MicroRNA-210-3p inhibits inflammatory pain in rats by regulation ten-eleven translocation 2 expression
Objective To investigate the roles and mutual regulatory mechanisms of microRNA-210-3p(miR-210-3p)and ten-eleven translocation 2(TET2)in a rat model of inflammatory pain induced by complete Freund's adjuvant(CFA).Methods Bioinformatics and dual-luciferase reporter assays were used to analyse and identify target genes regulated by miR-210-3p in rats.The combinations of plasmid and miR-210-3p cotransfection in the experiments were grouped as follows:pmirGLO+mimics-NC group,pmirGLO+mimics-miR-210-3p group,TET2-WT-pmirGLO+mim-ics-NC group,TET2-WT-pmirGLO+mimics-miR-210-3p group,TET2-MT-pmirGLO+mimics-NC group and TET2-MT-pmirGLO+mimics-miR-210-3p group;60 rats were divided into 4 groups according to the randomised numerical table method:Normal control(CON)group(n=20),Complete Freund's adjuvant(CFA)group(n=20),Complete Freund's adjuvant+adeno-associated virus vector negative control(CFA+AAV NC)group(n=10),Complete Fre-und's adjuvant+adeno-associated virus miR-210-3p inhibitor(CFA+AAVi)group(n=10).The rat inflammatory pain model was established by subcutaneous injection of CFA into the underside of the left hind paw;the intervention model was established by tail vein injection of AAV with miR-210-3p inhibitor;the behaviour of the rats was observed and measured;the expression of miR-210-3p was detected by RT-qPCR;Western blotting and immunofluorescence stai-ning were used to detect changes in the expression level and fluorescence intensity of TET2 protein in the spinal cord of lumbar extension segments from L4 to L6;and immunofluorescence staining was used to observe the cellular expression localisation of TET2 protein in the rat spinal cord.Results Dual-luciferase assays confirmed a negative regulatory rela-tionship between miR-210-3p and TET2,as evidenced by a binding site.CFA injection significantly decreased the me-chanical paw withdrawal mechanical threshold(PWMT)and the thermal paw thermal withdrawal latency(PTWL)(P<0.05).An increase in miR-210-3p and a concomitant decrease in TET2 protein expression were observed in the CFA group(P<0.05).Immunofluorescence showed that TET2 protein mainly colocalised with neuronal cells and its ex-pression in the spinal cord was decreased in the CFA group(P<0.05).After AAVi treatment,PWMT and PTWL were significantly higher in the CFA+AAVi group than in the CFA+AAV NC group(P<0.05),with increased TET2 protein levels(P<0.05).Conclusion miR-210-3p downregulates TET2 protein expression;its inhibition in rats with inflam-matory pain significantly alleviates pain symptoms.
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