坐骨神经预损伤后miR-124-3p调节GAP-43表达并促进轴突生长的实验研究

After sciatic nerve conditioning injury miR-124-3p regulates GAP-43 expression in dorsal root ganglion neurons and further promoting neurite growth via STAT3

目的 通过研究坐骨神经预损伤后背根神经节中microRNA表达谱变化,探讨坐骨神经预损伤促进脊髓后索损伤修复机制.方法 利用微阵列芯片技术和生物信息学方法,研究与坐骨神经预损伤促进脊髓后索损伤修复有关的microRNA,并用RT-qPCR技术、Western blot技术、免疫荧光染色、反义miRNA寡核苷酸抑制剂等技术验证.结果 单纯脊髓后索损伤组miR-124-3p在大鼠脊髓后索损伤后7d、14d明显上调;坐骨神经预损伤组,脊髓后索损伤后7d、14d背根神经节中miR-124-3p明显下调.与正常背根神经节神经元相比,抑制miR-124-3p后GAP-43表达增加,神经元轴突延长.与对照组相比,各组各时间窗STAT3 mRNA表达差异无统计学意义,坐骨神经预损伤组STAT3蛋白在脊髓后索损伤后7d和14d表达上调而单纯脊髓后索损伤组脊髓后索损伤后7d和14 d STAT3蛋白表达下调.与正常背根神经节神经元相比抑制miR-124-3p的神经元STAT3免疫荧光增强、轴突延长,p-STAT3、GAP-43蛋白表达明显上调.与正常背根神经节神经元相比AG490＋AMO-124共同处理的神经元轴突长度无明显差异,STAT3表达明显上调,而p-STAT3和GAP-43表达无明显差异.结论 背根神经节神经元中miR-124-3p 下调通过STAT3蛋白的上调促使GAP-43蛋白表达增加是坐骨神经预损伤促进脊髓后索损伤修复的机制之一.

Objective To study the effect of sciatic nerve conditioning injury on repairment of dorsal column lesion via investigating the alteration of miRNomes.Methods To study the microRNAs which are associated with dorsal column lesion whose repairment is promoted by sciatic nerve conditioning injury,the microRNA profiles of dorsal root ganglion in sciatic nerve conditioning injury group and simple dorsal column lesion group were investigated by Microarray and bioinformatics.And then RT-qPCR,western blot,immunofluorescence staining and antisense oligonucleotide （AMO-124） were applied to validate the result of microarray.Results miR-124-3p was significantly upregulated on 7 d and 14 d post dorsal column lesion in simple dorsal column lesion group,whereas it was downregulated on 7 d and 14 d post dorsal column lesion in sciatic nerve conditioning injury.Compared with normal dorsal root ganglion （DRG） neurons,expression was increased and neurite was prolonged in miR-124-3p inhibited neurons.Compared with control group,there was no statistical difference about the expression of signal transducer and activator of transcription 3 （STAT3） mRNA among each check point of every group.On 7 d and 14 d post dorsal column lesion,STAT3 protein expression was significantly upregulated in sciatic nerve conditioning injury group,whereas it was significantly downregulated in simple dorsal column lesion group.Compared with normal DRG neurons,immunofluorescence of STAT3 was enhanced,neurite was prolonged and the expression of p-STAT3 protein and protein was upregulated in miR-124-3p inhibited neurons.Compared with normal DRG neurons,in AG490＋AMO-124 co-inhibited neurons,the length of neurite and the expression of p-STAT3 protein and protein have no statistical difference whereas the expression of STAT3 was significantly upregulated.Conclusion It is one of the mechanisms of sciatic nerve conditioning injury promoting dorsal column lesion that the downregulation of miR-124-3p in DRG neurons increases the expression of GAP-43 protein via upregulating STAT3 protein.