Study On Screening And Regulation Of MiRNA And Protein Molecular Biomarkers In Traumatic Brain Injury

Background:Traumatic brain injury(TBI)is one of the most common diseases in neurosurgery and one of the main causes of death and disability.However,there is no recognized molecular marker for the diagnosis of TBI in clinic,especially for mild TBI,which has no obvious imaging changes and no obvious signs of nervous system localization.It has been a difficult point in clinical and forensic identification.Studies have found that TBI can cause changes in gene and protein expression profiles in brain tissues.Detection of gene and protein expression in body fluids reveals that some of them are closely related to the degree of brain injury and clinical prognosis.Therefore,genes and proteins with significant differences in expression before and after injury become potential biomarkers of TBI.MicroRNA is a class of highly conserved single-stranded RNA.90% of the microRNAs in the blood are in the form of nucleic acid-protein complexes.They are highly stable and resistant to degradation,and do not vary with individual differences,temperature,pH value,time and the presence or absence of enzymes.These characteristics make humoral microRNAs an ideal marker of disease.Protein,as the real executor and embodiment of life activities,shows a close and coordinated change with the process of life activities.In the search process of TBI biomarkers,many researchers focused on protein.Although many candidate biomarkers have been proposed,due to poor reproducibility,only a few of them have reference value for the diagnosis and prognosis of moderate to severe TBI with imaging changes.These biomarkers have not been used for clinical diagnosis,and biomarkers used for the diagnosis of mild TBI have not been reported till now.Furthermore,the relationship between marker expression changes and TBI damage mechanism and pathological prognosis is unknown so far.In our previous works,we used 2DE-MALDI-TOF/MS to analyze the protein expression profiles of brain tissue and serum after mild brain injury in rats,and identified 15 differentially expressed proteins before and after brain injury.Bioinformatics and network disturbance methods were used to preliminarily confirm the inconsistency of gene and protein expression levels related to brain injury,inhibiting transcription through miRNAs may be the potential meschanism.Aim:1.To screen and validate the biomarkers in plasma of TBI patients by using microarray and iTRAQ-based quantitative proteomics,in order to find the objective criteria for clinical diagnosis of TBI;2.To analyze the biological processes and signaling pathways involved in the pathological process after TBI by bioinformatics technology,in order to elucidate the pathogenesis and find the potential therapeutic targets of TBI.Methods:1.Sample collection:plasma from 90 patients with mild,moderate and severe TBI and 30 healthy controls were collected according to the criteria.2.miRNA microarray assay: Five samples from each group were detected by miRNA microarray,and the data were analyzed.The differential expression profiles of microRNAs in plasma samples from TBI patients with different degrees of injury were compared.RT-qPCR was performed to validate the candidate miRNAs acquired on microarrays.The receiver operating characteristic curve(ROC)analysis was conducted to analyze the specificity and sensitivity of candidate miRNA in diagnosing TBI.3.iTRAQ-base quantitative proteomics analysis: After enzymatic hydrolysis of high abundance protein-free plasma samples,labeled with iTRAQ reagent,separated and detected by mass spectrometry,then the data obtained were analyzedand enrichment analysis were conducted.Selected differential proteins and their upstream and downstream key proteins were validated by ELISA assay.Results:1.Clinical characteristics of the subjects: a total of 90 TBI patients and 30 healthy volunteers were enrolled in this study.miRNA microarray and iTRAQ-base quantitative proteomics analysis were performed in 5 patients with mild,moderate and severe TBI and 5 controls respectively.The sampling times in the mild,moderate,and severe TBI groups were 7.54±2.81,6.08±0.79,and 6.16±1.19,respectively.There were no statistical differences between these groups.The follow-up validation test was carried out in 25 patients with mild,moderate and severe TBI and25 in the control group.The time from injury to blood drawing in the patients with mild,moderate and severe TBI were 7.22±1.75,7.16±1.57and 6.53±1.56,respectively.There were no statistical differences between these groups.2.Microarray results and its verification:(1)Compared with the control group,plasma levels of 65,33 and 16 microRNAs were up-regulated,and 29,27 and 6 microRNAs were down-regulated in the mild,moderate and severe TBI groups,respectively.Among the three groups,13 miRNAs were significantly changed(7 of them were up-regulated and 6 were down regulated).(2)GO enrichment analysis of of the target genes of different microRNAs showed that there were 10 common biological processes in patients with mild and moderate TBI,but only the regulation of systemic arterial blood pressure by vasopressin was found in all three TBI groups.These results suggest that mild and moderate TBI may have similar pathological processes.(3)Pathway enrichment analysis showed that several important pathways were involved in the process of TBI injury,including p53 signaling pathway,mTOR signaling pathway,TGF-beta signaling pathway,SNARE interaction in vesicle transport,nicotinic acid and nicotinamide metabolism,and neurotrophin signaling pathway.In addition,two neurobiological pathways,pantothenate and CoA biosynthesis,long-term depression,were found only in the first 15pathways in the mild TBI group.