Correlation Analysis Of Serum Exosomes MiR-133a-3p And MiR-549a-3p With Diagnosis And Prognosis Of Patients With Traumatic Craniocerebral Injury

Objective:Traumatic brain injury(TBI)is a type of head injury caused by mechanical force with high morbidity and mortality worldwide,especially in low-income and middle-income countries.Our previous study found that the serum exosome miR-133a-3p and miR-549a-3p levels in TBI changed significantly compared with healthy controls,and further gene function enrichment analysis found that miR-133a-3p and miR-549a-3p were associated with oxidative stress,inflammatory response,angiogenesis,and neuronutrition after head trauma,but these results lacked effective experimental evidence.In this study,the expression levels of serum exosomes miR-133a-3p and miR-549a-3p in TBI and their correlation with the severity and prognosis of TBI were further investigated by quantitative analysis of serum exosomes miR-133a-3p and miR-549a-3p in TBI.By further determination of Superoxide dismutase(SOD)and brain-derived neurotrophic factor(BDNF),vascular endothelial growth factor(VEGF),and vascular endothelial growth inhibitor(VEGI),neuron specific enolase(NSE),Recombinant S100 Calcium Binding Protein β(S100β)content,to study the correlation of serum exosomes miR-133a-3p and miR-549a-3p with oxidative stress,inflammation,angiogenesis and neuronutrition.Methods:This prospective study used serum samples from patients with TBI and serum samples from healthy control donors.After informed consent,45 serum samples were obtained,including 15 severe TBI serum samples(3≤GCS≤8),15 mild or medium TBI serum samples(9≤GCS≤15),and 15 healthy control serum samples.Glasgow Coma Scale(GCS)was performed by more than two senior neurosurgical specialists using the criteria of Neurosurgery III.Clinical data of subjects were collected.Serum exosomes were extracted with exosome extraction kit.Nanoparticle Tracking Analysis(NTA),Transmission electron microscope(TEM),Western Blot(WB)were used to determine the size,mass and concentration of exosomes and the expressions of membrane specific proteins CD63 and TSG101.Then,Quantitative Real-time PCR(q PCR)was used to detect the expression levels of serum exosomes miR-133a-3p and miR-549a-3p in each sample.The correlation between the expression of serum exosomes miR-133a-3p and miR-549a-3p and TBI patients was analyzed.The contents of SOD,BDNF,VEGF,VEGI,NSE and S100β in serum of each group were determined by enzyme-linked immunosorbent assay.SPSS software was used to analyze the correlation between serum exosomes miR-133a-3p and miR-549a-3p and serum SOD,BDNF,VEGF,VEGI,NSE and S100β.receiver operating characteristic curve(ROC)was used to evaluate the diagnostic efficacy of serum exosomes miR-133a-3p and miR-549a-3p.Results:After adjusting for gender,age,hypertension,diabetes and other factors,there were no significant differences in the expression of serum exosome miR-133a-3p and miR-549a-3p in TBI group compared with the control group(P > 0.05).The TBI group was further divided into severe TBI(sTBI)group,mild or moderate TBI(m TBI)group and the control group.The results showed that,Pair-to-pair comparison showed no significant difference in the expression of miR-133a-3p in serum exosomes(P > 0.05).Compared with the control group,the expression of serum exosome miR-549a-3p in sTBI group was significantly up-regulated(P < 0.01),and the expression of serum exosome miR-549a-3p in sTBI group was significantly up-regulated compared with m TBI group(P < 0.05).There was no significant difference in the expression of serum exosome miR-549a-3p in m TBI group compared with control group(P > 0.05).Compared with the control group,the expressions of SOD and BDNF in the serum of sTBI were significantly down-regulated(P < 0.01),the expressions of VEGI,NSE and S100 β in the serum of sTBI were significantly up-regulated(P < 0.01),and the expression of VEGF in the serum of sTBI had no significant statistical difference(P >0.05).Further study on the correlation between serum exosome miR-549a-3p in sTBI and SOD,BDNF,VEGI,NSE and S100β showed that the expression of serum exosome miR-549a-3p in sTBI patients was positively correlated with the content of serum BDNF(P < 0.01).It was negatively correlated with the contents of NSE and S100β in serum(P < 0.01),but had no significant correlation with the contents of SOD and VEGI in serum(P > 0.05).ROC curve analysis showed that the area under curve(AUC)value of serum exosome miR-549a-3p for diagnosis of sTBI was 0.69,the sensitivity was 73%,the specificity was 76%,and the 95% confidence interval was 0.88-0.81.The area under curve(AUC)value of serum exosome miR-549a-3p for the diagnosis of poor prognosis in patients with sTBI was 0.80,the sensitivity was 58%,the specificity was 95%,and the95% confidence interval was 0.67-0.93.Conclusion:1.TEM,NTA and WB assay results showed that the extracts isolated from serum were consistent with the descriptions of exosome morphology and characteristics in the literature.2.The expression of serum exosome miR-549a-3p in sTBI group was significantly higher than that in control group and m TBI group.3.The expressions of SOD and BDNF in the serum of sTBI were significantly lower than those of the control group,while the expressions of VEGI,NSE and S100βwere significantly higher than those of the control group.4.The expression of serum exosome miR-549a-3p in sTBI is correlated with the serum contents of BDNF,NSE and S100β.5.Serum exosome miR-549a-3p has certain value in the diagnosis of sTBI and poor prognosis of sTBI.