RNA sequencing of exosomes secreted by fibroblast and Schwann cells elucidates mechanisms underlying peripheral nerve regeneration

Exosomes exhibit complex biological functions and mediate a variety of biological processes,such as promoting axonal regeneration and functional recove ry after injury.Long non-coding RNAs(IncRNAs)have been reported to play a crucial role in axonal regeneration.Howeve r,the role of the IncRNA-microRNAmessenger RNA(mRNA)-competitive endogenous RNA(ceRNA)network in exosome-mediated axonal regeneration remains unclear.In this study,we performed RNA transcriptome sequencing analysis to assess mRNA expression patterns in exosomes produced by cultured fibroblasts(FC-EXOs)and Schwann cells(SCEXOs).Diffe rential gene expression analysis,Gene Ontology analysis,Kyoto Encyclopedia of Genes and Genomes analysis,and protein-protein intera ction network analysis were used to explo re the functions and related pathways of RNAs isolated from FC-EXOs and SC-EXOs.We found that the ribosome-related central gene Rps5 was enriched in FC-EXOs and SC-EXOs,which suggests that it may promote axonal regeneration.In addition,using the miRWalk and Starbase prediction databases,we constructed a regulatory network of ceRNAs targeting Rps5,including 27 microRNAs and five IncRNAs.The ceRNA regulatory network,which included Ftx and Miat,revealed that exsosome-derived Rps5 inhibits scar formation and promotes axonal regeneration and functional recovery after nerve injury.Our findings suggest that exosomes derived from fibro blast and Schwann cells could be used to treat injuries of peripheral nervous system.

Evaluation of clustering algorithms for gene expression data using gene ontology annotations

Background Clustering is a useful exploratory technique for interpreting gene expression data to reveal groups of genes sharing common functional attributes. Biologists frequently face the problem of choosing an appropriate algorithm. We aimed to provide a standalone, easily accessible and biologically oriented criterion for expression data clustering evaluation. Methods An external criterion utilizing annotation based similarities between genes is proposed in this work. Gene ontology information is employed as the annotation source. Comparisons among six widely used clustering algorithms over various types of gene expression data sets were carried out based on the criterion proposed. Results The rank of these algorithms given by the criterion coincides with our common knowledge. Single-linkage has significantly poorer performance, even worse than the random algorithm. Ward＇s method archives the best performance in most cases. Conclusions The criterion proposed has a strong ability to distinguish among different clustering algorithms with different distance measurements. It is also demonstrated that analyzing main contributors of the criterion may offer some guidelines in finding local compact clusters. As an addition, we suggest using Ward＇s algorithm for gene expression data analysis.

A new gene ontology-based measure for the functional similarity of gene products

Background Although biomedical ontologies have standardized the representation of gene products across species and databases, a method for determining the functional similarities of gene products has not yet been developed. Methods We proposed a new semantic similarity measure based on Gene Ontology that considers the semantic influences from all of the ancestor terms in a graph. Our measure was compared with Resnik＇s measure in two applications, which were based on the association of the measure used with the gene co-expression and the protein- protein interactions. Results The results showed a considerable association between the semantic similarity and the expression correlation and between the semantic similarity and the protein-protein interactions, and our measure performed the best overall. Conclusion These results revealed the potential value of our newly proposed semantic similarity measure in studying the functional relevance of gene products.

Correlating Expression Data with Gene Function Using Gene Ontology

Clustering is perhaps one of the most widely used tools for microarray data analysis. Proposed roles for genes of unknown function are inferred from clusters of genes similarity expressed across many biological conditions. However, whether function annotation by similarity metrics is reliable or not and to what extent the similarity in gene expression patterns is useful for annotation of gene functions, has not been evaluated. This paper made a comprehensive research on the correlation between the similarity of expression data and of gene functions using Gene Ontology. It has been found that although the similarity in expression patterns and the similarity in gene functions are significantly dependent on each other, this association is rather weak. In addition, among the three categories of Gene Ontology, the similarity of expression data is more useful for cellular component annotation than for biological process and molecular function. The results presented are interesting for the gene functions prediction research area.

