| Objective:Bioinformatics softwares imply that miR-9a-5p may interact with Src tyrosine protein kinase as well as NMDA receptors(N-methyl-D-aspartic acid receptor,NMDAR)including GluN1,GluN2A and GluN2B.By investigation of the changes of Src,GluN1,GluN2A and GluN2B in the hypothalamic arcuate nucleus(ARC)of rats in inflammatory pain,we may clarify the role and mechanism of miR-9a-5p and hope to find out new targets for the clinical treatment of inflammatory pain.Methods:(1)Bioinformatics softwares show that the miR-9a-5p has the potential binding sites with Src,GluN1,GluN2A and GluN2B.(2)Inflammatory pain model was established by injection with 0.1 ml of Complete Freund’s adjuvant(CFA)in the right hind paws of rats.Normal saline(NS)and naive group were used as controls.(3)Von Frey tests and thermal radiation methods were respectively applied to detect the mechanical withdraw threshold(MWT)and thermal withdraw latency(TWL);(4)On the 1st,3rd,5th,7th and 14th day after CFA injection,quantitative real-time PCR(qPCR)was applied to detect the expression of Src,GluN1,GluN2A,GluN2B mRNA and miR-9a-5p in ARC.Then,the correlations between miR-9a-5p and Src,GluN1,GluN2A,GluN2B mRNA were analyzed;(5)Western blot analysis was used to detect the protein expression;(6)The long gene fragments containing the miR-9a-5p target site or complementary target site were synthesized and respectively constructed into plasmids called PGL3.The dual luciferase reporter gene assay was used to evaluated the kind/kinds of gene that miR-9a-5p regulates;(7)Immunostainging was applied to detect the GluN2A protein in ARC;(8)The miR-9a-5p agomir or miR-9a-5p antagomir was microinjected to change the content of miR-9a-5p in rats’ ARC,and then corresponding behavioral changes,mRNA and protein expression of GluN2A were detected.Results:After CFA injection,the mechanical withdraw threshold(MWT:0.47±0.13 g/1st day,0.77 ± 0.40 g/3rd day,1.61± 0.81 g/5th day,2.77± 0.75 g/7th day and 6.48± 1.03 g/14th day)and thermal withdraw latency(TWL:3.88± 0.46 s/1st day,6.38±1.03 s/3rd day,8.14±0.73 s/5th day,9.79 ± 1.44 s/7th day)of rats were significantly reduced compared with the control groups(P<0.001).On the 14th day,the TWL of CFA inflammatory rats returned to normal.(2)The mRNA expression of Src(2.04 ± 0.29),GluN2A(1.64 ± 0.26)and GluN2B(1.40± 0.24)in the ARC were significantly increased on the 5th day after CFA injection(P<0.001 or P<0.01).Contrastingly,the expression level of GluN1 mRNA(1.55± 0.38)was significantly increased only on the 1st day(P<0.01).Peripheral inflammation induced the significant increase in Src(2.31±0.70),GluN1(2.37 ± 0.59),GluN2A(2.38±1.04)and GluN2B(1.97±0.26)protein expression in ARC on the 5th day after CFA injection.(3)Bioinformatic analysis softwares indicate that miR-9a-5p has potential binding sites with Src,GluN1,GluN2A,GluN2B mRNA.(4)By applying qPCR,we found that the miR-9a-5p in the ARC of CFA inflammatory rats was significantly decreased on the 3rd day(0.75±0.11),5th day(0.51 ± 0.22),7th day(0.71 ± 0.09)and 14th day(0.79 ± 0.13)day.(5)Correlation analysis revealed that the miR-9a-5p had significant correlations with Src,GluN2A and GluN2B mRNA.(6)Dual luciferase reporter gene analysis figured out that miR-9a-5p directly target GluN2A 3’-UTR instead of Src and GluN2B;(7)The GluN2A in ARC mostly expressed in neurons and the nucleus of microglia rather than in astrocytes;(8)Microinjection of miR-9a-5p agomir into the rats1 ARC relieved mechanical pain in CFA-induced inflammatory pain(MWT were increased from 2.38 ± 1.95 g to 5.37 ± 2.37 g after the first injection and also increased to 7.16±2.43 g after the second injection),but had no significant effect on TWL.In addition,miR-9a-5p agomir significantly decreased the expression of GluN2A mRNA(0.76±0.19)and protein(0.78±0.33).(9)On the contrary,microinjection of miR-9a-5p antagomir into ARC of NS rats reduced MWT(from 12.84 ± 2.36 g to 7.07±1.48 g after the first injection and also reduce to 5.31± 1.31 g after the second injection),and TWL didn’t change a lot either.Besides,miR-9a-5p antagomir resulted in a significant increase in both GluN2A mRNA(1.41 ± 0.20)and protein(1.36 ± 0.26)expression.Conclusion:The miR-9a-5p of ARC can exert analgesic effect by targeting GluN2A in inflammatory pain,suggesting that miR-9a-5p has potential to be used as a new target for analgesia. |
