MicroRNAs dynamically remodel gastric smooth muscle cells

Smooth muscle cells (SMCs) display dynamic plasticity by changing phenotype under various pathophysiological conditions. Uncovering how SMCs regulate their phenotype is a key to understanding the molecular mechanisms of a number of gastrointestinal diseases. MicroRNAs (miRNAs), generated in cells by Dicer, have been identified as regulators of the differentiation state of SMCs. The goal of this study was to investigate the role of miRNAs during the development of gastrointestinal SMCs in a transgenic animal model. We generated SMC-specific Dicer (smDicer) null animals that express the reporter, green fluorescence protein (eGFP), in a SMC-specific manner. eGFP labeled SMCs were used for morphological, cytometric and genetic studies of smDicer null mutant and wild type control mice. The structure of the bowel wall was examined by confocal microscopy, and function was evaluated by recording spontaneous and evoked contractions. SMCs were purified with fluorescence-activated cell sorting and SM specific gene expression studies were performed with genechip arrays, PCR and quantitative PCR. Bioinformatic analyses were used to characterize the interaction between miRNAs and target genes. SMC-specific knockout of Dicer prevented SMC miRNA biogenesis, causing dramatic changes in phenotype, function, and global gene expression in SMCs. Profiling and bioinformatic analyses showed SMC phenotype is regulated by a complex network of positive and negative feedback by SM miRNAs, serum response factor (SRF), and other transcriptional factors. SM miRNAs play an important role in growth, development and survival of SMCs. Phenotypic changes of SMCs may be regulated through multiple pathways in an interaction network of SM miRNAs, SRF, and additional transcriptional factors. We used microarrays to identify genetic changes during the development of gastric smooth muscle cells in the smDicer null mice. Mouse mRNA microarrays were performed on GeneChip Mouse Genome 430 2.0 Arrays (Affymetrix). Total RNAs isolated from the jejunal muscularis of the smDicer knockout (KO) and the wild type (WT) control mice at the ages of ~3 weeks were used for the arrays. The cDNA synthesis, hybridization, cDNA labeling, and scanning were performed. Gene expression between the smDicer KO and the WT controls were compared.平滑肌细胞（smooth muscle cells, SMCs）可在多种病理生理条件下发生表型转换，展现出动态可塑性。阐明平滑肌细胞调控自身表型的机制，是解析诸多胃肠道疾病分子机制的关键所在。 由迪切酶（Dicer）在细胞内合成的微小RNA（miRNAs），已被证实为平滑肌细胞分化状态的重要调控因子。本研究旨在探究微小RNA在转基因动物模型的胃肠道平滑肌细胞发育过程中的作用。我们构建了平滑肌细胞特异性迪切酶（SMC-specific Dicer, smDicer）敲除动物模型，该模型可通过平滑肌细胞特异性方式表达报告基因绿色荧光蛋白（eGFP）。借助eGFP标记的平滑肌细胞，我们对smDicer敲除突变小鼠与野生型对照小鼠开展了形态学、细胞计量学及遗传学研究。 利用共聚焦显微镜观察肠壁结构，通过记录自发及诱发收缩评估肠道功能。采用荧光激活细胞分选术（fluorescence-activated cell sorting）纯化平滑肌细胞，并通过基因芯片阵列、聚合酶链反应（PCR）及定量聚合酶链反应（qPCR）开展平滑肌细胞特异性基因表达分析。借助生物信息学分析表征微小RNA与靶基因之间的相互作用。 平滑肌细胞特异性敲除迪切酶会阻断细胞内微小RNA的生物合成途径，进而引发平滑肌细胞表型、功能及全局基因表达的显著改变。表达谱分析与生物信息学研究显示，平滑肌细胞表型受微小RNA、血清反应因子（serum response factor, SRF）及其他转录因子构成的正负反馈复杂网络调控。 微小RNA在平滑肌细胞的生长、发育及存活过程中发挥关键作用。平滑肌细胞的表型改变可能通过微小RNA、血清反应因子及其他转录因子的相互作用网络中的多条通路实现调控。 本研究利用基因芯片技术鉴定smDicer敲除小鼠胃平滑肌细胞发育过程中的遗传改变。实验采用Affymetrix公司的GeneChip Mouse Genome 430 2.0阵列开展小鼠mRNA基因芯片检测。提取约3周龄smDicer敲除（KO）小鼠与野生型（WT）对照小鼠空肠肌层的总RNA，用于芯片实验。实验流程涵盖cDNA合成、杂交、cDNA标记及扫描。对smDicer敲除组与野生型对照组的基因表达水平进行了比较分析。