Reduced non-CpG methylation is a potential epigenetic target after spinal cord injury

DNA methylation is a critical epigenetic regulator in the occurrence and development of diseases and is closely related to various functional responses in relation to spinal cord injury.To investigate the role of DNA methylation in spinal cord injury,we constructed a library with reduced-representation bisulfite sequencing data obtained at various time points(day 0-42)after spinal cord injury in mice.Global DNA methylation levels,specifically non-CpG(CHG and CHH)methylation levels,decreased modestly following spinal cord injury.Stages post-spinal cord injury were classified as early(day 0-3),intermediate(day7-14),and late(day 28-42)based on similarity and hie rarchical cluste ring of global DNA methylation patterns.The non-CpG methylation level,which included CHG and CHH methylation levels,was markedly reduced despite accounting for a minor proportion of total methylation abundance.At multiple genomic sites,including the 5’untranslated regions,promoter,exon,intron,and 3’untranslated regions,the non-CpG methylation level was markedly decreased following spinal cord injury,whereas the CpG methylation level remained unchanged at these locations.Approximately one-half of the differentially methylated regions were located in intergenic areas;the other differentially methylated regions in both CpG and non-CpG regions were cluste red in intron regions,where the DNA methylation level was highest.The function of genes associated with differentially methylated regions in promoter regions was also investigated.From Gene Ontology analysis results,DNA methylation was implicated in a number of essential functional responses to spinal cord injury,including neuronal synaptic connection creation and axon regeneration.Notably,neither CpG methylation nor non-CpG methylation was implicated in the functional response of glial or inflammatory cells.In summary,our work elucidated the dynamic pattern of DNA methylation in the spinal co rd following injury and identified reduced nonCpG methylation as an epigenetic target after spinal cord injury in mice.

Transcriptomic and bioinformatics analysis of the mechanism by which erythropoietin promotes recovery from traumatic brain injury in mice

Recent studies have found that erythropoietin promotes the recovery of neurological function after traumatic brain injury.However,the precise mechanism of action remains unclea r.In this study,we induced moderate traumatic brain injury in mice by intrape ritoneal injection of erythro poietin for 3 consecutive days.RNA sequencing detected a total of 4065 differentially expressed RNAs,including 1059 mRNAs,92 microRNAs,799 long non-coding RNAs,and 2115circular RNAs.Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses revealed that the coding and non-coding RNAs that were differentially expressed after traumatic brain injury and treatment with erythropoietin play roles in the axon guidance pathway,Wnt pathway,and MAPK pathway.Constructing competing endogenous RNA networks showed that regulatory relationship between the differentially expressed non-coding RNAs and mRNAs.Because the axon guidance pathway was repeatedly enriched,the expression of Wnt5a and Ephb6,key factors in the axonal guidance pathway,was assessed.Ephb6 expression decreased and Wnt5a expression increased after traumatic brain injury,and these effects were reversed by treatment with erythro poietin.These findings suggest that erythro poietin can promote recove ry of nerve function after traumatic brain injury through the axon guidance pathway.

Hub genes and key pathways of traumatic brain injury: bioinformatics analysis and in vivo validation

The exact mechanisms associated with secondary brain damage following traumatic brain injury(TBI)remain unclear;therefore,identifying the critical molecular mechanisms involved in TBI is essential.The m RNA expression microarray GSE2871 was downloaded from the Gene Expression Omnibus(GEO)repository.GSE2871 comprises a total of 31 cerebral cortex samples,including two post-TBI time points.The microarray features eight control and seven TBI samples,from 4 hours post-TBI,and eight control and eight TBI samples from 24 hours post-TBI.In this bioinformatics-based study,109 and 66 differentially expressed genes(DEGs)were identified in a Sprague-Dawley(SD)rat TBI model,4 and 24 hours post-TBI,respectively.Functional enrichment analysis showed that the identified DEGs were significantly enriched in several terms,such as positive regulation of nuclear factor-κB transcription factor activity,mitogen-activated protein kinase signaling pathway,negative regulation of apoptotic process,and tumor necrosis factor signaling pathway.Moreover,the hub genes with high connectivity degrees were primarily related to inflammatory mediators.To validate the top five hub genes,a rat model of TBI was established using the weight-drop method,and real-time quantitative polymerase chain reaction analysis of the cerebral cortex was performed.The results showed that compared with control rats,Tnf-α,c-Myc,Spp1,Cxcl10,Ptprc,Egf,Mmp9,and Lcn2 were upregulated,and Fn1 was downregulated in TBI rats.Among these hub genes,Fn1,c-Myc,and Ptprc may represent novel biomarkers or therapeutic targets for TBI.These identified pathways and key genes may provide insights into the molecular mechanisms of TBI and provide potential treatment targets for patients with TBI.This study was approved by the Experimental Animal Ethics Committee of the First Affiliated Hospital of Nanchang University,China(approval No.003)in January 2016.

