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

Abstract

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 neuro-degeneration 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.

Analysis of piRNA-Like Small Non-coding RNAs Present in Axons of Adult Sensory Neurons

“…The muscle and skin overlying the nerve were closed in layers and animals were placed in prewarmed cages. Animals were euthanized at 1, 4, 7, and 14 days post-injury (DPI), and injured and uninjured sciatic nerves were collected and processed as previously described (16). …”

“…A mechanical squeezing method was used to isolate total RNA from nerves as previously described (16). Briefly, 10 mm segments of nerve were cut into small pieces, transferred into Eppendorf tubes, and manually squeezed with a plastic pestle on ice in QIAzol lysis reagent (Qiagen) according to the manufacturer’s instructions.…”

“…A total of 15 samples of sciatic nerves collected at different time points (1, 4, 7, and 14 DPI) including uninjured nerves were processed to Illumina HiSeq 2500 sequencing for generating small RNA libraries by the University of Delaware DNA Sequencing and Genotyping Center (UDSGC) located in the Delaware Biotechnology Institute (DBI) in Newark, DE, as previously described (16). Sequence data were analyzed by the CLC Genomics Workbench (Qiagen) software package with support from the University of Delaware Center for Bioinformatics and Computational Biology (CBCB) Bioinformatics Core.…”

“…Illumina deep sequencing data were validated by quantitative PCR (qPCR) analysis using piRNA-specific LNA primers (Exiqon) as previously described (16). In brief, using NCode ™ VILO ™ miRNA cDNA Synthesis Kit (Invitrogen) enabling poly(A) tailing of the small RNAs according to the manufacturer’s instructions with a universal RT primer, the purified small RNA was converted to cDNA.…”

“…We recently reported profiles for miRNAs in distal axons of sciatic nerve after crush injury (16). From deep sequencing of small RNAs isolated from the sciatic nerve axoplasm, we also detected a set of sncRNAs in the size of mammalian piRNAs (piRNA-like sncRNAs).…”