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Autologous nerve grafting is the golden standard therapeutic approach of peripheral nerve injury. However, the clinical effect of autologous nerve grafting is still unsatisfying. To achieve better clinical functional recovery, it is of an impending need to expand our understanding of the dynamic cellular and molecular changes after nerve transection and autologous nerve transplantation. To address this aim, in the current study, rats were subjected to sciatic nerve transection and autologous nerve grafting. Rat sciatic nerve segments were collected at 4, 7, and 14 days after surgery and subjected to antibody array analysis to determine phosphoprotein profiling patterns. Compared with rats that underwent sham surgery, a total of 48, 19, and 75 differentially expressed phosphoproteins with fold changes > 2 or < -2 were identified at 4, 7, and 14 days after autologous nerve grafting, respectively. Several phosphoproteins, including STAM2 (Phospho-Tyr192) and Tau (Phospho-Ser422), were found to be differentially expressed at multiple time points, suggesting the importance of the phosphorylation of these proteins. Western blot validation of the expression patterns of STAM2 (Phospho-Tyr192) indicated the accuracy of antibody array assay. Bioinformatic analysis of these differentially expressed proteins suggested that cellular behavior and organ morphology were significantly involved biological functions while cell behavior and immune response-related signaling pathways were significantly involved canonical signaling pathways. These outcomes contributed to the illumination of the molecular mechanisms underlying autologous nerve grafting from the phosphoprotein profiling perspective.

Axon guidance helps growing neural axons to follow precise paths to reach their target locations. It is a critical step for both the formation and regeneration of neuronal circuitry. Netrin-1 (Ntn1) and its receptor, deleted in colorectal carcinoma (Dcc) are essential factors for axon guidance, but their regulation in this process is incompletely understood. In this study, using quantitative real-time RT-PCR (qRT-PCR) and biochemical and reporter gene assays, we found that the Ntn1 and Dcc genes are both robustly up-regulated in the sciatic nerve stump after peripheral nerve injury. Moreover, we found that the microRNA (miR) let-7 directly targets the Ntn1 transcript by binding to its 3'-untranslated region (3'-UTR), represses Ntn1 expression, and reduces the secretion of Ntn1 protein in Schwann cells. We also identified miR-9 as the regulatory miRNA that directly targets Dcc and found that miR-9 down-regulates Dcc expression and suppresses the migration ability of Schwann cells by regulating Dcc abundance. Functional examination in dorsal root ganglion neurons disclosed that let-7 and miR-9 decrease the protein levels of Ntn1 and Dcc in these neurons, respectively, and reduce axon outgrowth. Moreover, we identified a potential regulatory network comprising let-7, miR-9 , Ntn1, Dcc, and related molecules, including the RNA-binding protein Lin-28 homolog A (Lin28), SRC proto-oncogene nonreceptor tyrosine kinase (Src), and the transcription factor NF-κB. In summary, our findings reveal that the miRs let-7 and miR-9 are involved in regulating neuron pathfinding and extend our understanding of the regulatory pathways active during peripheral nerve regeneration., (© 2019 Wang et al.)

Peripheral nerve injury is a complex biological process that involves the expression changes of various coding and non-coding RNAs. Previously, a number of novel miRNAs that were dysregulated in rat sciatic nerve stumps after peripheral nerve injury were identified and functionally annotated by Solexa sequencing. In the current study, we studied one of these identified novel miRNAs, miR-3075, in depth. Results of transwell-based cell migration assay showed that increased expression of miR-3075 suppressed the migration rate of Schwann cells while decreased expression of miR-3075 elevated the migration rate of Schwann cells, demonstrating that miR-3075 inhibited Schwann cell migration. Results of BrdU cell proliferation assay showed that neither miR-3075 mimic nor miR-3075 inhibitor would affect Schwann cell proliferation. We further studied candidate target genes of miR-3075 by using bioinformatic tools and analyzing gene expression patterns and found that miR-3075 might target contactin 2 (Cntn2). Previous study showed that Cntn2 regulated cell migration and myelination. Our current observation suggested that the biological effects of miR-3075 on Schwann cell phenotype might by through the negative regulation of Cntn2. Overall, our study revealed the function of a novel miRNA, miR-3075, and expanded our current understanding of the molecular mechanisms underlying peripheral nerve injury and regeneration.

