Profile of MicroRNAs following Rat Sciatic Nerve Injury by Deep Sequencing: Implication for Mechanisms of Nerve Regeneration

et al. (2011) Profile of MicroRNAs following Rat Sciatic Nerve Injury by Deep Sequencing: Implication for Mechanisms of Nerve Regeneration. PLoS ONE 6(9): e24612. doi:10.1371/journal.pone.0024612 Profile of MicroRNAs following Rat Sciatic Nerve Injury by Deep Sequencing: Implication for Mechanisms of Nerve Regeneration Bin Yu 0 Songlin Zhou 0 Yongjun Wang 0 Guohui Ding 0 Fei Ding 0 Xiaosong Gu 0 Cheng-Xin Gong, New York State Institute for Basic Research, United States of America 0 1 Jiangsu Key Laboratory of Neuroregeneration, Nantong University , Nantong , China , 2 Key Lab of Systems Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , Shanghai , China Unlike the central nervous system, peripheral nerves can regenerate when damaged. MicroRNA (miRNA) is a novel class of small, non-coding RNA that regulates gene expression at the post-transcriptional level. Here, we report regular alterations of miRNA expression following rat sciatic nerve injury using deep sequencing. We harvested dorsal root ganglia tissues and the proximal stumps of the nerve, and identified 201 and 225 known miRNAs with significant expression variance at five time points in these tissues after sciatic nerve transaction, respectively. Subsequently, hierarchical clustering, miRNA expression pattern and co-expression network were performed. We screened out specific miRNAs and further obtained the intersection genes through target analysis software (Targetscan and miRanda). Moreover, GO and KEGG enrichment analyses of these intersection genes were performed. The bioinformatics analysis indicated that the potential targets for these miRNAs were involved in nerve regeneration, including neurogenesis, neuron differentiation, vesicle-mediated transport, homophilic cell adhesion and negative regulation of programmed cell death that were known to play important roles in regulating nerve repair. Finally, we combined differentially expressed mRNA with the predicted targets for selecting inverse miRNA-target pairs. Our results show that the abnormal expression of miRNA may contribute to illustrate the molecular mechanisms of nerve regeneration and that miRNAs are potential targets for therapeutic interventions and may enhance intrinsic regenerative ability. - Funding: This study was supported by the National Natural Science Foundation of China (Grant No. 30870811), the Jiangsu Provincial Natural Science Foundation (Grant No. BK2008010), the Basic Research Program of Jiangsu Education Department (Grant No. 08KJA310002), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. The peripheral nervous system (PNS), differing from the central nervous system (CNS), has the intrinsic capacity to regenerate. Previous studies have demonstrated that severed peripheral nerves are able to re-grow and re-connect to their targets, even if their previous functions were seriously compromised [1]. As we know, nerve regeneration is a complex biological phenomenon incorporating multiple cells, growth factors and extracellular matrices [2,3]. In order to achieve successful nerve repair, neuronal loss has to be prevented, axons have to re-grow and arrive at their correct target cells [4]. There is, of particular interest, a growing consensus that the distinct ability of peripheral nerves to re-grow to their targets hinges on the regenerative properties of its glia, the Schwann cells (SCs). Following adult peripheral nerve axotomy, dedifferentiated SCs can replenish lost or damaged tissue by proliferating, and produce various trophic factors and adhesion molecules to facilitate axon outgrowth [5]. In addition, two-way communication between neuron and SC is essential for axonal conduction in axon regeneration. SCs can regulate synapse formation, can control synaptic strength, and may participate in information processing by coordinating activity among sets of neurons. Conversely, neural impulse activity regulates a wide range of SC activities, including proliferation, differentiation, and myelination [6]. However, the widely elucidatory molecular mechanisms that are responsible for PNS injury and the subsequent restoration of nerve remain largely unclear. MiRNAs are endogenous, non-coding 21- to 23-nucleotide small RNA molecules that regulate gene expression by binding to the 39-untranslated region of target mRNAs, leading to their translational inhibition or degradation [7]. Many studies have indicated miRNAs are attractive candidates as upstream regulators, because miRNAs can post-transcriptionally regulate the entire set of genes [8]. The importance of miRNA in neural development and neurodegeneration is starting to be recognized [9,10], but their roles in nerve injury and repair currently remains largely unknown. It was reported that miRNA expression profiles were significantly altered in the spinal cord injury model of adult rats [11,12]. Recently, miRNA-144, 145, and 214 are identified to be down-regulated in primary neurons responding to sciatic nerve transection, and miR-145 inhibited neurite growth of dorsal root ganglia (DRG) neuron through Slit-Robo-srGAP signaling pathway [13]. In particular, through microarray we also found that abnormal expression of miRNA in DRG may contribute to illustrate the molecular mechanisms of nerve regeneration during the early phase after sciatic nerve transection [14]. Since deep sequencing overcomes the technical drawbacks of microarray and traditional small RNA library sequencing, and has several additional advantages, such as high resolution, highthroughput, high-accuracy and reduced complexity of experimental procedures, it has dramatically changed the speed of all aspects of sequencing in a rapid and cost-effective fashion. This innovation can permit unbiased, quantitative and in-depth investigation of the small RNA transcriptome, and has been widely employed to reveal the expression profiles of miRNA in different species and understand the role of miRNA in fundamental processes [1517]. In this work, we have designed an integrative strategy with bioinformatic analysis to identify miRNAs in the DRG and the proximal stump response to resection of the sciatic nerve in a rat model by deep sequencing (Figure 1). We found 201 and 225 known miRNAs with significant expression variance in the DRG and the proximal stump after nerve injury, respectively. More importantly, our study screened out some key miRNAs, through bioinformatics analysis, that may be involved in many aspects of nerve repair, and provided an opportunity to decipher what molecules they target to regulate nerve regeregation by integration of predicted targets with differentially expressed mRNAs, (...truncated)