Yang Johnson Chia-Shen, Wu Shao-Chun, Rau Cheng-Shyuan, Chen Yi-Chun, Lu Tsu-Hsiang, Wu Yi-Chan, Tzeng Siou-Ling, Wu Chia-Jung, Hsieh Ching-Hua
Department of Plastic and Reconstructive Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, No. 123, Ta-Pei Road, Niao-Song District, Kaohsiung City 833, Taiwan.
Department of Anesthesiology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, No. 123 Ta-Pei Road, Niao-Song District, Kaohsiung City 833, Taiwan.
Biomed Res Int. 2015;2015:410721. doi: 10.1155/2015/410721. Epub 2015 Jan 26.
The aim of this study was to profile TLR4/NF-κB-responsive microRNAs (miRNAs) and their potential target genes in the skeletal muscles of mice following ischemia-reperfusion injury.
Thigh skeletal muscles of C57BL/6, Tlr4(-/-), and NF-κB(-/-) mice isolated based on femoral artery perfusion were subjected to ischemia for 2 h and reperfusion for 0 h, 4 h, 1 d, and 7 d. The muscle specimens were analyzed with miRNA arrays. Immunoprecipitation with an argonaute 2- (Ago2-) specific monoclonal antibody followed by whole genome microarray was performed to identify mRNA associated with the RNA-silencing machinery. The potential targets of each upregulated miRNA were identified by combined analysis involving the bioinformatics algorithm miRanda and whole genome expression.
Three TLR4/NF-κB-responsive miRNAs (miR-15a, miR-744, and miR-1196) were significantly upregulated in the muscles following ischemia-reperfusion injury. The combined in silico and whole genome microarray approaches identified 5, 4, and 20 potential target genes for miR-15a, miR-744, and miR-1196, respectively. Among the 3 genes (Zbed4, Lrsam1, and Ddx21) regulated by at least 2 of the 3 upregulated miRNAs, Lrsam1 and Ddx21 are known to be associated with the innate immunity pathway.
This study profiled TLR4/NF-κB-responsive miRNAs and their potential target genes in mouse skeletal muscle subjected to ischemia-reperfusion injury.
本研究旨在分析缺血再灌注损伤后小鼠骨骼肌中TLR4/NF-κB反应性微小RNA（miRNA）及其潜在靶基因。
基于股动脉灌注分离C57BL/6、Tlr4（-/-）和NF-κB（-/-）小鼠的大腿骨骼肌，进行2小时缺血及0小时、4小时、1天和7天再灌注。用miRNA芯片分析肌肉标本。用特异性针对AGO2的单克隆抗体进行免疫沉淀，随后进行全基因组芯片分析，以鉴定与RNA沉默机制相关的mRNA。通过结合miRanda生物信息学算法和全基因组表达分析，确定每个上调miRNA的潜在靶标。
缺血再灌注损伤后，三种TLR4/NF-κB反应性miRNA（miR-15a、miR-744和miR-1196）在肌肉中显著上调。计算机模拟和全基因组芯片相结合的方法分别鉴定出miR-15a、miR-744和miR-1196的5个、4个和20个潜在靶基因。在这三种上调miRNA中至少由两种调控的3个基因（Zbed4、Lrsam1和Ddx21）中，Lrsam1和Ddx21已知与天然免疫途径相关。
本研究分析了缺血再灌注损伤小鼠骨骼肌中TLR4/NF-κB反应性miRNA及其潜在靶基因。
