Transcriptome Profile Of Rat Genes In Injured Spinal Cord At Different Stages By RNA-sequencing

Background: Spinal cord injury(SCI)results in a devastating loss ofmotor and sensory functions.Despite advances insurgical techniques for the spinal cord,there are still noeffective treatments for this devastating neurological disorder.Therefore,it is very important to further understandthe molecular changes in SCI in order to developa better therapeutic program.In this study,genome-wide transcriptional profiling of spinal cord samples from injured rats at different time points after SCI was performed by RNA-Sequencing(RNA-Seq).The transcriptomes were systematically characterized to identify the critical genes and pathways that are involved in SCI pathology.Objective: 1.To clarify the changes of local gene transcription level in spinal cord injury in rats;2.To identify the key molecules and signaling pathways that can be used as targets for drug therapy.Method:(1)Rat contusive SCI model was established using a New York University impactor.(2)Beattie Bresnahan Basso(BBB)motor function score was used to observe the exercise function of SCI rats.(3)RNA-Seq technique was used to analyze the changes of the expression of local genes at different time points after SCI.(4)The genes with significant changes were selected and verified by fluorescent quantitative reverse transcription polymerase chain reaction(q RT-PCR).(5)Bioinformatics analysis of differentially expressed genes was performed by Gene Ontology(GO)and Kyoto Encyclopedia genesand genomes(KEGG).(6)Western Blot was used to verify the expression of target molecules at the protein level.(7)Cell location of selected proteins was performed by immunofluorescence staining.Results: RNA-Seq analysis was carried out on total RNA harvested from the rat spinal cords of sham control,acute,subacute,and chronic phases(1 day,6 days,and 28 days after injury,n = 3 in every group).Compared with sham-controlled group,1797(1223 up-regulated and 574 down-regulated),6590(3460 up-regulated and 3130 downregulated),and 3499(1866 up-regulated and 1633 down-regulated)differentially expressed genes(adjusted P-value <0.05 by DESeq)were obtained in acute,subacute,and chronic phases,respectively.Gene ontology(GO)enrichment analysis showed that the significantly differentially expressed genes were most enrichment in immune response,MHC protein complex,antigen processing and presentation,translationrelated genes,structural constituent of ribosome,ion gated channel activity,small GTPase mediated signal transduction,cytokine and/or chemokine activity,etc.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis showed that the top enriched pathways included ribosome,antigen processing and presentation,retrograde endocannabinoid signaling,axon guidance,dopaminergic synapses,glutamatergic synapses,GABAergic synapses,and TNF,HIF-1,NF-kappa B,Toll-like receptor,NOD-like receptor,c AMP,calcium,oxytocin,Rap1,B cell receptor,and chemokine signaling pathway.Ncf1 and Plau,which are highly expressed in RNA-Seq,were selected to further investigate their expression and cellular localization after SCI.The results showed that both the two molecules were increased after SCI.Ncf1 was highly expressed in subacute and chronic phase,while Plau was increased in acute,subacute and chronic stages.They were co-localized with infiltrated leukocytes.Conclusion:1.RNA-seq analysis showed that there were 1797,6590 and 3499 differentially expressed genes in the acute,subacute and chronic phases of spinal cord injury compared with the sham operation group,respectively.2.In acute phase,ribosome,TNF signaling pathway,proteoglycans in cancer,malaria,and staphylococcus aureus infection were enriched pathways;In subacute phase,glutamatergic synapse,circadianentrainment,GABAergic synapse and retrograde endocannabinoid signaling were enriched pathways;In chronic SCI,retrograde endocannabinoid signaling,oxytocin signaling,Rap1 signaling and thesynaptic vesicle cycle were also enriched pathways.3.The expression and localization of Ncf1 and Plau after spinal cord injury were further studied.The results confirmed that their expression levels were both increased after spinal cord injury.Ncf1 was increased in the subacute and chronic phases;and Plau in the acute,subacute and chronic phases.The increased Ncf1 and plau were distributed in the infiltratied white blood cells.These suggest that they are involved in the inflammatory process of SCI.