MRNA Profiling For MiR-124-mediated Repair In Spinal Cord Injury

Objective:Spinal cord injury(SCI)is a destructive neurological disease.Microribonucleic acid(miRNA)-124 is one of the potential targets for repairing of spinal cord injury.This study aims to research the messenger RNA(mRNA)expression profiling of miR-124-mediated SCI repair and analyze its potential molecular mechanism.Methods:①SCI rat models were established by allen’s method,SCI+miR-124 overexpression models were constructed by intrathecal injection of agomiR-124,BBB score was used to assess the behavioral changes of rats in control group,sham operation group(sham group),spinal cord injury group(SCI group)and spinal cord injury+miR-124 group(treat group),the level of NeuN and GFAP was detected by immunofluorescence,the level of NF-200 was detected by immunohistochemistry.②Collect total RNA of spinal cord of rats in each experimental group,mRNA sequencing library preparation was followed by purification using AMPure XP system,use agilent Bioanalyzer 2100 system to evaluate the library quality.The index-encoded samples were clustered on the cBot cluster generation system,and the differential expression analysis is completed by the edgeR software package.③Use GOseqR software package to analyze the GO enrichment of DE gene and KOBAS software to analyze the KEGG pathway of DE gene based on database resources.④QPCR was used to verify the eight significantly differential genes.⑤TargetScan predicted targets of miR-124 online,use qPCR to detect changes of Tall expression in the spinal cord tissue of rats after SCI,and then transfect agomiR-124 or miR-124 inhibitor into SH-SY5Y cells,qPCR detected the expression level of miR-124,Tall,NeuN,Ki-67,CCK-8 test to detect cell viability.Results:①AgomiR-124 can effectively improve the BBB score in rats after SCI,increase the number of neurons in the spinal cord tissue,reduce astrocytes,and promote the expression of NF-200 in the spinal cord of rats.②After SCI in rats,there are a total of 210 up-regulated mRNAs and 78 down-regulated mRNAs in the spinal cord.After agomiR-124 treatment,there are a total of 85 up-regulated mRNAs and 80 down-regulated mRNAs.Among them,48 mRNAs are affected by both SCI and agomiR-124.③There is no significant difference between SCI group and treat group in the GO analysis comparison.However,the DE genes between SCI group and sham group are significantly enriched in GO terms.There are 39 terms related to ’biological processes’and 7 terms related to ’cell components’ while no gene enrichment in the GO category of ’molecular function’ is found.④In the KEGG analysis,the DE genes between SCI group and sham group are mainly enriched in phagosome,complement and coagulation cascades,lysosome,cell adhesion molecules,ECM-receptor interaction,FcyR-mediated phagocytosis,transcriptional misregulation in cancer,endocytosis,primary immunodeficiency,etc.While the DE genes between SCI group and treat group are enriched in alanine,aspartate and glutamate metabolism,VEGF signaling pathway,complement and coagulation cascades,endocytosis,ubiquitin mediated proteolysis,spliceosome,Wnt signaling pathway,GnRH signaling pathway,mRNA surveillance pathway,natural killer cell mediated cytotoxicity,Hippo signaling pathway,oxytocin signaling pathway,transcriptional misregulation in cancer,MAPK signaling pathway,etc.⑤The mRNA expressions of Nploc4,Yme111,LOC103693564 and Aspa are up-regulated,while the mRNA expressions of Epb4112,LOC100911685,LOC100910833 and Smarccl are down-regulated after agomiR-124 treatment.The results are consistent with the sequencing results.⑥Online prediction find that Nploc4,Yme111,Aspa,Smarccl and Tall are target mRNAs of miR-124.The expression of Tall after spinal cord injury is negatively related with miR-124.In SH-SY5Y cells,agomiR-124 can reverse the increasing of Tall induced by SCI,increase Ki-67 expression and decrease NeuN expression,and increase cell viability,suggesting that low levels of Tall gene can promote the proliferation of neuron precursor cells and inhibit Their differentiationConclusions:①Overexpression of miR-124 can protect and repair the spinal cord.②miR-124 mediates SCI repair by effecting the expression level of various mRNAs in rats,especially the nine DE mRNAs that have been identified:Nploc4,Yme11,LOC103693564,Aspa,Epb4112,LOC100911685,LOC100910833,Smarcc1 and Tal1.③miR-124/Tal1 axis can participate in the repair of SCI by supplementing neural stem cells.