Expression Alteration Of Long Non-coding RNA After Spinal Cord Injury And Its Regulatory Effect On Neurite Outgrowth

Objective Spinal cord injury(SCI)is a devastating condition following compression or rupture of the spinal cord which can result in abnormal or absent motor and sensory functions.As a serious central nervous system injury,SCI can seriously affect the quality of life of patients and bring heavy economic burden to patients.In recent years,the incidence of SCI at home and abroad increases year by year.However,the pathological and molecular mechanism is very complex,and the effective methods to treat SCI are still scarce.Long non-coding RNA(lncRNA)is a type of non-coding RNA which can regulate expression of genes.In recent years,more and more studies focused on lncRNA,and their roles in the pathological changes of SCI needs to be clarified.In this study,we use the microarray chip,the high-throughput techniques,to identify the messenger RNAs(mRNAs)and lncRNAs expression alteration in rats at different time points after SCI,and we also performed bioinformatics analysis to identify the key differentially expressed mRNAs and lncRNAs and reveale their functions.Moreover,the study aimed to verify the function of key lncRNA,and these results may provide potencial targets for clinical treatment of SCI.Methods In this study,the spinal cord contusion model of rats was first built,and then the mRNA and lncRNA expressions at 2 hours,2 days and 7 days after SCI were detected by microarray.Subsequently,Bioinformatics analysis was performed to analyze the differentially expressed mRNAs and lncRNAs.GO analysis was performed to identify the key biological processes involved in the pathological process of SCI.KEGG analysis was performed to analyze the important signaling pathways involved in the pathological process of SCI.Furthermore,protein-protein interaction(PPI)analysis was performed to identify the modules with similar biological function and the key interacting genes.After that,cerebral cortex neurons in rat were isolated and cultured,and we transfected the lentivirus and siRNA into neurons to upregulate and downregulate the lncRNA expression respectively.The expression levels of key lncRNA and its target genes were detected by qRT-PCR and Western blot.Immunofluorescence was performed to detecte the effect of lncRNAs on neurite outgrowth.Results A total of 992 mRNAs were differentially expressed at 2 hours after SCI,among which 730 mRNAs were up-regulated and 262 mRNAs were down-regulated.A total of 4,308 mRNAs were differentially expressed at 2 days after injury,including 2,229 mRNAs with up-regulated expression and 2,079 mRNAs with down-regulated expression.A total of 4,113 mRNAs were differentially expressed at 7 days after injury,including 2,339 up-regulated and 1,774 down-regulated mRNAs.GO analysis results suggested that the different mRNAs were involved in neuron neurite outgrowth,immune response,inflammatory response,myelin formation,apoptosis and other biological processes.KEGG results showed that the main enrichment of signaling pathways after SCI were cell apoptosis signaling pathways,p53 signaling pathway,MAPK signaling pathways,NOD sample receptor signal pathways,toll-like receptors signaling pathways and so on.After SCI,772 lncRNAs were differentially expressed in the 2-hour group compared with the control group,and 528 lncRNAs were up-regulated and 244 lncRNAs were down-regulated.Compared with the control group,the expression of 3,193 lncRNAs in the 2-day group was differentially expressed,and 1,332 lncRNAs were up-regulated and 1,861 lncRNAs were down-regulated.Compared with the control group,a total of 3,093 lncRNAs were differentially expressed in the 7-day group,and 1,290 lncRNAs were up-regulated and 1,803 lncRNAs were down-regulated.The differentially expressed lncRNAs were associated with some biological processes such as protein phosphorylation,intracellular signal transduction,regulation of ion transmembrane transport,axon generation,axon guidance,cell proliferation,cell migration and so on.In addition,the differentially expressed lncRNAs were also likely to be associated with mitogen-activated protein kinase signaling pathways,axon guidance and cAMP signaling pathways.Furthermore,the results showed that the expression of NONRATT006058 was increased after SCI,peaked at day 2,and keep the high expressionat at day 7.Bioinformatics predicted that Brca1 was a target gene of NONRATT006058,and qRT-PCR and Western blot results showed that NONRATT006058 and Brca1 had the same change trend of expression.Rat neurons were successfully cultured in vitro,and we also established the cell models of overexpression and downexpression of NONRATT006058 in neuron.The results showed that NONRATT006058 could inhibit neurons neurite growth by regulating the expression of Brca1.Conclusion This study systematically described the expression changes of mRNAs and lncRNAs in different phases after SCI in the perspective of histology,the biological functions and signal pathways,which provided a basis for the overall understanding of the molecular mechanism of the occurrence and development of SCI.In addition,the function of NONRATT006058 was first identified in this study.In vitro studies,the results showed that NONRATT006058 inhibited neurite growth by upregulating the expression of Brca1.The results further revealed the molecular mechanism of SCI and provided a new target for clinical treatment of SCI.