As the major glia in PNS, Schwann cells play a critical role in peripheral nerve injury repair. Finding an efficient approach to promote Schwann cell activation might facilitate peripheral nerve repair. Long noncoding RNAs (lncRNAs) have been shown to regulate gene expression and take part in many biological processes. However, the role of lncRNAs in peripheral nerve regeneration is not fully understood. In this study, we obtained a global lncRNA portrayal following sciatic nerve injury in male rats using microarray and further investigated one of these dys-regulated lncRNAs, TNXA-PS1, confirming its vital role in regulating Schwann cells. Silencing TNAX-PS1 could promote Schwann cell migration and mechanism analyses showed that TNXA-PS1 might exert its regulatory role by sponging miR-24-3p/miR-152-3p and affecting dual specificity phosphatase 1 (Dusp1) expression. Systematic lncRNA expression profiling of sciatic nerve segments following nerve injury in rats suggested lncRNA TNXA-PS1 as a key regulator of Schwann cell migration, providing a potential therapeutic target for nerve injury repair. SIGNIFICANCE STATEMENT The PNS has an intrinsic regeneration capacity after injury in which Schwann cells play a crucial role. Therefore, further exploration of functional molecules in the Schwann cell phenotype modulation is of great importance. We have identified a set of dys-regulated long noncoding RNAs (lncRNAs) in rats following sciatic nerve injury and found that the expression of TNXA-PS1 was significantly downregulated. Mechanically analyses showed that TNXA-PS1 might act as a competing endogenous RNA to affect dual specificity phosphatase 1 (Dusp1) expression, regulating migration of Schwann cells. This study provides for the first time a global landscape of lncRNAs following sciatic nerve injury in rats and broadens the known functions of lncRNA during nerve injury. The investigation of TNXA-PS1 might facilitate the development of novel targets for nerve injury therapy., (Copyright © 2018 the authors 0270-6474/18/386574-12$15.00/0.)

Following peripheral nerve injury, hypoxia is formed as a result of defects in blood supply at the injury site. Despite accumulating evidence on the effects of microRNAs (miRNAs) on phenotype modulation of Schwann cells (SCs) after peripheral nerve injury, the impact of hypoxia on SC behaviors through miRNAs during peripheral nerve regeneration has not been estimated. In this study, we confirmed our previous microarray data on the upregulation of miR-132 after sciatic nerve injury in rats and observed that overexpression of miR-132 significantly promoted cell migration of primary cultured SCs. Interestingly, hypoxia-increased expression of miR-132 also enhanced SC migration while inhibition of miR-132 suppressed hypoxia-induced increase in SC migration. miR-132 downregulated PRKAG3 through binding to its 3'-UTR, and PRKAG3 knockdown compromised the reducing effect of miR-132 inhibition on SC migration under normal or hypoxia condition. Moreover, in vivo injection of miR-132 agomir into rats with sciatic nerve transection accelerated SC migration from the proximal to distal stump. Overall, our results suggest that the hypoxia-induced upregulation of miR-132 could promote SC migration and facilitate peripheral nerve regeneration.

The regulative effects of microRNAs (miRNAs) on responses of Schwann cells to a nerve injury stimulus are not yet clear. In this study, we noted that the expression of eight miRNAs was downregulated at different time points following rat sciatic nerve transection, and found that 368 potential targets of these eight miRNAs were mainly involved in phenotypic modulation of Schwann cells. Of these miRNAs, miR-9 was identified as an important functional regulator of Schwann cell migration that was a crucial regenerative response of Schwann cells to nerve injury. In vitro, upregulated expression of miR-9 inhibited Schwann cell migration, whereas silencing of miR-9 promoted Schwann cell migration. Intriguingly, miR-9 exerted this regulative function by directly targeting collagen triple helix repeat containing protein 1 (CTHRC1), which in turn inactivated downstream Rac1 GTPase. Rac1 inhibitor reduced the promotive effects of anti-miR-9 on Schwann cell migration. In vivo, high expression of miR-9 reduced Schwann cell migration within a regenerative nerve microenvironment. Collectively, our results confirmed the role of miR-9 in regulating Schwann cell migration after nerve injury, thus offering a new approach to peripheral nerve repair.

