microRNA profiling of extraocular muscles

The extraocular muscles (EOMs) are a unique group of muscles that are anatomically and physiologically distinct from other skeletal muscles. Previously, we and others have shown that EOMs have a unique transcriptome and proteome. Here, we investigated the expression pattern of microRNAs (miRNAs) in EOM, as they may play a role in generating the unique EOM allotype. We screened LC Sciences miRNA microarrays covering the sequences of miRBase 10.0 to define the microRNAome of normal mouse EOM and tibialis anterior (TA) limb muscle. 74 miRNAs were found to be differentially regulated (p-value < 0.05) and 31 miRNAs (14 up-regulated and 17 down-regulated) were found to be differentially regulated at a signal strength > 500 including the muscle-specific miR-206, miR-1, miR-133a, miR-133b and miR-499. qPCR analysis was used to validate the differential expression. Bioinformatic tools were used to identify potential miRNA-mRNA-protein interactions and integrate data with previous transcriptome and proteomic profiling data. Luciferase assays using co-transfection of precursor miRNAs (pre-miRNAs) along with reporter constructs containing the 3'-untranslated region (3'-UTR) of their predicted target genes were used to validate targeting by identified miRNAs. The definition of the EOM microRNAome complements existing transcriptome and proteome data about the molecular make-up of EOM and provides further insight into regulation of muscle genes. These data will also help to further explain the unique EOM muscle allotype and its differential sensitivity to diseases such as Duchenne's muscular dystrophy (DMD) and may assist in development of therapeutic strategies. Total RNA from four EOM and four TA tissue samples dissected from four adult male C57/Bl10 mice were used (TA served as control) to screen four LC Sciences microRNA Microarray chips. The chips contained microRNA sequences based on miRBase content 10.0 totalling 568 different miRNAs. Samples were labelled with Cy3 and Cy5 using dye-swap. Relative differences of miRNA expression was expressed as fold-changes EOM/TA, which were calculated after normalization across all four arrays.眼外肌（extraocular muscles, EOMs）是一类独特的肌肉群，在解剖学与生理学层面均有别于其他骨骼肌。此前本团队与其他研究团队均已证实，眼外肌拥有独特的转录组（transcriptome）与蛋白质组（proteome）。本研究聚焦于眼外肌内微小核糖核酸（microRNAs, miRNAs）的表达模式，因其可能在塑造眼外肌独特表型的过程中发挥关键作用。本研究采用覆盖miRBase 10.0序列的LC Sciences miRNA微阵列，对正常小鼠眼外肌与胫前肌（tibialis anterior, TA）的微小RNA组（microRNAome）进行了表征分析。经筛选，共有74种miRNAs呈现差异表达（p值<0.05）；当信号强度大于500时，另有31种miRNAs呈现差异表达，其中14种上调、17种下调，涵盖肌肉特异性miR-206、miR-1、miR-133a、miR-133b及miR-499。本研究采用实时定量聚合酶链式反应（qPCR）对上述差异表达结果进行了验证。通过生物信息学工具预测潜在的miRNA-mRNA-蛋白质相互作用，并将本研究数据与既往转录组、蛋白质组表达谱数据进行整合分析。通过共转染前体miRNAs（pre-miRNAs）与携带预测靶基因3'非翻译区（3'-untranslated region, 3'-UTR）的报告基因构建体的荧光素酶报告实验，验证了所鉴定miRNAs的靶向调控作用。眼外肌微小RNA组的表征，补充了现有关于眼外肌分子组成的转录组与蛋白质组数据，为肌肉基因的调控机制提供了更深层次的解析。本数据集还有助于进一步阐释眼外肌的独特表型，及其对杜氏肌营养不良症（Duchenne's muscular dystrophy, DMD）等疾病的差异化易感性，并可为相关治疗策略的开发提供参考依据。本研究采集4只成年雄性C57/Bl10小鼠的眼外肌与胫前肌组织样本各4份（胫前肌作为对照），使用4张LC Sciences miRNA微阵列芯片完成筛选实验。该芯片基于miRBase 10.0版本构建，共收录568种不同的miRNAs序列。实验采用染料交换法，以Cy3与Cy5荧光染料对样本进行标记。所有4张芯片经标准化处理后，以EOM/TA的倍数变化表示miRNA表达的相对差异。