Identify the signature genes for diagnose of uveal melanoma by weight gene co-expression network analysis

AIM: To identify and understand the relationship between co-expression pattern and clinic traits in uveal melanoma, weighted gene co-expression network analysis(WGCNA) is applied to investigate the gene expression levels and patient clinic features. Uveal melanoma is the most common primary eye tumor in adults. Although many studies have identified some important genes and pathways that were relevant to progress of uveal melanoma, the relationship between co-expression and clinic traits in systems level of uveal melanoma is unclear yet. We employ WGCNA to investigate the relationship underlying molecular and phenotype in this study.METHODS: Gene expression profile of uveal melanoma and patient clinic traits were collected from the Gene Expression Omnibus(GEO) database. The gene co-expression is calculated by WGCNA that is the R package software. The package is used to analyze the correlation between pairs of expression levels of genes.The function of the genes were annotated by gene ontology(GO).RESULTS: In this study, we identified four co-expression modules significantly correlated with clinictraits. Module blue positively correlated with radiotherapy treatment. Module purple positively correlates with tumor location(sclera) and negatively correlates with patient age. Module red positively correlates with sclera and negatively correlates with thickness of tumor. Module black positively correlates with the largest tumor diameter(LTD). Additionally, we identified the hug gene(top connectivity with other genes) in each module. The hub gene RPS15 A, PTGDS, CD53 and MSI2 might play a vital role in progress of uveal melanoma.CONCLUSION: From WGCNA analysis and hub gene calculation, we identified RPS15 A, PTGDS, CD53 and MSI2 might be target or diagnosis for uveal melanoma.

Comparative Analysis of Gene Expression Profiling Between Resistant and Susceptible Varieties Infected With Soybean Cyst Nematode Race 4 in Glycine max

Soybean cyst nematode （SCN） is one of the most devastating pathogen for soybean. Therefore, identiifcation of resistant germplasm resources and resistant genes is needed to improve SCN resistance for soybean. Soybean varieties Huipizhiheidou and Wuzhaiheidou were distributed in China and exhibited broad spectrums of resistance to various SCN races. In this study, these two resistant varieties, combined with standard susceptible varieties （Lee and Essex）, were utilized to identify the differentially expressed transcripts after infection with SCN race 4 between resistant and susceptible reactions by using the Affymetrix Soybean Genome GeneChip. Comparative analyses indicated that 21 common genes changed signiifcantly in the resistant group, of which 16 increased and 5 decreased. However, 12 common genes changed signiifcantly in the susceptible group, of which 9 increased and 3 decreased. Additionally, 27 genes were found in common between resistant and susceptible reactions. The 21 signiifcantly changed genes in resistant reaction were associated with disease and defense, cell structure, transcription, metabolism, and signal transduction. The fold induction of 4 from the 21 genes was conifrmed by quantitative RT-PCR （qRT-PCR） analysis. Moreover, the gene ontology （GO） enrichment analyses demonstrated the serine family amino acid metabolic process and arginine metabolic process may play important roles in SCN resistance. This study provided a new insight on the genetic basis of soybean resistance to SCN race 4, and the identiifed resistant or resistant-related genes are potentially useful for SCN-resistance breeding in soybean.

