Effect of miR-124 on neuropathic pain induced by paclitaxel via regulating early growth response1 EGR1
Objective To understand the effect of the expression of microRNA-124(miR-124)on paclitaxel-induced neuropathic pain(NPP)and the regulating mechanism on early growth response 1(EGR1).Methods The P12 cells were exposed to paclitaxel at the doses of 0 μmol/L,0.5 μmol/L,1 μmol/L and 2 μmol/L respectively,for 48 h.Injury model of P12 cells induced by paclitaxel was established real-time quantitative polymerase chain reaction(qRT-PCR)and double luciferase test were used to detect the expression of mRNA of miR-124 and the mRNA expression of EGR1 and the relationship between them were detected though PCR test,double luciferase test and protein co-immunoprecipitation test.Totally 50 SD male rats aged 8 to 10 weeks were intraperitoneally injected 2 mg/kg of paclitaxel on day 1,day 3,day 5 and day 7,respectively,to establish the NPP models.The rats were divided into sham operation group(Sham),model group(Model),agomiR-124-NC+pcDNA3.1-EGR1-NC group(Control),agonist group(agomiR-124 group)and over expression group(pcDNA3.1-EGR1),with 10 rats in each group.The rats in the sham operation group were intraperitoneally injected with equal dose of normal saline during the modeling process.After successful modeling,the rats in the control group were injected with agomiR-124-NC+pcDNA3.1-EGR1-NC through tail vein.the rats in agonist group were injected with agomiR-124.The rats in the over expression group were injected with agomiR-124+pcDNA3.1-EGR1 through the tail vein once a day for 14 days..The mechanical withdrawal threshold(MWT)and thermal withdrawal latency(TWL)were detected.TdT-mediated dUTPnick end labelling(TUNEL)staining was used to detect the apoptosis rate of neurons in spinal cord tissue.enzyme Enzyme linked immunosorbent assay was used to detect the levels of interleukin-1 β(IL-1β),IL-6,IL-4 and IL-10 in serum of rats in each group.Immunofluorescence was used to detect the phenotypic transition of microglia in spinal cord tissue.Immunohistochemistry was used to detect the expression of B cell lymphoma/leukemia-2(Bcl-2)and Bcl-2-associated X protein(Bax)in spinal cord tissues,and Western blot was used to detect the expression of EGR1 in spinal cord tissues of each group.Results In the P12 cell injury model induced by paclitaxel,the expression of miR-124 was significantly decreased(P＜0.05)and the mRNA expression of EGR1 was significantly increased(P＜0.05).miR-124 targeted to regulate the expression of EGR1.In animal experiment,compared with sham group,the levels of MWT and TWL in model group,control group,agonistagomiR-124 group and over expressionpcDNA3.1-EGR1 group,the proportion of M2 microglia in spinal cord tissue and the expression of Bcl-2 in spinal cord tissue were significantly decreased(P＜0.05),while the apoptosis rate,serum the levels of IL-1β,IL-6,IL-4 and IL-10 in serum,the proportion of M1 microglia in spinal cord tissue and the expression of Bax and EGR1 in spinal cord tissue were significantly increased(P＜0.05).Compared with Model group,the levels of MWT and TWL in agonistagomiR-124 group and overexpressionpcDNA3.1-EGRl group,the levels of IL-4 and IL-10 in serum,the proportion of M2 microglia in spinal cord tissue and the expression of Bcl-2 in spinal cord tissue were significantly increased(P＜0.05),while the apoptosis rate,the level of IL-1 β and IL-6 in serum,the proportion of M1 microglia in spinal cord tissue and the expression of Bax and EGR1 in spinal cord tissue were significantly decreased(P＜0.05).Compared with agonistagomiR-124 group,the levels of MWT and TWL in overexpressionpcDNA3.1-EGR1 group,the level of IL-4 and IL-10 in serum,the proportion of M2 microglia in spinal cord and the expression of Bcl-2 in spinal cord were significantly decreased(P＜0.05).While the apoptosis rate,the level of IL-1β and IL-6 in serum,the proportion of M1 microglia in spinal cord and the expression of Bax and EGR1 in spinal cord were significantly increased(P＜0.05).the difference was statistically significant(all P＜0.05).The difference of related parameters between model group and control group was not statistically significant(P＞0.05).Conclusion In the model of neuropathic pain induced by paclitaxel,the expression of miR-124 significantly decreased,the mRNA expression of EGR1 significantly increased,and miR-124 targeted the expression of EGR1.Over expression of miR-124 can significantly promote the conversion of microglia to M2 in spinal cord tissue,reduce the apoptosis rate of neurons,reduce the pathological injury of spinal cord tissue and relieve the pain.
MicroRNA-124Early growth response 1PaclitaxelNeuropathic painPhenotypic transformation of microglia
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