Differential microRNA expression patterns in CCR2-/- versus wild type mice following skeletal muscle injury
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Dynamic events in injured skeletal muscle lead to a robust regenerative response that is critically dependent on macrophages; however, the regulatory role of these inflammatory cells remains to be established. Given the important role of microRNA (miRNA) in macrophage biology, it seems likely that these small, non-coding RNAs are involved in macrophage-specific biological events. The present study was designed to explore this possibility by exploiting the known defect in macrophage recruitment and activation in CCR2-/- mice. Thus, the temporal expression of macrophage-related miRNA in muscle regenerative events in vivo was examined in cardiotoxin-injured muscle derived from wild type (WT, C57Bl/6J) or CCR2-/- mice. Total RNA was extracted from muscle and miRNA expression determined by qRT-PCR in TaqMan assays (Applied Biosystems). As compared to baseline (uninjured) muscle, log fold change in miRNA expression at 1, 3, or 7 days following injury (n=4 mice/group/timepoint) was determined. In a separate group of animals studied at similar timepoints, monocyte/macrophage numbers in injured muscle were determined by flow cytometry and confirmed a significant reduction in macrophage infiltration in CCR2-/- animals. In both mouse strains, considerable changes in diverse miRNAs were observed following injury. But, most importantly, in comparisons of the temporal expression patterns of miRNA from WT and CCR2-/- mice, cluster analysis revealed 60 miRNAs with different patterns of expression, including miRNAs that have been reported to be highly expressed in macrophages. Interestingly, 15 miRNAs with no known relationship to macrophages were matched with the temporal expression patterns of macrophage-related miRNAs. Thus, it is conceivable that this latter group of miRNAs may be unique, macrophage-related miRNA that regulate the muscle regenerative response. Given the parallel development of sarcopenia and altered macrophage biology during aging, the miRNAs identified in the present study may be useful in the elucidation of new targets for therapeutic intervention of sarcopenia.
