Tianmei Qian

miR-221 and miR-222 promote Schwann cell proliferation and migration by targeting LASS2 after sciatic nerve injury.

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.

Gekko japonicus genome reveals evolution of adhesive toe pads and tail regeneration

Reptiles are the most morphologically and physiologically diverse tetrapods, and have undergone 300 million years of adaptive evolution. Within the reptilian tetrapods, geckos possess several interesting features, including the ability to regenerate autotomized tails and to climb on smooth surfaces. Here we sequence the genome of Gekko japonicus (Schlegels Japanese Gecko) and investigate genetic elements related to its physiology. We obtain a draft G. japonicus genome sequence of 2.55u2009Gb and annotated 22,487 genes. Comparative genomic analysis reveals specific gene family expansions or reductions that are associated with the formation of adhesive setae, nocturnal vision and tail regeneration, as well as the diversification of olfactory sensation. The obtained genomic data provide robust genetic evidence of adaptive evolution in reptiles.

Early changes of microRNAs expression in the dorsal root ganglia following rat sciatic nerve transection

MicroRNAs (miRNAs) are a novel class of small non-coding RNAs that regulate gene expression at the post-transcriptional level. Here we report early alterations of miRNAs expression following rat sciatic nerve injury using microarray analysis. We harvested dorsal root ganglia (DRG) tissues and identified 19 miRNAs that showed significant changes at four early time points after sciatic nerve transection. Subsequently, miR-188 and miR-500 microarray results were verified by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The bioinformatics analysis indicated that the potential targets for these miRNAs were involved in the intracellular signaling cascade, the regulation of signal transduction, the regulation of cellular process and the response to cAMP that were known to play important roles in mobilizing the inherent capacity for neurite outgrowth and promoting regeneration during the early phase of sciatic nerve injury. Our results show that abnormal expression of miRNAs may contribute to illustrate the molecular mechanisms of nerve regeneration and miRNAs are potential targets for therapeutic interventions that may enhance intrinsic regenerative ability.

miR-9 inhibits Schwann cell migration by targeting CTHRC1 following sciatic nerve injury

ABSTRACT 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.

Altered long noncoding RNA expressions in dorsal root ganglion after rat sciatic nerve injury.

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.

Altered microRNA expression following sciatic nerve resection in dorsal root ganglia of rats

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.

Hypoxia-Induced Upregulation of miR-132 Promotes Schwann Cell Migration After Sciatic Nerve Injury by Targeting PRKAG3

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.

Fibroblast-derived tenascin-C promotes Schwann cell migration through β1-integrin dependent pathway during peripheral nerve regeneration.

Peripheral nerve regeneration requires precise coordination and dynamic interaction among various types of cells in the tissue. It remains unclear, however, whether the cellular crosstalk between fibroblasts and Schwann cells (SCs) is related to phenotype modulation of SCs, a critical cellular process after peripheral nerve injury. In this study, microarray analysis revealed that a total of 6,046 genes were differentially expressed in the proximal nerve segment after sciatic nerve transection in rats, and bioinformatics analysis further identified tenascin‐C (TNC), an extracellular matrix (ECM) protein, as a key gene regulator. TNC was abundantly produced by nerve fibroblasts accumulating at the lesion site, rather than by SCs as usually expected. TNC significantly promoted SC migration without effects on SC proliferation in primary culture. In co‐culture of fibroblasts and SCs, inhibition of TNC expression either by siRNA transfection or antibody blockade could suppress SC migration, while TNC‐stimulated SC migration was mediated by TNC binding to β1‐integrin receptor in SCs through activation of Rac1 effectors. The in vivo evidence showed that exogenous TNC protein enhanced SC migration and axonal regrowth. Our results highlight that TNC‐mediated cellular interaction between fibroblasts and SCs may regulate SC migration through β1‐integrin‐dependent pathway during peripheral nerve regeneration. GLIA 2016;64:374–385

Signaling pathways regulating dose-dependent dual effects of TNF-α on primary cultured Schwann cells.

After peripheral nerve injury, Schwann cells are rapidly activated to participate in the regenerative process and modulate local immune reactions. Tumor necrosis factor-α (TNF-α), one of the major initiators of the inflammatory cascade, has been known to exert pleiotropic functions during peripheral nerve injury and regeneration. In this study, we aimed to investigate the in vitro effects of TNF-α on peripheral neural cells. First, gene-microarray analysis was applied to the RNA samples extracted from injured peripheral nerves, providing the information of gene interactions post nerve injury. Then, after primary cultured Schwann cells were treated with increasing dosages (0–40xa0ng/ml) of TNF-α, cell proliferation and migration were examined by EdU incorporation and a transwell-based assay, and cell apoptosis was observed and quantified by electron microscopy and Annexin V-FITC assay, respectively. The results showed that lower dosages of TNF-α increased cell proliferation and migration, whereas higher dosages of TNF-α decreased cell proliferation and migration and enhanced cell apoptosis. The tests using a chemical inhibitor of TNF-α further confirmed the above effects of TNF-α. To understand how TNF-α produced the dose-dependent dual effects on primary cultured Schwann cells, we performed co-immunoprecipitation, Western blot analysis, and immunocytochemistry to decipher the complex network of biochemical pathways involving many signaling molecules, i.e., TNF receptor-associated death domain, Fas-associated death domain, receptor interacting protein, JNK, NF-κB p65, and caspases, thus assuming the mechanisms by which TNF-α activated the death and survival pathways and achieved a balance between the two opposite actions in primary cultured Schwann cells.

Long non-coding RNA uc.217 regulates neurite outgrowth in dorsal root ganglion neurons following peripheral nerve injury.

The intrinsic regeneration capacity of dorsal root ganglion (DRG) neurons can be activated after sciatic nerve injury, and peripheral nerve regeneration is a complex process regulated by multiple molecular responses and signaling pathways. Long non‐coding RNAs (lncRNAs) are RNA transcripts > 200 nucleotides in length without protein‐coding potential. They regulate gene expression at epigenetic, transcriptional and post‐transcriptional levels, and are thus involved in many biological processes and human diseases. However, the role and mechanisms of lncRNAs in regulating the responses of DRG neurons to sciatic nerve injury are not fully investigated. We have previously analysed the expression profiles of lncRNAs and mRNAs in L4‐6 DRGs, following rat sciatic nerve transection, by microarray analysis, and constructed a coexpression network of dysregulated lncRNAs and coding genes. In this study, one of these dysregulated lncRNAs, uc.217, was chosen for detailed examination of its expression changes and regulative functions in regenerative DRG neuronal outgrowth. Quantitative real‐time PCR and in situ hybridisation confirmed that the expression of uc.217 was down‐regulated in DRG neurons after sciatic nerve injury. Silencing of uc.217 expression by small interfering RNA could significantly promote neurite outgrowth in cultured DRG neurons. Moreover, bioinformatic analysis and experimental validation were performed to identify several potential targets of uc.217, which were involved in the regulation of DRG neuron outgrowth. Collectively, our results suggested that a new lncRNA, uc.217, played an important regulative role in peripheral nerve regeneration.