Spatiotemporal microRNA profile in peripheral nerve regeneration:miR-138 targets vimentin and inhibits Schwann cell migration and proliferation

While the peripheral nervous system has regenerative ability,restoration of sufficient function remains a challenge.Vimentin has been shown to be localized in axonal growth fronts and associated with nerve regeneration,including myelination,neuroplasticity,kinase signaling in nerve axoplasm,and cell migration;however,the mechanisms regulating its expression within Schwann cell（SC） remain unexplored.The aim of this study was to profile the spatial and temporal expression profile of micro RNA（mi RNA） in a regenerating rat sciatic nerve after transection,and explore the potential role of mi R-138-5 p targeting vimentin in SC proliferation and migration.A rat sciatic nerve transection model,utilizing a polyethylene nerve guide,was used to investigate mi RNA expression at 7,14,30,60,and 90 days during nerve regeneration.Relative levels of mi RNA expression were determined using microarray analysis and subsequently validated with quantitative real-time polymerase chain reaction.In vitro assays were conducted with cultured Schwann cells transfected with mi RNA mimics and assessed for migratory and proliferative potential.The top seven dysregulated mi RNAs reported in this study have been implicated in cell migration elsewhere,and GO and KEGG analyses predicted activities essential to wound healing.Transfection of one of these,mi RNA-138-5 p,into SCs reduced cell migration and proliferation.mi R-138-5 p has been shown to directly target vimentin in cancer cells,and the luciferase assay performed here in rat Schwann cells confirmed it.These results detail a role of mi R-138-5 p in rat peripheral nerve regeneration and expand on reports of it as an important regulator in the peripheral nervous system.

Gene and protein expression profiles of olfactory ensheathing cells from olfactory bulb versus olfactory mucosa

Olfactory ensheathing cells(OECs) from the olfactory bulb(OB) and the olfactory mucosa(OM) have the capacity to repair nerve injury. However, the difference in the therapeutic effect between OB-derived OECs and OM-derived OECs remains unclear. In this study, we extracted OECs from OB and OM and compared the gene and protein expression profiles of the cells using transcriptomics and non-quantitative proteomics techniques. The results revealed that both OB-derived OECs and OM-derived OECs highly expressed genes and proteins that regulate cell growth, proliferation, apoptosis and vascular endothelial cell regeneration. The differentially expressed genes and proteins of OB-derived OECs play a key role in regulation of nerve regeneration and axon regeneration and extension, transmission of nerve impulses and response to axon injury. The differentially expressed genes and proteins of OM-derived OECs mainly participate in the positive regulation of inflammatory response, defense response, cytokine binding, cell migration and wound healing. These findings suggest that differentially expressed genes and proteins may explain why OB-derived OECs and OM-derived OECs exhibit different therapeutic roles. This study was approved by the Animal Ethics Committee of the General Hospital of Ningxia Medical University(approval No. 2017-073) on February 13, 2017.

An Integrated Quantitative Trait Locus Map of Oil Content in Soybean,Glycine max(L.) Merr.,Generated Using a Meta-Analysis Method for Mining Genes

Soybean is a major cash crop in the world, and its oil content was one of the very important traits. Therefore, the study of gene mapping for oil content in soybean is very important for breeding application. At present, at least 130 QTL loci for soybean oil content have been published; however, the mapping results of oil content were dispersed and a coalescent public map should be established to integrate the published QTLs, and to more efficiently mine genes based on the meta- analysis method of the bioinformatics tools. This study was to construct an integrated map of QTLs for soybean oil content and accelerate the application of bioinformation resource related to oil content improvement in the practice of soybean breeding. We collected information of 130 QTLs reported over the past 20 yr for soybean oil content and used the Software BioMercator 2.1 to project QTLs from their own maps onto a reference map, which was an early-integrated map constructed by Song （2004） for oil-content quantitative trait loci （QTLs） in soybean. Gene mining was performed based on the meta-analysis by running the local ver. GENSCAN and InterProScan. The confidence interval of QTLs was efficaciously narrowed using the meta-analysis method, and 25 consensus QTLs were mapped on the reference map. Using a local version of GENSCAN, 12 805 sequences in the consensus QTL intervals were predicted. With BLAST, these predicted sequences were aligned to gene sequences from the International Protein Index database using InterProScan locally. Thirteen predicted genes were in the class of the geme ontology （GO） accession （0006631）, which were involved in the fatty acid metabolic process. These genes were analyzed using BLAST at the NCBI website to examine whether they were related to oil content. Six genes were found in the oil-synthesis pathway. Twenty-five consensus QTLs and six genes were found in the oil-synthesis pathway. These results would lay the foundation for marker-assisted selection and mapping QTL precisely, and these genes will facilitate the researches on the gene mining of oil synthesis and molecular breeding in soybean.