(4)The up-regulated expression of miRNAs(miR-6867-5p,miR-3665,miR-328-5p,miR-762,miR-3195,miR-4669 and miR-2861)in the plasma of the injured patients was verified by qRT-PCR.The results of qRT-PCR were consistent with the results of microarray.Compared with the control group,the levels of seven candidated microRNAs in plasma of patients with mild,moderate and severe TBI groups were significantly increased.The levels of miR-3195and miRNA-328-5p in severe TBI group were significantly higher than those in mild and moderate TBI groups,and the levels of miRNA-6867-5p in severe and moderate TBI were significantly higher than those in mild TBI group.(5)ROC analysis was conducted to calculate the area under the curve(AUC)to identify the diagnostic accuracy of the miRNAs.The AUC values were as following:miR-6867-5p(0.854,P<0.001),miR-3665(0.877,P<0.001),miR-328-5p(0.888,P<0.001),miR-762(0.916,P<0.001),miR-3195(0.899,P<0.001),miR-4669(0.907,P<0.001),and miR-2861(0.913,P<0.001).All miRNAs showed good diagnostic accuracy.(6)ROC analysis was conducted to evaluate whether these miRNAs could be used to diagnose mild TBI.AUC values were as follows:miR-6867-5p(0.765,P<0.001),miR-3665(0.916,P<0.001),miR-328-5p(0.855,P<0.001),14 miR-762(0.921,P<0.001),miR-3195(0.859,P<0.001),miR-4669(0.894,P<0.001),and miR-2861(0.898,P<0.001),all of which had a certain diagnostic accuracy in identifing mild TBI patients.miR-3665 and miR-762 were more accurate in diagnosing mild TBI patients.3.iTRAQ results and its verification:(1)5035 peptides and 1109 proteins were identified in the iTRAQ quantitative assay.Compared with healthy control group,the up-regulated protein numbers in mild,moderate and severe TBI groups were 90,92 and 103,and the down-regulated protein numbers were 42,58 and 58 respectively.(2)GO enrichment analysis showed that the differentially expressed proteins after TBI injury were involved in the biological processes of acute-phase response,acute inflammatory response,injury response to wounding,stress response to stress,platelet activation,wound healing and blood coagulation.Lipid metabolism-related biological processes such as regulation of triglyceride catabolic process,regulation of lipid catabolic process,and regulation of triglyceride metabolic process in the biological process of severe TBI ranked relatively higher.(3)Pathway enrichment analysis showed that Hippo signaling pathway-fly,complement and coagulation cascades,and Salmonella infection were invovled in all three TBI groups.(4)ELISA assay validated the expression levels of C4,C4 b,C3,C3 b,C4BP and C5 involved in the complement cascade and Lectin pathway.The results showed that the levels of C4 and C4 b were up-regulated significantly in all15 TBI groups(P < 0.05),and the expression level of C4 was also significantly different between mild to severe TBI groups(P < 0.05).There were no significant differences in the expression of C3 and C3 b between all groups.C4 BP,an inhibitory factor of the cascade pathway,was significantly down-regulated in the three TBI groups(P < 0.05).The expression of C5 was up-regulated in all TBI groups after injury,and the expression level of C5 in severe TBI group was higher than which in mild TBI group(P < 0.05).(5)ROC curve analysis was used to evaluate the accuracy of C4,C4 b and C5 in the diagnosis of TBI.AUC values were as following: C4(0.882,P < 0.001),C4b(0.910,P < 0.001),C5(0.924,P < 0.001).(6)ROC analysis of C4,C4 b and C5 in the diagnosis of mild TBI showed that AUC values were as following: C4(0.779,P < 0.001),C4b(0.893,P < 0.001),C5(0.874,P < 0.001),C4 b and C5 showed better diagnostic accuracy in identifing mild TBI patients.Conclusion:1.Seven microNAs,miR-6867-5p,miR-3665,miR-328-5p,miR-762,miR-3195,miR-4669 and miR-2861,were identified as diagnostic markers of TBI.miR-3195 and miR-328-5p may be utilized to distinguish mild and moderate TBI from severe TBI.2.GO enrichment analysis showed that there were 10 common biological processes in mild and moderate TBI groups.Only one biological process named vasopressin regulating systemic arterial pressure appeared in mild,moderate and severe TBI groups.3.Several important pathways are involved in the process of TBI injury,including p53 signaling pathway and mTOR signaling pathway.4.The results of GO enrichment analysis of differentially expressed proteins showed that the differentially expressed proteins after TBI injury were more correlated with acute phase reaction,acute inflammation reaction,injury reaction,stress reaction,platelet activation,injury repair,coagulation and other biological processes.5.The pathway enrichment analysis of differentially expressed proteins showed that the three groups of TBI involved signal transduction pathways including Hippo signal transduction pathway,complement coagulation cascade reaction and Salmonella infection.6.Differential microRNA and protein enrichment analysis showed that the pathological process of mild TBI was closer to that of moderate TBI,while that of severe TBI was relatively independent.7.Complement and coagulation cascade pathways were activated in TBI.The expression of key proteins C4,C4 b and C5 in severe TBI group was significantly higher than that in mild and moderate TBI group,suggesting that they were more activated in severe TBI.C4,C4 b and C5 can be used as potential diagnostic markers of TBI.