Dynamic Change and Target Prediction of Axon-Specific MicroRNAs in Regenerating Sciatic Nerve

RESEARCH ARTICLE Dynamic Change and Target Prediction of Axon-Specific MicroRNAs in Regenerating Sciatic Nerve Monichan Phay1,2, Hak Hee Kim1, Soonmoon Yoo1* 1 Nemours Biomedical Research, Alfred I DuPont Hospital for Children, Wilmington, Delaware, United States of America, 2 Department of Biological Sciences, University of Delaware, Delaware, Newark, United States of America * Abstract OPEN ACCESS Citation: Phay M, Kim HH, Yoo S (2015) Dynamic Change and Target Prediction of Axon-Specific MicroRNAs in Regenerating Sciatic Nerve. PLoS ONE 10(9): e0137461. doi:10.1371/journal. pone.0137461 Editor: Simone Di Giovanni, Hertie Institute for Clinical Brain Research, University of Tuebingen., GERMANY Received: May 6, 2015 Accepted: August 17, 2015 Published: September 2, 2015 Copyright: © 2015 Phay et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was funded by awards from the National Institutes of Health (P20-GM103464 and R21-NS085691 to SY). This project was also partially supported by Delaware INBRE Core Center Access Award (SY) from an Institutional Development Award (IDeA) Network of Biomedical Research Excellence program (INBRE; P20-GM103446) of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Injury to axons in the peripheral nervous system induces rapid and local regenerative responses to form a new growth cone, and to generate a retrogradely transporting injury signal. The evidence for essential roles of intra-axonal protein synthesis during regeneration is now compelling. MicroRNA (miRNA) has recently been recognized as a prominent player in post-transcriptional regulation of axonal protein synthesis. Here, we directly contrast temporal changes of miRNA levels in the sciatic nerve following injury, as compared to those in an uninjured nerve using deep sequencing. Small RNAs (<200 nucleotides in length) were fractionated from the proximal nerve stumps to improve the representation of differential miRNA levels. Of 141 axoplasmic miRNAs annotated, 63 rat miRNAs showed significantly differential levels at five time points following injury, compared to an uninjured nerve. The differential changes in miRNA levels responding to injury were processed for hierarchical clustering analyses, and used to predict target mRNAs by Targetscan and miRanda. By overlapping these predicted targets with 2,924 axonally localizing transcripts previously reported, the overlapping set of 214 transcripts was further analyzed by the Gene Ontology enrichment and Ingenuity Pathway Analyses. These results suggest the possibility that the potential targets for these miRNAs play key roles in numerous neurological functions involved in ER stress response, cytoskeleton dynamics, vesicle formation, and neurodegeneration and-regeneration. Finally, our results suggest that miRNAs could play a direct role in regenerative response and may be manipulated to promote regenerative ability of injured nerves. Introduction Axonal injury to the peripheral nervous system (PNS) triggers active translation of the localized transcripts that have been transported into axons from the cell body [1–4]. Intra-axonal translation of the mRNAs has been directly linked to spontaneous regenerative responses of PNS neurons [5–11]. For example, PNS nerve injury triggers a rapid translation of PLOS ONE | DOI:10.1371/journal.pone.0137461 September 2, 2015 1 / 12 Alterations of Axonal MicroRNA Levels after Injury Competing Interests: The authors have declared that no competing interests exist. importin-β1, vimentin, RanBP1, and Stat3 mRNAs in axons [5, 8–10, 12]. The proteins encoded by these axonal mRNAs provide for retrograde injury signals to the cell body. Furthermore, Verma et al [13] has shown that axonally synthesized proteins are needed for the reformation of growth cones, which are critical for subsequent regeneration of injured axons. These localized mRNAs are translationally dormant in the axon until recruited into translation machinery following injury. However, it is unclear how the activated local translation following injury becomes silent when it is no longer needed. MicroRNAs (miRNAs) are a class of small regulatory non-coding RNAs (~22 nt long). They have been proposed to negatively regulate translation in the nervous system at the posttranscriptional level in a sequence-specific manner [14–20]. Recent evidence shows numerous miRNAs in the mammalian central nervous system (CNS) and PNS neurons, suggesting that they could play important roles as molecular switches to alter expression of protein cohorts through binding to multiple mRNAs in neurons [21–23]. Interestingly, several groups have further shown temporal changes of miRNA expression in the spinal cord, as well as in the sciatic nerve following injury [20, 24–26]. These studies suggest direct roles of miRNAs in nerve regeneration via regulating protein synthesis. Although a recent study reported altered levels of miRNA in the proximal stumps of the sciatic nerve to the injury site at five post-injury time points [26], a big question still remains when it comes to axon-specific miRNAs directly responsible for the involvement in regenerative responses to injury. The contamination from non-neuronal cells during microdissection of nerve tissues and axonal RNA isolation from whole nerve lysate (axoplasm) possibly argue that the RNA impurities may have impacted on the subsequent bioinformatics analyses for the differential miRNA expression profiling, leading to errors in analytical measurement results. Here, we attempt to purify axoplasmic lysate from the proximal stump of the sciatic nerve to generate new insight into temporal responses of axon-specific miRNAs in response to injury through deep sequencing studies. We find that axoplasmic miRNA levels in rat sciatic nerve are altered, upon injury. We identify a total of 141 rat miRNAs from the proximal stump of the sciatic nerve to the injury site. We further focus on bioinformatic analyses of seven most up-regulated and eight most downregulated miRNAs with at least a mean fold change of 2 in regenerating nerve, compared to those in naïve nerve. By integrating predicted potential target mRNAs and 2,924 transcripts known to be localized to distal axons of DRG neurons [27], we suggest the possibility that the targets of the intra-axonal miRNAs are directly related to multiple biological and neurological functions including regenerative responses following injury. Materials and Methods Animal Surgery and Tissue Preparation Animal procedures were approved (Approved protocol number (...truncated)