miR-135a-5p in alleviating neuropathic pain in rats by targeted downregulation of FTO protein
Objective To observe the expression of FTO protein in dorsal root ganglion(DRG)after miR-135a-5p mimic treatment in rats with neuropathic pain,and to investigate the targeting relationship between miR-135a-5p and FTO.Methods Sixty male SD rats,weighing 200-240 g,were randomly divided into sham-operation group(no L5 nerve root ligation+pump with 10 μL of normal saline at L5 DRG),model group[spinal nerve ligation(SNL)+pump with 10 μL of normal saline at L5 DRG],low-dose group(SNL+pump with 10 μL of 10 μmol/L miR-135a-5p mimic at L5 DRG)and high-dose group(SNL+pump with 10 μL of 20 μmol/L miR-135a-5p mimic at L5 DRG),with 15 rats in each group.Five rats in each group were taken to measure paw withdrawal threshold(PWT)and paw withdrawal latency(PWL)by day 0,1,7,14 and 21 after operation.Ten rats were sacrificed by day 14 after operation to obtain DRG.The relative expressions of miR-135a-5p and FTO mRNA were detected by real-time fluorescence quantitative PCR,and the relative expression of FTO protein was detected by Western blot.The adrenal pheochromocytoma PC12 cells were harvested,and dual luciferase reporter gene experiment was used to verify the targeting relationship between miR-135a-5p and FTO.Results(1)There were significant differences in the PWT and PWL values by day 1,7,14 and 21 after operation among sham-operation group[PWT:(23.76±1.79),(23.52±1.49),(23.10±1.74),(23.18±1.57)g;PWL:(11.84±0.40),(11.78±0.47),(12.04±0.58),(12.04±0.58)s],model group[PWT:(8.56±1.90),(8.44±1.45),(7.78±0.80),(7.20±0.99)g:PWL:(7.76±0.64),(5.78±0.43),(6.02±0.24),(4.26±0.83)s],low-dose group[PWT:(13.16±0.52),(11.42±0.68),(10.10±0.69),(9.42±0.66)g;PWL:(8.90±0.75),(8.20±0.57),(7.18±0.61),(5.36±0.50)s],and high-dose group[PWT:(17.02±1.05),(14.58±0.62),(12.34±0.40),(11.58±0.67)g;PWL:(9.84±0.87),(9.02±0.94),(8.46±0.32),(6.12±1.33)s](F=101.100,164.500,212.200,235.600,all P values＜0.05;F=31.500,75.530,69.850,79.980,all P values＜0.05).The PWT and PWL values at all time points after operation increased sequentially in model group,low-dose group,high-dose group and sham-operation group(P＜0.05).(2)There were significant differences in the relative expression of miR-135a-5p(1.03±0.13,0.50±0.09,3.82±0.30,5.17±1.34),FTO mRNA(1.01±0.10,4.13±1.20,3.64±0.96,2.68±0.78)and FTO protein(100.00±6.77,228.80±78.26,198.10±28.38,178.50±38.47)by day 14 after operation among sham-operation group,model group,low-dose group and high-dose group(F=52.150,12.710,7.140;all P values＜0.05).The relative expression of miR-135a-5p was lower in model group,low-dose group and high-dose group than that in sham-operation group(P＜0.05),was higher in low-and high-dose groups than that in model group(P＜0.05),and showed no significant difference between high-and low-dose groups(P＞0.05).The relative expressions of FTO mRNA and protein were higher in model group,low-dose group and high-dose group than those in sham-operation group(P＜0.05),were lower in low-and high-dose groups than those in model group(P＜0.05),and showed no significant differences between high-and low-dose groups(P＞0.05).(3)The luciferase activity was lower in miR-135a-5p mimic+FTO-WT group(0.39±0.21)than that in miR-135a-5p scramble+FTO-WT group(1.00±0.24)(t=0.230,P=0.002),and showed no significant difference between miR-135a-5p mimic+FTO-Mut group(0.96±0.12)and miR-135a-5p scramble+FTO-Mut group(1.02±0.17)(t=0.650,P=0.530).miR-135a-5p targeted FTO.Conclusion miR-135a-5p can alliviate neuropathic pain by inhibiting the expression of FTO in DRG of rats.
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