Identification of differentially expressed metastatic genes and their signatures to predict the overall survival of uveal melanoma patients by bioinformatics analysis

AIM:To identify metastatic genes and mi RNAs and to investigate the metastatic mechanism of uveal melanoma(UVM).METHODS:GSE27831,GSE39717,and GSE73652 gene expression profiles were downloaded from the Gene Expression Omnibus(GEO)database,and the limma R package was used to identify differentially expressed genes(DEGs).Gene Ontology(GO)term enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis were performed using the DAVID online tool.A comprehensive list of interacting DEGs was constructed using the Search Tool for the Retrieval of Interacting Genes(STRING)database and Cytoscape software.The Cytoscape MCODE plug-in was used to identify clustered sub-networks and modules of hub genes from the proteinprotein interaction network.GEPIA online software was used for survival analysis of UVM patients(n=80)from the The Cancer Genome Atlas(TCGA)cohort.Oncomi R online software was used to find that the mi RNAs were associated with UVM prognosis from the TCGA cohort.Target Scan Human 7.2 software was then used to identify the mi RNAs targeting the genes.RESULTS:There were 1600 up-regulated genes and 1399 down-regulated genes.The up-regulated genes were mainly involved in protein translation in the cytosol,whereas the down-regulated genes were correlated with extracellular matrix organization and cell adhesion in the extracellular space.Among the 2999 DEGs,five genes,Znf391,Mrps11,Htra3,Sulf2,and Smarcd3 were potential predictors of UVM prognosis.Otherwise,three mi RNAs,hsa-mi R-509-3-5 p,hsa-mi R-513 a-5 p,and hsa-mi R-1269 a were associated with UVM prognosis.CONCLUSION:After analyzing the metastasis-related enriched terms and signaling pathways,the up-regulated DEGs are mainly involved in protein synthesis and cell proliferation by ribosome and mitogen-activated protein kinase(MAPK)pathways.However,the down-regulated DEGs are mainly involved in processes that reduced cell-cell adhesion and promoted cell migration in the extracellular matrix through PI3 K-Akt signaling pathway,focal adhesion,and extracellular matrix-receptor interactions.Bioinformatics and interaction analysis may provide new insights on the events leading up to the development and progression of UVM.

Differential Expression of Genes in HepG2 Cells Caused by UC001kfo RNAi as Shown by RNA-seq

The differential expression of genes in HepG2 cells caused by UC001 kfo RNAi was investigated using RNA-seq. HepG2 cells were infected by Lenti-sh UC001 kfo lentivirus particles. The expression of UC001 kfo m RNA in the HepG2-sh UC001 kfo cell line was detected by real-time PCR. RNA-seq technology was used to identify the difference in the expression of genes regulated by lnc RNA UC001 kfo in the HepG2 cell line. Gene ontology and signaling pathway analysis were performed to reveal the biological functions of the genes encoding of significantly different m RNAs. The results showed that m RNAs were differentially expressed between the HepG2-sh UC001 kfo cell line and the HepG2 cell line. The UC001 kfo m RNA was significantly down-regulated in the stable cell line HepG2-sh UC001kfo（P〈0.001）. Additionally, we found 19 signaling pathways or functional classifications encompassing 30 genes that played a role in cancer characteristics, cell adhesion, invasion and migration. The results also showed that the expression of many genes associated with cancer cell invasion and metastasis was decreased with the down-regulation of the lnc RNA UC001 kfo. Lnc RNA UC001 kfo may play a role in regulating cancer cell invasion and metastasis. It was suggested that m RNAs were differentially expressed in the HepG2 cell line after the down-regulation of lnc RNA-UC001 kfo. Some took part in the extracellular matrix, cell adhesion, motility, growth, and localization. The genes encoding of differentially expressed m RNAs may participate in cell invasion and metastasis.

