Effect of alteration of miRNAomes in rat dorsal root ganglia after sciatic nerve conditioning injury on the repairment of dorsal column lesion

English Keywords:【Key words】 Sciatic nerve conditioning injury  Dorsal column lesion  miRNA  Dusp4  p38 protein  Rat

English Abstract:

  【Abstract】 Objectives: To study the effect of alteration of miRNomes after sciatic nerve conditioning injury on the repairment of dorsal column lesion. Methods: Thirty-nine female Wistar rats were divided randomly into four groups, group A, B, C and D. Group A(n=12) underwent dorsal column lesion on the 10th thoracic vertebra at the 7th day after sciatic nerve conditioning injury. Group B(n=12) underwent only dorsal column lesion. Group C(n=12) underwent only sciatic nerve injury. Group D(n=3) was used as blank control. The dorsal root ganglias of group A and B were excised for total RNA isolation and western blot at 4 hours, 3 days, 7 days and 14 days after dorsal column lesion. The spinal cord tissues of lesion sited in group A and B were harvested at 14 days after dorsal column lesion for immunohistochemistry of neurofilament-200(NF-200) and hematoxylin-eosin staining. The dorsal root ganglias of group C were harvested at same time points for total RNA isolation and Western blot. The dorsal root ganglias of group D were harvested for total RNA isolation and Western blot. To study its mechanism, the miRNA profiles of dorsal root ganglias in group A and B were investigated by Microarray and bioinformatics. The significantly changed miRNA miR-199a-5p whose target was Dusp4 was screened out. RT-qPCR was used to detect the expression of miR-199a-5p in each group and the expression of Dusp4 mRNA in group A, B and D. Western blot was applied to test the expression of Dusp4 protein in each group and the expression of p38 protein and p-p38 protein in group A and D. Immunohistochemistry of NF-200 and hematoxylin-eosin staining were used to investigate the repairment of dorsal column lesion. Results: Microarry revealed that miR-199a-5p downregulated at each time point of group A and upregulated at 4h of group B, however the expression of miR-199a-5p in group B did not alter significantly at 3d, 7d, and 14d after dorsal column lesion compared with that of group D. RT-qPCR showed that miR-199a-5p downregulated at each time point of group A(P＜0.05) and upregulated at 4h of group B(P＜0.05), while the expression of miR-199a-5p at 3d, 7d and 14d of group B and the expression at each time point of group C showed no statistical difference compared with that of group D. The Dusp4 mRNA expressions of group A and B showed no statistical difference at each time point compared with that of group D. Statistical difference was noted for Dusp4 protein at each time point of group A with group D(P＜0.05). Compared with group D, the Dusp4 protein downregulated significantly at 4h of group B(P＜0.05) and then raised to the level of group D at 3d, 7d and 14d. Compared with group D, there was no statistical difference regarding to the expression of Dusp4 protein at each time point of group C. p38 protein and phosphorylated p38 protein level was consistent with the level of miR-199a-5p in group A. At 14 days, compared with group B, the expression level of neurofilament protein increased significantly and the shape of nerve fiber bundle was more regular in caudal lesion site of group A. Conclusions: The downregulation of the expression of miR-199a-5p after sciatic nerve conditioning injury can promote the repairment of dorsal column lesion.