MiR-124 Involves In Neuropathic Pain By Regulating The Expression Of EGR1 In The Dorsal Root Ganglion And Spinal Cord Of Rats

Background:Neuropathic pain is a pain caused by nervous system injury or disease.Its characteristic is that the pain after primary injury healing is still strong,stubborn and persistent,which causes great burden to society and great pain to patients physically and mentally.Although some mechanisms have been recognized,there are still many problems to be solved in the practical application of clinical treatment,and further research needs to be carried out.Early growth response 1(EGR1)regulates the transcriptional activation of many target genes in vivo,and is also regulated and controlled by other transcription factors.Previous studies have reported that epigenetic factor miR-124 may have a negative regulatory effect on the expression of EGR1 protein.Recent studies have shown that EGR1 is involved in learning and memory-related long-term potentiation and synaptic plasticity,and EGR1 has also been shown to play an important role in inflammatory pain,post-operative pain and neuropathic pain.However,whether miR-124 is involved in the regulation of EGR1 expression in neuropathic pain after lumbar 5 spinal nerve ligation(L5 SNL)in rats has not been reported.ObjectiveIn this study,we used L5 SNL model,combined with pain behavior experiments and molecular biology techniques.First,we observed the effects of L5 SNL on the expression of EGR1 and miR-124 in dorsal root ganglion and spinal cord of rats,and proved the role of EGR1 in neuropathic pain.To verify the relationship between miR-124 and EGR1,and then observe the regulation of miR-124 on EGR1 induced by L5 SNL by intrathecal injection of microRNA mimics.The results will provide a new target for the treatment of neuropathic pain from the epigenetic direction.Methods and Results 1 Changes of expression of miR-124 and EGR1 in DRG and spinal cord of rats after L5 SNLThe male SD rats were randomly divided into experimental group(L5 SNL group)and control group(sham group).The mechanical withdrawal threshold(PWT)and thermal withdrawal latency(PWL)were measured 3,1 day before operation and 1,3,5,7,10,14 days after operation.It was found that,consistent with the literature,the PWT and PWL of the hind paw of the rats began to decrease significantly,the lowest on the 7th day and lasted until the 14 th day after operation.The result indiated that the model was successfully established.The expression of EGR1 was detected by Western blot,RT-PCR and q-PCR in L4-5 DRG and L4-5 spinal cord of rats at 0,1,3,7 and 14 days after operation.The expression of miR-124 was detected by q-PCR.Western blot results showed that the expression of DRG and EGR1 in spinal cord increased after L5 SNL,and reached the highest level on day 3.The results of RT-PCR and q-PCR showed that the expression of DRG and EGR1 in spinal cord increased after L5 SNL and reached the highest level on day 3.q-PCR results showed that the expression of miR-124 in DRG and spinal cord decreased after L5 SNL,and was the lowest on day 3.These results suggest that L5 SNL induces up-regulation of EGR1 protein and mRNA expression and down-regulation of miR-124 in L4-5 DRG and L4-5 spinal cord of rats.2 Cell types of EGR1 expression in DRG and spinal dorsal horn after L5 SNL in ratsL4-5 DRG and spinal cord of L5 SNL and sham rats were stained and photographed by immunofluorescence histochemistry.The results showed that L5 SNL could increase the expression of EGR1 in DRG and spinal dorsal horn.Immunofluorescence double staining technique was used to observe the cell types of EGR1 expression in DRG and spinal cord.The results showed that EGR1 was mainly distributed in myelinated A fibers and unmyelinated C fibers in DRG,astrocytes and neurons and astrocytes in spinal cord.3 Validation of Target Relationship between miR-124 and EGR1Bioinformatics technology and Target Scan Human 7.2 were used to predict the existence of target relationship between miR-124 and EGR1.PC12 cells were cultured in vitro and double luciferase assay experiment was used to verify whether miR-124 binds to EGR1 and negatively regulates the expression of EGR1.Different doses of miR-124 agomir(10 pM,50 pM,100 pM)and wild type(pmirGLO EGR1 3’UTR)were co-transfected into PC12 cells.Compared with scramble transfection,the results showed that the luciferase activity of miR-124 agomir at 50 pM was significantly decreased,indicating that there was a target relationship between miR-124 and EGR1.Wild-type(pmirGLO EGR1 3’UTR)vector co-transfected with miR-124 agomir N.C and miR-124 antagomir into PC12 cells respectively.Mutant double luciferase reporter gene vector(pmirGLO MUT EGR1 3’UTR)co-transfected with miR-124 agomir,miR-124 agomir N.C and miR-124 antagomir,respectively,showed no significant change in luciferase activity,indicating that miR-124 agomir had sequence specificity.These results fully demonstrate that EGR1 is the target gene of miR-124.4 Intrathecal injection of miR-124 agomir could partly relieve neuropathic pain caused by L5 SNL,and also down-regulate the expression of EGR1.MiR-124 agomir(20uM,10ul)was injected intrathecally immediately after L5 SNL operation in rats for 4 consecutive days.PWT and PWL were detected.Compared with L5 SNL + veh rats,the mechanical withdrawal threshold and thermal withdrawal latency of hindpaw in L5 SNL + agomir group increased on the 2nd day and lasted until the 4th day,partially alleviating neuropathic pain.There was no significant change in contralateral values.Western Blot results showed that compared with SNL + veh group,the expression of EGR1 protein in DRG and spinal dorsal horn decreased in SNL + agomir group.5 Painful behavior and up-regulation of EGR1 expression in normal rats after intrathecal injection of miR-124 antagomirIntrathecal injection miR-124 antagomir(20uM,10ul)of normal rats was progressed for 4 consecutive days.PWL and P WT were detected at different time points.Compared with naive rats,the PWL and PWT in the naive + antagomir group decreased significantly on the 1st day after intrathecal catheterization,and lasted until the 4th day after operation.Western Blot results showed that compared with naive group,the level of EGR1 protein in naive + antagomir group increased in DRG and spinal dorsal horn.6 Mitigation of neuropathic pain in L5 SNL rats by intrathecal injection of specific EGR1 decoy AYX1To further verify the role of EGR1 in neuropathic pain,we injected EGR1-specific decoy AYX1(200 nmol,15ul)immediately after L5 SNL operation in rats.The latency of PWL and PWT were measured at different time points after operation.Compared with the SNL+veh group,the PWL and PWT in SNL+AYX1 group were increased to some extent at different time points after operation,which partially alleviated the neuropathic pain in rats.ConclusionMiR-124 is required for the expression of EGR1 in the dorsal root ganglion and spinal cord in the development of neuropathic pain following L5 SNL.