Expression And Research Of Serum Exosomes MicroRNA As Biomarkers In Patients With Traumatic Brain Injury

Objective:Combined comparative analysis of the sequencing results of serum exosome(Exo)micro RNA(miRNA or miR)in patients with traumatic brain injury(TBI)and related data of the GEO dataset,using bioinformatics and statistical methods,the results could be used to search for differentially expressed miRNAs after TBI,and to predict possible biological functions and signaling pathways involved in the process of mechanisms affecting TBI.Not only that,the results could also be used to explore the changes in the expression levels of differentially expressed miRNAs in TBI patients,and explore their efficacy,assessment and prognosis of TBI in early diagnosis,in order to obtain the clinical application value of specific miRNAs.Methods:1.A total of 14 TBI patients admitted to the First Hospital of Shanxi Medical University were selected as the experimental group.According to the Glasgow coma score(GCS)at the time of consultation,the patients were divided into mild to moderate TBI group(8 < GCS ≤ 15)and severe TBI group(3 ≤ GCS ≤ 8),with seven subjects in each group.Six healthy subjects with similar age and gender were selected as the control group.The peripheral blood of the experimental group and the control group were collected and the differentially expressed serum Exo miRNAs were obtained by high-throughput sequencing(Serum sample group of this unit: Data 1).All TBI patients were followed up for 6 months by way of outpatient or telephone,and they were divided into good prognosis group(4≤GOS ≤5)and poor prognosis group(1≤GOS ≤3)according to the Glasgow Outcome Scale(GOS).2.TBI patient serum miRNA dataset named GSE131695(GEO Database Group: Data2)was retrieved from the GEO gene chip database.After exclusion and screening,a total of 17 TBI patients were included as the experimental group.According to the GCS score,they were divided into mild to moderate TBI group(8<GCS≤15)and severe TBI group(3≤GCS≤8),the number of subjects in the two groups were four and thirteen respectively.At the same time,six subjects in the healthy control group were included.Using DEseq software to screen differentially expressed serum miRNAs.3.Venn diagrams were used to jointly analyze the sequencing results of the Data 1and the Data 2 to search miRNAs with common differential expression between the above two sets of data.Then,bioinformatics methods were used to perform GO annotation and KEGG pathway analysis on its target genes,in order to preliminarily reveal its target gene loci and important signaling pathways involved in regulation.4.In the Data 1 and Data 2,we performed statistical analysis on the previously screened miRNA sequencing data with common differential expression,in order to further explore their expression differences in patients with different degrees of TBI.Then in Data1,the diagnostic value of different serum Exo miRNAs in TBI patients was compared,by calculating its sensitivity,specificity,Youden index and area under the curve(AUC).We selected Exo miRNA,which had the best diagnostic performance for TBI,to explore the correlation between its expression and prognosis of TBI patients by using Spearman’s correlation coefficient and ROC curve,which coulde valuate its predictive value of the prognosis of TBI.Results:1.Through the preliminary comparative analysis and screening of Data 1 and Data 2,a total of 5 differentially expressed miRNAs were obtained,namely miR-106b-5p,miR-3613-5p,miR-451 a,miR-499a-5p,miR-6740-5p,which predicted that there are a total of 4866 target genes would be obtained.The enrichment analysis showed that its target genes are not only involved in regulatory processes such as body signal transduction,cell connection,nervous system development,cell communication,and metabolic processes,but are also closely related to(cell)autophagy,AMPK,CAMP,Fox O and other signaling pathways.2.Statistical analysis showed that compared with the healthy control group,the expressions of miR-106b-5p,miR-3613-5p,miR-451 a,and miR-499a-5p in TBI patients were significantly up-regulated,and the differences were statistically significant(P<0.05).Among them,the differential expressions of miR-451 a in each group of Data 1 and Data 2are completely consistent.The differential expressions of miR-106b-5p,miR-3613-5p,miR-499a-5p in each group of Data 1 and Data 2 are partially consistent.The differential expression of miR-6740-5p in Data 1 was inconsistent with that of each group in the Data2.3.The areas under the receiver operating characteristic(ROC)curves of serum EXO miR-106b-5p,miR-3613-5p,miR-451 a,and miR-499a-5p within 24 hours after TBI were0.869(95%CI 0.644～0.976),0.905(95%CI 0.689～0.989),0.964(95%CI 0.77～1.000),and 0.845(95%CI 0.615～0.966)respectively,all of which have good diagnostic value for TBI,among which miRNA-451 a has the best diagnostic performance.4.Spearman correlation analysis showed that serum Exo miRNA-451 a was negatively correlated with GOS score of 6-month prognosis in patients with TBI(r=-0.551,P<0.05).The expression level of serum Exo miRNA-451 a 24 hours after injury in the good prognosis group(4 ≤ GOS ≤ 5)was significantly lower than that in the poor prognosis group(1≤GOS≤3),and the differences were statistically significant(P<0.05).And the area under ROC curve(AUC)of serum Exo miRNA-451 a in predicting the prognosis of TBI patients after 6 months was 0.844(95% CI 0.557 ～ 0.978).Conclusion:1.EXO miRNAs are differentially expressed in the serum of TBI patients,and in different degrees of injury,the expression differences are different.2.There are a series of miRNAs that may be key miRNAs in a series of pathophysiological mechanisms after TBI,including miR-106b-5p,miR-3613-5p,miR-451 a,miR-499a-5p,and miR-6740-5p,which could play a role in regulating inflammatory response,immune stress,angiogenesis,nerve damage repair and other processes after TBI.3.The expression levels of miR-106b-5p,miR-3613-5p,miR-451 a,and miR-499a-5p in serum Exo of TBI patients were significantly increased,and the difference was significant,and they all had good value for the diagnosis of TBI patients.Among them,Exo miR-451 a had the best diagnostic performance.4.Exo miR-451 a was significantly negatively correlated with the 6-month prognosis score(GOS)of TBI patients,which means that the higher the serum exosomal miR-451 a expression in TBI patients,the worse the patient’s prognosis.The results also found that the serum Exo miR-451 a expression has a certain predictive value for the prognosis of TBI patients.Therefore,Exo miR-451 a is expected to be a serum biomarker for TBI diagnosis,injury severity assessment and prognosis prediction because of its potential clinical application value.