Expression Of Lnc Rna In Exosome From Extracellular Space Of Mouse Brain After TBI And Analysis Of LncRNA-miRNA-mRNA Network

Objective: The exosomes of mouse brain extracellular space were isolated and the expression of long non-coding RNA(lncRNA)in exosomes after traumatic brain injury(TBI)was analyzed by high-throughput whole transcriptome sequencing.Bioinformatics was used to predict the function of differentially expressed lncRNA,and the lncRNA-miRNA-mRNA network was constructed to clarify the regulatory function of exosome and lncRNA in order to provide a new idea for the study of the molecular biological mechanism of TBI,to provide a new molecular marker for the clinical diagnosis of TBI and a new strategy for the treatment of TBI.Methods:(1)Method of isolating exosome: A method was established to isolate exosomes from the brain extracellular space by papain digestion and exosome isolation kit.The morphological characteristics of exosomes were observed with the negative staining under transmission electron microscope.Exosomal surface markers,such as CD63?TSG101 and HSP70 were identified by Western Blot.(2)Experimental group: 24 mice were randomly divided into TBI group and Sham group.Exosomes were isolated from the brain extracellular space and exosome samples were obtained.(3)RNA high-throughput sequencing: Total RNA was extracted from exosome for RNA quality control,ribosomal RNA(rRNA),was removed to construct RNA library and library quality control was carried out.Sequencing with a gene chip,lncRNA,miRNA and mRNA were detected,identified and then annotated after the high-quality reads were obtained.And then,the differentially expressed lncRNA,miRNA and mRNA were screened.(4)Verification of RNA high-throughput sequencing results: Respectively random selection 5 differentially expressed lncRNA,miRNA and mRNA to verify the accuracy of RNA high-throughput sequencing results by quantitative real-time polymerase chain reaction(qRT-PCR).(5)Bioinformatics analysis of differentially expressed lncRNAs: Gene ontology(GO)and kyoto encyclopedia of genes and genomes(KEGG)pathway analyses were performed for the differentially expressed lncRNA.(6)The lncRNA-miRNA-mRNA interaction network was constructed with the related genes.Results:(1)A method was successfully established to isolate exosomes from the brain extracellular space by papain digestion and exosome isolation kit.Under TEM,these exosomes were irregular spheres ranging of 30-100 nm in diameter.The exosomes highly expressed the typical exosomal markers,such as CD63,TSG101 and HSP70.(2)A heat map of differentially expressed RNAs was generated to illustrate the distinguishable RNA expression profile of the samples.Cluster analysis shows that the biological duplication of the sample was good.There were siginificant differences between 442 lncRNAs(fold change ? 2.0 and P-values ? 0.0),among which 255 were up-regulated and 187 were down-regulated.There were siginificant differences between 50 miRNAs(fold change ? 2.0 and P-values ? 0.0),among which 5 were up-regulated and 45 were down-regulated.There were siginificant differences between 1496 mRNAs(fold change ? 2.0 and P-values ? 0.0),among which 118 were up-regulated and 1378 were down-regulated.(3)Go analysis showed that differential lncRNA is involved in cellular localization,cognition,learning or memory,and closely related to the composition of the nucleus and organelles,and is involved in the binding process of proteins,oxygen and kinases,etc.These results suggest that lncRNA is related to the repair of neurons after brain injury,the prognosis of the organism and the recovery of learning ability.MAPK signal pathway and ARVCsignal pathway are the signal pathways with the highest degree of correlation among the differentially expressed lncRNA.(4)In lncRNA-miRNA-mRNA network,there were close relationship between ENSMUST00000147847 and mRNA Npas4 or miRNA mmu-miR-200a-3p,and between AK016241 and mRNA Bdnf,miRNA mmu-mir-200a-3p as well.Interaction can be found among ENSMUST00000152569,Emp1 and Rhobtb2 through miRNA mmu-mir-30b-3p.These complexities of RNA network contribute to our study of the role of lncRNAs in TBI and how lncRNAs interacts with miRNA and mRNA to regulate the formation and maintenance of inhibitory synapses,the survival of neurons and apoptosis,which provides a new way of thinking and entry point.Conclusion:(1)Traumatic brain tissue after TBI can release exosome into the brain extracellular space.The changes of biomaterial in exosome can actually reflect the physiological and pathological process after TBI.(2)In this study,we reported and annotated a series of differentially expressed lncRNA,in the exosome of brain extracellular space after TBI,which broadened the idea of gene research on the exosome of brain extracellular space after TBI.It provides a new target for the further study on the molecular mechanism of TBI,early diagnosis and intervention therapy,and provides a basis for the study of the mechanism of the effects of exosome and its its contents onTBI.(3)The differential expression of lncRNA in the exosome of brain extracellular space after TBI is involved in the specific biological process after TBI,and may be involved in the pathophysiological regulation process after TBI through lncRNAmiRNA-mRNA network,thus participating in the injury repair process after TBI.