The Study Of LncRNAs In Schwann Cells After Peripheral Nerve Injury

ObjectivePeripheral nervous injuries(PNI)caused by compression,entrapment,or ischemia can lead to functional limitations that can severely affect quality of life.After PNI in mammals,the distal nerve to the site of injury may undergo a process called Wallerian Degeneration(WD),during which schwann cells lose contact with axons and proliferate to form a bundle that provides a permissive microenvironment for axon regeneration.However,The molecular mechanism of the regenerative process has not yet been fully elucidated.In this study,the expression of long noncoding RNAs(lncRNAs)and messenger RNAs(mRNAs)in the distal segment of the sciatic nerve after PNI was profiled using microarray analysis.Bioinformatic approaches,cell biology combined with molecular biological techniques were performed to investigate key lncRNAs predicted to participate peripheral nerve regeneration.We are aimed to promote the overall understanding of WD and thus provide a basis and new direction for repairing peripheral nerve injury.Methods1.The microarray was used to detect the m RNAs and lncRNAs expression in the distal nerve at 0,3,7,and 14 days after sciatic nerve crush.Bioinformatics approaches were used to investigete the important biological processes and signaling pathways related to nerve regeneration after PNI,qRT-PCRwas used to validate the predictions.2.We also applied bioinformatic approaches to predict the target genes and the function of the differentially expressed lncRNAs.The key lncRNAs predicted to participate the peripheral nerve regeneration were chosen for further analysis.3.Mice schwann cells(SCs)were isolated and cultured,candidate lncRNAs were overexpressed in SCs.CCK-8 technique was applied to evaluate the proliferation abilty of SCs after lncRNA overexpression.PCR and western were performed to investigate the target genes of the lncRNAs and the downstream pathways.Results1.There were 5,041 genes that were differentially expressed between the control and 3 d sample.4,273 genes were differentially expressed between the control and 7 d samples.3,228 genes were differentially expressed between the control and 14 d samples.Biological processes related to response to stimulus,inflammatory response,cell proliferation,cell migration,axon guidance and extracellular matrix predominate at the early stage of peripheral nerve regeneration after PNI,the biological processes related to immune response and extracellular matrix predominate at 7th day after PNI,growth factors were secreted and kept a high level through the whole peripheral nerve regenerative process after PNI.The results of KEGG analysis indicated that the differentially expressed mRNAs were closely associated with cytokine-cytokine receptor interaction,axon guidance,p53 signaling pathway,cell cycle and chemokine signaling pathway.2.There were 3,788 lnc RNAs that were differentially expressed between the control and 3 d samples.3,314 lncRNAs were differentially expressed between the control and 7 d samples.2,400 lnc RNAs were differentially expressed between the control and 14 d samples.31 lncRNAs and six LncRNA-mRNA pairs were predicted to participate peripheral nerve injury and regeneration.Considering that NONMMUG014387-Cthrc1 changes significantly after PNI.We chose NONMMU G014387-Cthrc1 for further analysis.3.PCR showed that the expression of Cthrc1 was consistent with that of NONMMUG014387 after PNI.Further investigation revealed that overexpression of NONMMUG014387 promoted proliferation of SCs.Compared to the control group,NONMMUG014387 overexpression promoted proliferation of SCs.PCR and western showed that NONMMUG014387 overexpression increased the expression of Cthrc1 and activated Wnt/PCP signaling pathway.Conclusion1.We have drew a comprehensive and systematic map on the whole process of peripheral nerve regeneration after PNI.2.In addition,we screened out 31 noval lnc RNAs and 6 lncRNA-mRNA pairs which are predicted to participate peripheral nerve regeneration.3.At last,we investigated the function of lnc RNA NON MMUG014387,after sciatic nerve injury,the expression of NONMMUG014387 was increased,as a result,the expression of Cthrc1 was increased.The increased Cthrc1 activated Wnt/PCP pathway which promote the proliferation of SCs.Our study adds to the new understanding of regeneration after nerve injury,elucidating the molecular-biological basis for repairing peripheral nerve injury.