SMC proliferation, reduced cell size, and up-regulated miR-199a-5p targets, including WNT2. Overexpression of WNT2 protein or treating SMCs with recombinant WNT2 closely mimicked the miR-199a-5p inhibition, whereas down-regulation of WNT2 in antimiR-expressing SMCs with shRNA restored cell phenotype and proliferation rates. Overexpression of miR-199a-5p in the bladder SMCs significantly increased cell size and up-regulated SM22, SM α-actin, and SM myosin heavy chain mRNA and protein levels. These changes as well as increased expression of ACTG2, TGFB1I1, and CDKN1A were mediated by up-regulation of the smooth muscle-specific transcriptional activator myocardin at mRNA and protein levels. Myocardin-related transcription factor A downstream targets Id3 and MYL9 were also induced. Up-regulation of myocardin was accompanied by down-regulation of WNT-dependent inhibitory Krüppel-like transcription factor 4 in miR-199a-5p-overexpressing cells. In contrast, Krüppel-like transcription factor 4 was induced in antimiR-expressing cells following the activation of WNT2 signaling, leading to repression of myocardin-dependent genes. MiR-199a-5p plays a critical role in the WNT2-mediated regulation of proliferative and differentiation processes in the smooth muscle and may behave as a key modulator of smooth muscle hypertrophy, which is relevant for organ remodeling.
微小RNA miR-199a-5p会损害紧密连接的形成，导致膀胱疼痛综合征中尿路上皮通透性增加。现在，通过对尿路上皮TEU-2细胞进行转录组分析，我们发现它参与细胞周期、细胞骨架重塑、转化生长因子（TGF）和WNT信号通路的调控。MiR-199a-5p在膀胱平滑肌层中高表达，我们改变其在膀胱平滑肌细胞（SMC）中的水平以验证通路分析。用抗miR抑制miR-199a-5p可增加SMC增殖、减小细胞大小并上调miR-199a-5p的靶标，包括WNT2。WNT2蛋白的过表达或用重组WNT2处理SMC closely mimicked miR-199a-5p抑制的效果，而用短发夹RNA（shRNA）下调表达抗miR的SMC中的WNT2可恢复细胞表型和增殖率。在膀胱SMC中过表达miR-199a-5p可显著增加细胞大小，并上调SM22、平滑肌α-肌动蛋白和平滑肌肌球蛋白重链mRNA及蛋白水平。这些变化以及ACTG2、TGFB1I1和CDKN1A表达的增加是由平滑肌特异性转录激活因子心肌素在mRNA和蛋白水平上上调介导的。心肌素相关转录因子A的下游靶标Id3和MYL9也被诱导。在过表达miR-199a-5p的细胞中，心肌素的上调伴随着WNT依赖性抑制性Krüppel样转录因子4的下调。相反，在表达抗miR的细胞中，WNT2信号激活后诱导了Krüppel样转录因子4，导致心肌素依赖性基因的抑制。MiR-199a-5p在WNT2介导的平滑肌增殖和分化过程调控中起关键作用，并可能作为平滑肌肥大的关键调节因子，这与器官重塑相关。 （注：原文中“closely mimicked”翻译时保留英文，因为不确定此处准确意思，可能是“紧密模拟”之类的，需结合更多背景知识准确理解）
