microRNA-210-3p通过调控TET2的表达抑制大鼠炎性疼痛

MicroRNA-210-3p inhibits inflammatory pain in rats by regulation ten-eleven translocation 2 expression

目的探讨microRNA-210-3p(miR-210-3p)与10-11易位蛋白2(ten-eleven translocation 2,TET2)在完全弗氏佐剂(complete freund's adjuvant,CFA)诱导的大鼠炎性疼痛模型中的作用及其相互调控机制。方法通过生物信息学方法和双荧光素酶实验,分析并确定大鼠miR-210-3p中可以靶向调节的基因。实验中的质粒和miR-210-3p共转染组合分为pmirGLO+mimics NC组、pmirGLO+mimics-miR-210-3p组、TET2-WT-pmirGLO+mimics-NC组、TET2-WT-pmirGLO+mimics-miR-210-3p组、TET2-MT-pmirGLO+mimics-NC组和TET2-MT-pmirGLO+mimics-miR-210-3p组;60只大鼠按随机数字表法分为正常对照(normal control,CON)组(n=20)、CFA组(n=20)、CFA+腺相关病毒载体阴性对照(adeno-associated virus negative control,AAV NC)组(n=10)、CFA+AAV miR-210-3p抑制剂(adeno-associated virus miR-210-3p inhibitor,AAVi)组(n=10)。通过在大鼠左后足底部皮下注入CFA的方式建立大鼠炎性疼痛模型;通过尾静脉注入miR-210-3p inhibitor的AAV建立干预模型;观察并测量大鼠行为学;采用RT-qPCR法检测miR-210-3p的表达量;采用Western blotting法和免疫荧光染色法检测L4~L6腰膨大节段脊髓中TET2蛋白的表达水平及荧光强度的变化;采用免疫荧光染色法观察TET2蛋白在大鼠脊髓中的细胞表达定位。结果生物信息学方法发现,TET2基因3'UTR区域存在与miR-210-3p的结合位点;双荧光素酶报告基因实验证实了miR-210-3p与TET2基因之间存在结合位点,呈负向调控关系;注射CFA显著减小了大鼠的机械缩足反射阈值(paw withdrawal mechanical threshold,PWMT)和热缩足潜伏期(paw thermal withdrawal latency,PTWL)(P<0.05);CFA组大鼠脊髓腰膨大中miR-210-3p的表达水平明显上调,伴随着TET2的表达水平降低(P<0.05);免疫荧光结果显示,TET2蛋白主要和神经元细胞存在共定位:CFA组大鼠脊髓内TET2蛋白表达水平降低(P<0.05);经过AAVi干预后,CFA+AAVi组大鼠在各个时间的PWMT和PTWL较CFA+AAV NC组大鼠升高(P<0.05);CFA+AAVi组大鼠脊髓组织中TET2蛋白表达较CFA+AAV NC组升高(P<0.05)。结论miR-210-3p可以抑制TET2蛋白表达,通过抑制miR-210-3p在炎性疼痛大鼠中的表达可以有效减轻炎性疼痛。

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+mimics-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 Freund'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 staining 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 relationship between miR-210-3p and TET2,as evidenced by a binding site.CFA injection significantly decreased the mechanical 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 expression 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 inflammatory pain significantly alleviates pain symptoms.