Identification of shared and unique gene families associated with oral clefts

Oral clefts, the most frequent congenital birth defects in humans, are multifactorial disorders caused by genetic and environmental factors. Epidemiological studies point to different etiologies underlying the oral cleft phenotypes, cleft lip(CL),CL and/or palate(CL/P) and cleft palate(CP). More than 350 genes have syndromic and/or nonsyndromic oral cleft associations in humans. Although genes related to genetic disorders associated with oral cleft phenotypes are known, a gap between detecting these associations and interpretation of their biological importance has remained. Here, using a gene ontology analysis approach, we grouped these candidate genes on the basis of different functional categories to gain insight into the genetic etiology of oral clefts. We identified different genetic profiles and found correlations between the functions of gene products and oral cleft phenotypes. Our results indicate inherent differences in the genetic etiologies that underlie oral cleft phenotypes and support epidemiological evidence that genes associated with CL/P are both developmentally and genetically different from CP only, incomplete CP, and submucous CP. The epidemiological differences among cleft phenotypes may reflect differences in the underlying genetic causes. Understanding the different causative etiologies of oral clefts is important as it may lead to improvements in diagnosis, counseling, and prevention.

A comparative analysis of differentially expressed genes in rostral and caudal regions after spinal cord injury in rats

The initial mechanical damage of a spinal cord injury(SCI)triggers a progressive secondary injury cascade,which is a complicated process integrating multiple systems and cells.It is crucial to explore the molecular and biological process alterations that occur after SCI for therapy development.The differences between the rostral and caudal regions around an SCI lesion have received little attention.Here,we analyzed the differentially expressed genes between rostral and caudal sites after injury to determine the biological processes in these two segments after SCI.We identified a set of differentially expressed genes,including Col3a1,Col1a1,Dcn,Fn1,Kcnk3,and Nrg1,between rostral and caudal regions at different time points following SCI.Functional enrichment analysis indicated that these genes were involved in response to mechanical stimulus,blood vessel development,and brain development.We then chose Col3a1,Col1a1,Dcn,Fn1,Kcnk3,and Nrg1 for quantitative real-time PCR and Fn1 for immunostaining validation.Our results indicate alterations in different biological events enriched in the rostral and caudal lesion areas,providing new insights into the pathology of SCI.

Gene Expression Profiling Related to Hyphal Growth in a Temperature-Sensitive Mutant of Magnaporthe oryzae

The rice blast,caused by fungus Magnaporthe oryzae,is a major constraint to the world food security.Hyphal growth is the foundation of fungal development and proliferation of fungi.To investigate genes involved in hyphal growth of this fungus,digital gene expression tag profiling was used to compare a previously generated temperature-sensitive mutant which defect at hyphae growth and reduction on pathogenicity,with its related wildtype strain.416 genes were detected as differential expression,178 of which were specifically expressed in Guy-11 but down-regulated expression in the mutant.Functional classification analysis revealed the phenotype mutation may be mainly caused by a defection in translational and vacuole- related processes.The results and the protocol used will improve our knowledge on morphogenesis and promote the further study on M.oryzae pathogenesis.

Early Detection of Temporal Lobe Epilepsy: Identification of Novel Candidate Genes and Potential Biomarkers Using Integrative Genomics Analysis

Currently afflicting more than 50 million people worldwide, epilepsy is the spectrum disorder characterizing seizures that occur without other plausible medical explanations. Temporal lobe epilepsy (TLE) is one of the most common forms of epilepsy. Current clinical methods;including MRI scans, EEG tests, and doctor visits;can take upwards of several months to confirm a TLE diagnosis;during this time, patients may experience additional seizures and are at an increased risk for other psychiatric disorders. The purpose of this study is to identify candidate genetic biomarkers to facilitate the earlier detection and diagnosis of TLE through gene-based testing (e.g., genomic heatmap analysis or genetic and/or microarray testing). It was hypothesized that potential biomarkers could be identified by analyzing genes that are normally significantly overexpressed in the temporal lobe relative to the gray matter. Statistical and functional analysis was performed on significantly overexpressed genes (≥3.000 fold change) in the gene expression profiles of four donors without epilepsy. The experimental-evidence-based STRING protein interactions analysis showed associations between genes found in DAVID keyword search and other genes facilitating network interconnectivity. After evaluation of the genes’ STRING enriched functions, changes in the expression of the genes CAMK2A, NPY, DLG4, MEF2C, and MAPK7 were concluded to be potential biomarkers for TLE, confirming the original hypothesis. Specifically, the identification of MEF2C and MAPK7 for this purpose is relatively novel in the fields of bioinformatics and neurogenetics. Future work includes investigating the utility of the candidate genes in real-world gene-based diagnostic methods.