Dorsal root ganglia (DRG) neurons spontaneously undergo robust neurite growth after axotomy. Long noncoding RNAs (lncRNAs) are an important class of pervasive genes involved in a variety of biological functions. However, the functions of lncRNAs in the regulation of responses of DRG neurons to injury stimuli remain untested. Here, lncRNA microarray analysis was performed to profile the lncRNAs in L4-L6 DRGs following rat sciatic nerve resection. The 105 lncRNAs were identified to be differentially expressed at 0, 1, 4, 7 d post injury. A coexpression network of 24 down-regulated lncRNAs and coding genes was constructed, and 115 targets of these 24 lncRNAs were found to be mainly involved in cell phenotype modulation, including glial cell migration, purinergic nucleotide receptor signaling pathway, vasodilation, regulation of multi-organism process, and neuropeptide signaling pathway, and also to be potentially associated with several key regeneration signaling pathways, including MAPK signaling pathway, and neuroactive ligand-receptor interaction. LncRNA BC089918 was selected from 24 down-regulated lncRNAs for validation by quantitative real-time polymerase chain reaction and in situ hybridization. And silencing of BC089918 with small interfering RNAs indicted that the lncRNA had a particular promoting effect on neurite outgrowth. Our data demonstrated a distinct involvement of lncRNAs in DRGs after nerve injury, thus contributing to illustration of molecular mechanisms responsible for nerve regeneration., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The regulation of Schwann cell (SC) responses to injury stimuli by microRNAs (miRNAs) remains to be explored. Here, we identified 17 miRNAs that showed dynamic expression alterations at five early time points following rat sciatic nerve resection. Then we analyzed the expression pattern of 17 miRNAs, and integrated their putative targets with differentially expressed mRNAs. The resulting 222 potential targets were mainly involved in cell phenotype modulation, including immune response, cell death and cell locomotion. Among 17 miRNAs, miR-182 expression was up-regulated. The enhanced expression of miR-182 was correlated with nerve injury-induced phenotype modulation of SCs. Further investigation revealed that fibroblast growth factor 9 (FGF9) and neurotrimin (NTM) were two direct targets of miR-182 in SCs, with miR-182 binding to the 3'-untranslated region of FGF9 and NTM. Silencing of FGF9 and NTM recapitulated the inhibiting effect of miR-182 mimics on SC proliferation and migration, respectively, whereas enforced knockdown of FGF9 and NTM reversed the promoting effect of miR-182 inhibitor on SC proliferation and migration, respectively. Our data indicate that nerve injury inhibits SC proliferation and migration through rapid regulation of miR-182 by targeting FGF9 and NTM, providing novel insights into the roles of miRNAs in nerve injury and repair.

microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level. Their roles in regulating the responses of Schwann cells (SCs) to injury stimuli remain unexplored. Here we report dynamic alteration of miRNA expression following rat sciatic nerve injury using microarray analysis. We harvested the proximal nerve stumps and identified 77 miRNAs that showed significant changes at four time points after nerve transection. Subsequently, we analyzed the expression pattern of miRNA, selected one significant profile, and then integrated putative miRNA targets with differentially expressed mRNA yielding 274 potential targets. The 274 targets were mainly involved in cell proliferation, cell locomotion and cellular homeostasis that were known to play important roles in modulating cell phenotype. The upregulation of the miR-221 and miR-222 cluster (miR-221/222) was found to correlate with the injury-induced SC phenotypic modulation. Enhanced expression of miR-221/222 could promote SC proliferation and migration in vitro, whereas silencing their expression resulted in a reduced proliferation and migration. Further studies revealed that longevity assurance homologue 2 (LASS2) was a direct target of miR-221/222 in SCs because miR-221/222 bound directly to the 3'-untranslated region of LASS2, thus reducing both mRNA and protein levels of LASS2. Silencing of LASS2 recapitulated the effects of miR-221/222 mimics, whereas enforced knockdown of LASS2 reversed the suppressive effects of miR-221/222 inhibitors. Our findings indicate that injury promotes SC proliferation and migration through the regulation of miR-221/222 by targeting LASS2, and provide new insights into the role of miRNAs in nerve regeneration.

MicroRNAs (miRNAs) are a class of small, non-coding RNAs (∼22 nucleotides) that negatively regulate gene expression post-transcriptionally, either through translational inhibition or degradation of target mRNAs. We uncovered a previously unknown alteration in the expression of miRNAs in the dorsal root ganglia (DRG) at 1, 4, 7, and 14 days after resection of the sciatic nerve in rats using microarray analysis. Thirty-two significantly upregulated and 18 downregulated miRNAs were identified in the DRG at four time points following sciatic nerve injury. The expression of four consecutively deregulated miRNAs, analyzed by real-time Taqman polymerase chain reaction, was in agreement with the microarray data (upregulated: miR-21, miR-221; downregulated: miR-500, miR-551b). The potential targets for these miRNAs, altered after sciatic nerve resection, are involved mainly in nervous system development, multi-cellular organismal development, and the regulation of cellular processes. This study demonstrated a different involvement of miRNAs in the DRG after resection of the sciatic nerve in a rat model, and it may also contribute in illustrating the molecular mechanisms responsible for nerve regeneration.

MicroRNAs (miRNAs) are non-coding RNAs that regulate various tissues and organs, including the nervous system. Peripheral nerve injury is a common pathology of the nervous system and leads to differential expressions of a variety of miRNAs. Previously, a group of novel miRNAs have been identified in rat proximal nerve segments after sciatic nerve transection. However, the biological functions of these novel miRNAs remain undetermined. The aim of the current study was therefore to identify the function of a novel miRNA, miR-sc6, following nerve injury. Its target genes and effects on phenotypic modulation of Schwann cells were determined using a miR-sc6 mimic transfection. These observations contribute to the understanding of miRNA involvement in peripheral nerve injury and the cognition of regulatory mechanisms in peripheral nerve regeneration. [ABSTRACT FROM AUTHOR]