Lab of Tissue Engineering, College of Life Sciences , Northwest University , Xi'an 710069 , PR China.
Provincial Key Laboratory of Biotechnology of Shaanxi , Xi'an 710069 , PR China.
J Proteome Res. 2019 Mar 1;18(3):1088-1098. doi: 10.1021/acs.jproteome.8b00778. Epub 2019 Jan 17.
Salamanders completely regenerate their limbs after amputation. Thus, these animals are unique models to investigate the mechanisms modulating regeneration in vertebrates. To investigate the influence of microRNAs (miRNAs) on newt limb regeneration, the miRNAs and mRNAs were simultaneously profiled using Illumina HiSeq 2500 System during limb regeneration of Cynops orientalis at 3, 7, 14, 30 and 42 days postamputation. A total of 203 miRNAs and 4230 mRNAs were identified to be differentially expressed. Together with the proteomic data obtained from our previous study, integrative analysis of multiple profiling data sets was performed to construct an interaction network of differentially expressed miRNAs, mRNAs and proteins. Results of GO and KEGG analyses showed that the differentially expressed miRNA targets were mainly directed to cytoskeletal remodeling and carbohydrate metabolism. The stage-specific regulation of miRNAs on their targets was analyzed by hierarchical clustering analysis and validated by qRT-PCR. The negative regulation of miR-223 and miR-133a on their targets was tested by performing dual luciferase reporter assay. The integration analysis will provide a powerful tool to identify the regulatory mechanisms of miRNAs and their targets. The results may have implications in understanding the complex mechanisms underlying newt limb regeneration.
蝾螈在截肢后会完全再生肢体。因此，这些动物是研究脊椎动物再生调节机制的独特模型。为了研究 microRNAs（miRNAs）对蝾螈肢体再生的影响，我们使用 Illumina HiSeq 2500 系统在东方蝾螈截肢后 3、7、14、30 和 42 天期间同时对其 miRNA 和 mRNA 进行了分析。共鉴定出 203 个 miRNA 和 4230 个 mRNA 存在差异表达。结合我们之前研究中获得的蛋白质组学数据，对多个分析数据集进行了综合分析，构建了差异表达 miRNA、mRNA 和蛋白质的相互作用网络。GO 和 KEGG 分析的结果表明，差异表达 miRNA 的靶标主要指向细胞骨架重塑和碳水化合物代谢。通过层次聚类分析对 miRNA 对其靶标的阶段特异性调控进行了分析，并通过 qRT-PCR 进行了验证。通过双荧光素酶报告基因检测实验验证了 miR-223 和 miR-133a 对其靶标的负调控作用。整合分析将为识别 miRNA 及其靶标的调控机制提供有力工具。这些结果可能有助于理解蝾螈肢体再生的复杂机制。
