Analysis Of The Differential Expression Profile Of Serum Exosomes MiRNA In Patients With Traumatic Brain Injury

ObjectiveTraumatic brain injury(TBI) is a common disease of central nervous system with the highest death rate and disability rate,which is common in both ordinary and wartime.After TBI,the Exosome(Exo)miRNA(micro RNA)in peripheral blood circulation can be altered.Bioinformatics analysis was used to mine the differential Exo miRNAs and related pathways closely related to brain injury,to predict the action targets of the differential genes,and to search for potential new biomarkers that can be used in the diagnosis and treatment of TBI.Methods:1.This study is a prospective study.Fourteen patients with TBI who were admitted to the Department of Neurosurgery and Emergency Medicine of the First Clinical College of Shanxi Medical University were selected.The patients were classified into mild or moderate TBI(mTBI)according to the GCS score(8 points<GCS<15 points)7 cases,severe TBI(s TBI)(GCS≤8 points)7 cases,the control group was selected as the healthy physical examination during the same period of 6 cases,the sample group was injured Within 24 hours,8-10 ml of peripheral blood before the operation,8-10 ml of peripheral blood samples of the physical examination subjects in the control group,stand for 2 hours,centrifuge to extract serum,and cryopreserve at-80℃.2.Use Exo extraction kit to extract Exo in the peripheral blood serum of TBI patients,and identify the size,concentration and quality of Exo with TM,NTA and WB.3.Trizol reagent was used to extract total RNA in Exo,and Illumina’s Truseq RNA method was used to build a library and perform sequencing analysis to screen known miRNAs and predict new miRNAs.Focus on the analysis of known miRNA expression in each sample.4.DESeq(version 1.18.0,Anders S and Huber W,2010)was used to analyze the differential expression of miRNA,and to screen the statistically significant differential expression of Exo miRNA in TBI(|log2FC|>1 and P<0.05).5.Use miranda,mi RWalk,mi RTar Base and Targetscan to predict the target genes of differentially expressed miRNAs.6.Go functional annotation and KEGG pathway analysis were used to perform functional enrichment analysis on the target genes of differentially expressed miRNAs.GO functional annotations include: BP,CC and MF,to understand the role of differentially expressed Exo miRNA in the pathogenesis of TBI.7.The String database and Cytoscape3.6.1 software used to construct and visualize the PPI relationship network among target genes to further understand the relationship between differentially expressed target genes of miRNA.8.Statistical methods: All statistical data analysis was performed in SPSS 25.0.The data are expressed as mean ± standard deviation.The t test is used to assess the difference between the two groups.Using one-way analysis of variance and selecting the two-tailed range,the differential expression of Exo miRNA between the control group,mTBI and s TBI was calculated.When P<0.05,the difference is considered to be statistically significant.Results:1.Exo and Exo miRNAs extracted from serum samples of Control group and TBI group were qualified,and high-throughput sequencing and data analysis were performed.2.Through the differential expression analysis of the miRNA of each group of samples,it was found that compared with the control group,there were 113 miRNAs in the mTBI group with significant differences(P<0.05;|log2FC|>1),of which up-regulated and down-regulated miRNAs were 63 and 50 respectively;there were significant differences in71 miRNAs in the s TBI group,of which 14 miRNAs were down-regulated and 57 miRNAs were up-regulated;compared with mTBI group(P<0.05;|log2FC|>1),There were61 differentially expressed miRNAs in the s TBI group,of which 45 miRNAs were up-regulated and 16 miRNAs were down-regulated.MTBI and s TBI screened 27co-expressed miRNAs with significant differences.The control group and mTBI and s TBI screened out 3 co-expressed miRNAs with significant differences.3.In the field of TBI,it mainly involves cell proliferation,aging,Rap1 signaling pathway,MAPK signaling pathway,cell adhesion molecules(CAMs),calcium signaling pathway and other inflammation-related signaling pathways,as well as γ-aminobutyric acid synapses,dopaminergic synapses,Serotonin-containing nerve synapses,neurotrophic factor signaling pathways and other pathways related to neurovascular remodeling.4.miRNA-206,miRNA-21-3p,miRNA-9-3p and miRNA-124-3p et al are key genes in the signaling pathway such as Exo vesicular transport,cellular aging,neurotrophic factors,and inflammatory changes after TBI.5.CDC5L,CDC42,MAPK,FRS2 et al play an important role in pathophysiological changes in TBI.Conclusion:1.Exo miRNA varies significantly with the degree of injury in TBI patients.2.miRNA-206,miRNA-21-3p,miRNA-9-3p and miRNA-124-3p et al.are key genes in TBI involving inflammatory-related pathways and neurovascular factor signaling pathways.3.CDC5 L,CDC42,MAPK,FRS2 et al.are the main target genes in the development of the pathophysiogenesis of TBI.