The Role And Mechanism Of MiR-124-3p In Inhibiting Neuronal Apoptosis After Injury By Targeting SPTLC2

Objective:Traumatic brain injury(TBI)is commonly seen in multiple types of trauma,associates with high incidence,disability and mortality.The current fundamental researches on the prognosis of TBI aim at secondary brain injuries(nerve apoptosis,autophagy,necrosis)due to inflammation responses in TBI,endogenous nerve regeneration(neural stem cell proliferation,differentiation,migration),and the process of neuronal axon regeneration and targeting.In this study,the mouse TBI model and mouse cortical primary neuron scratch model were taken as the basic research objects.Expression changes of micro RNA-124-3p(miR-124-3p),serine palmitoyltransferase long chain base subunit 2(SPTLC2)and key proteins of toll like receptor 4(TLR4)signaling pathway after injury were analyzed.The relationships among miR-124-3p,SPTLC2,TLR4 signaling pathway and neuronal apoptosis were verified by in vivo and in vitro experiments in order to investigate the possible molecular mechanism of miR-124-3p in regulating neuronal apoptosis through SPTLC2 and TLR4 signaling pathway.MethodsPrecision cortical impactor-3000(PCI-3000)was used to establish TBI model of mouse in vivo.The primary neurons in mouse cortex were isolated and cultured in vitro,then a scratch injury was made to simulate the model of neuronal damage.In vivo experimental research,lateral ventricle injection of miR-124-3p agomir was used to intervene on the miR-124-3p expression level in mouse brain.Real-time quantitative polymerase chain reaction(RT-q PCR)and western blot were used to detect the relative expression changes of miR-124-3p,SPTLC2,apoptosis-related proteins and key proteins of TLR4 signaling pathway to analyze the potential relationship of expression changes between miR-124-3p and SPTLC2,as well as their potential relationship with TLR4 signaling pathway and neuronal cell apoptosis.Immunofluorescence staining was adopted to study the changes of apoptotic cells after various interventions,Neurological severity score(NSS)and water maze experiment were taken to explore the effects of the above interventions on motor,spatial location learning and memory ability of mice.In vitro experimental study,transfection,scratch model,RT-q PCR,western blot,lactate dehydrogenase(LDH)activity detection and TUNEL staining were applied to observe the effects of miR-124-3p and SPTLC2 on the apoptosis of primary cortical neurons in mice.Transfection,bioinformatics prediction,luciferase report gene system,rescue experiment,immune coprecipitation and western blot methods were used to verify and clarify the relationships among the miR-124-3p,SPTLC2 and TLR4 signaling pathways.Results:In this study,a relatively stable C57BL/6 mouse TBI model was established.Cortical primary neurons were extracted and purified,and a neuron mechanical scratch model was well set up.In vivo study,it was found that: 1)The natural expression of miR-124-3p was significantly increased in the hippocampus of mice after TBI;2)After the injection of miR-124-3p agomir into the lateral ventricle,the expression level of miR-124-3p in the brain tissue of mice could be interfered and increased significantly;3)The expression level of miR-124-3p was negatively correlated with SPTLC2 and key proteins of TLR4 signaling pathways;4)High expression of miR-124-3p could significantly reduce the proportion of nerve cell apoptosis caused by TBI,suggesting a neuroprotective effect;5)The degree of apoptosis was positively correlated with the expression level of SPTLC2;6)The high expression of miR-124-3p did not significantly improve the short term motor ability of mice after TBI,but it could improve the spatial memory and orientation ability.In vitro study,it was observed that: 1)The natural expression of miR-124-3p in neurons decreased significantly after mechanical injury;2)Transfection with miR-124-3p mimic and SPTLC2 plasmid could change the expression levels of miR-124-3p and SPTLC2 in neurons respectively;3)Transfection of SPTLC2 plasmid could increase the expression of SPTLC2 protein but did not affect the expression level of miR-124-3p;4)In vitro,expression levels of miR-124-3p and SPTLC2,key proteins of TLR4 signaling pathway showed a negative correlation,this finding was consistent with experimental result in vivo;5)High expression of SPTLC2 promoted apoptosis,however,the high expression of miR-124-3p inhibited the pro-apoptosis effect of SPTLC2;6)SPTLC2 m RNA was the direct target gene of miR-124-3p,which could inhibit the expression of SPTLC2 protein;7)SPTLC2 could interact with myeloid differentiation factor 88(MYD88)protein;8)miR-124-3p could affect the TLR4 signaling pathway by down-regulating the expression of SPTLC2 protein,inhibit the occurrence of post-traumatic nerve cell apoptosis,and played a neuroprotective role.Conclusion:Taken together,these results suggest that high expression of SPTLC2 has the effect of promoting apoptosis after neuronal injury,and it is a new target gene of miR-124-3p for inhibiting neuronal apoptosis.After neuronal injury,miR-124-3p plays a protective role by reducing the expression of SPTLC2 and inhibiting neuron apoptosis through the TLR4 signaling pathway.SPTLC2 as an enzyme has been known by many scholars,but as suggested in this study,it can directly or indirectly interact with MYD88 protein,thus affect apoptosis through TLR4 signaling pathway.It indicates that SPTLC2 might not only work as a key enzyme of sphingomyelin synthesis but also as a messenger in the cell,which still needs further experimental evidence.This study provides a new neurobiological basis for exploring the relevant mechanism of neuronal protection after TBI,and also puts forward some new hypotheses,provides a new research idea for improving the prognosis of TBI.