Bioinformatics analysis of ferroptosis in spinal cord injury

Ferroptosis plays a key role in aggravating the progression of spinal cord injury(SCI),but the specific mechanism remains unknown.In this study,we constructed a rat model of T10 SCI using a modified Allen method.We identified 48,44,and 27 ferroptosis genes that were differentially expressed at 1,3,and 7 days after SCI induction.Compared with the sham group and other SCI subgroups,the subgroup at 1 day after SCI showed increased expression of the ferroptosis marker acyl-CoA synthetase long-chain family member 4 and the oxidative stress marker malondialdehyde in the injured spinal cord while glutathione in the injured spinal cord was lower.These findings with our bioinformatics results suggested that 1 day after SCI was the important period of ferroptosis progression.Bioinformatics analysis identified the following top ten hub ferroptosis genes in the subgroup at 1 day after SCI:STAT3,JUN,TLR4,ATF3,HMOX1,MAPK1,MAPK9,PTGS2,VEGFA,and RELA.Real-time polymerase chain reaction on rat spinal cord tissue confirmed that STAT3,JUN,TLR4,ATF3,HMOX1,PTGS2,and RELA mRNA levels were up-regulated and VEGFA,MAPK1 and MAPK9 mRNA levels were down-regulated.Ten potential compounds were predicted using the DSigDB database as potential drugs or molecules targeting ferroptosis to repair SCI.We also constructed a ferroptosis-related mRNA-miRNA-lncRNA network in SCI that included 66 lncRNAs,10 miRNAs,and 12 genes.Our results help further the understanding of the mechanism underlying ferroptosis in SCI.

Charcot-Marie-Tooth-1A and sciatic nerve crush rat models:insights from proteomics

The sensorimotor and histological aspects of peripheral neuropathies were already studied by our team in two rat models:the sciatic nerve crush and the Charcot-Marie-Tooth-1A disease.In this study,we sought to highlight and compare the protein signature of these two pathological situations.Indeed,the identification of protein profiles in diseases can play an important role in the development of pharmacological targets.In fact,Charcot-Marie-Tooth-1A rats develop motor impairments that are more severe in the hind limbs.Therefore,for the first time,protein expression in sciatic nerve of Charcot-Marie-Tooth-1A rats was examined.First,distal sciatic nerves were collected from Charcot-Marie-Tooth-1A and uninjured wild-type rats aged 3 months.After protein extraction,sequential window acquisition of all theoretical fragment ion spectra liquid chromatography and mass spectrometry was employed.445 proteins mapped to Swiss-Prot or trEMBL Uniprot databases were identified and quantified.Of these,153 proteins showed statistically significant differences between Charcot-Marie-Tooth-1A and wild-type groups.The majority of these proteins were overexpressed in Charcot-Marie-Tooth-1A.Hierarchical clustering and functional enrichment using Gene Ontology were used to group these proteins based on their biological effects concerning Charcot-Marie-Tooth-1A pathophysiology.Second,proteomic characterization of wild-type rats subjected to sciatic nerve crush was performed sequential window acquisition of all theoretical fragment ion spectra liquid chromatography and mass spectrometry.One month after injury,distal sciatic nerves were collected and analyzed as described above.Out of 459 identified proteins,92 showed significant differences between sciatic nerve crush and the uninjured wild-type rats used in the first study.The results suggest that young adult Charcot-Marie-Tooth-1A rats(3 months old)develop compensatory mechanisms at the level of redox balance,protein folding,myelination,and axonogenesis.These mechanisms seem insufficient to hurdle the progress of the disease.Notably,response to oxidative stress appears to be a significant feature of Charcot-Marie-Tooth-1A,potentially playing a role in the pathological process.In contrast to the first experiment,the majority of the proteins that differed from wild-type were downregulated in the sciatic nerve crush group.Functional enrichment suggested that neurogenesis,response to axon injury,and oxidative stress were important biological processes.Protein analysis revealed an imperfect repair at this time point after injury and identified several distinguishable proteins.In conclusion,we suggest that peripheral neuropathies,whether of a genetic or traumatic cause,share some common pathological pathways.This study may provide directions for better characterization of these models and/or identifying new specific therapeutic targets.