TLR4/NF-κ B-responsive MicroRNAs and their potential target genes: A mouse model of skeletal muscle ischemia-reperfusion injury

Abstract

Background.The aim of this study was to profile TLR4/NF-κ B-responsive microRNAs (miRNAs) and their potential target genes in the skeletal muscles of mice following ischemia-reperfusion injury. Methods. Thigh skeletal muscles of C57BL/6, Tlr4-/- and NF-κ B-/-mice isolated based on femoral artery perfusion were subjected to ischemia for 2h and reperfusion for 0h, 4h, 1d, 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. Results.Three TLR4/NF-κ B-responsive miRNAs (miR-15a, miR-744, and miR- 1196)were significantly upregulated in themuscles following ischemia-reperfusion injury.Thecombined in silico andwhole 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 upregulatedmiRNAs, Lrsam1 and Ddx21 are known to be associated with the innate immunity pathway. Conclusions. This study profiled TLR4/NF-κ B-responsive miRNAs and their potential target genes in mouse skeletal muscle subjected to ischemia-reperfusion injury.