The significance of mirna pathway in peripheral nerve regeneration following sciatic nerve injury.

Abstract

microRNAs (miRNAs) are small (21-23 nucleotides) single-stranded RNAs that play important roles in post-transcriptional regulation of gene expression. The binding of miRNA to its specific target mRNAs at 3' UTR causes either degradation of the mRNAs or inhibition of protein translation. Increasing evidence suggests that miRNA participate in the regulation of almost every cellular process investigated and changes of their expressions are observed in human pathologies. In neurons, the functions of miRNA pathway are just beginning to emerge. Recent studies have elucidated that miRNAs are dysregulated in neurological disorders and that they serve important roles in neural development. Subcellular localization of miRNA and miRNA machinery has also been identified in axons, which suggests its involvement in axonal functions. In the current study, we tested the hypothesis that miRNA pathway is involved in the post-transcriptional regulation of gene expression in sciatic nerve after injury, and that it plays a critical role for peripheral nerve regeneration. We performed sciatic nerve crush as our injury model on adult mice. A corresponding injury-regulated expression of miRNA machinery, including components of RNA induced silencing complex (RISC) and Processing bodies (P-bodies) were observed in both western blot analysis and immuofluorescent staining. Microarray analysis followed by RT-qPCR confirmation revealed a group of miRNAs following injury-regulated expression patterns as well. The physiological importance of miRNA pathway in peripheral nerve regeneration was evaluated in inducible Dicer knockout mice. Blocking Dicer-dependent miRNA biogenesis significantly delayed peripheral nerve regeneration in behavioral tests, electrophysiological studies, and histological evaluations. The detrimental effect of Dicer ablation on regenerative axon growth was also shown in dissociated dorsal root ganglion (DRG) neurons. A significant decrease in axon length and arborization was observed in vitro. To find the specific miRNAs involved in mediating axon growth, 19 miRNAs differentially expressed after nerve injury were identified with miRNA array analyses. Functional analysis for injury-induced miRNAs has shown that miR-431 promoted axon growth in DRG neurons. Bioinformatics prediction followed by experimental approaches proved Kremen1 as a target gene for miR-431. A negative correlation between miR-431 and Kremen1 expression was confirmed at both mRNA and protein levels. Since Kremen1 inhibits the Wnt signaling, enhanced axon growth after overexpressing miR-431 might be mediated through Wnt signaling. The findings of this study improve our understanding of the significant role that miRNA pathway plays in regenerative axon growth.