微小RNA-137-3p通过靶向下调赖氨酸特异性去甲基化酶4A基因缓解大鼠神经病理性痛

MicroRNA-137-3p alleviates neuropathic pain in rats by targeting downregulation of lysine specific demethylase 4A

目的观察微小RNA(miR)-137-3p对赖氨酸特异性去甲基化酶4A基因(KDM4A)的调控在坐骨神经慢性压迫损伤(CCI)引起的神经病理性痛中的作用。方法将大鼠随机分为假手术组(Sham)、CCI模型组、2 mg/kg miR-137-3p模拟物(mimic)鞘内注射组、4 mg/kg miR-137-3p mimic鞘内注射组。术后的第14天,实时定量聚合酶链反应(Real-time PCR)法检测大鼠背根神经节(DRG)和脊髓(SC)中miR-137-3p及KDM4A的表达变化。于注射前及注射后的第1、3、7、14天,采用von Frey检测大鼠机械撤足阈值(PWT)和热撤足潜伏期(PWL)。蛋白质印迹法(Western blot)检测DRG和SC中KDM4A的表达变化;酶联免疫吸附试验(ELISA)法检测脊髓L4~L5组织中脑源性神经营养因子(BDNF)的含量;双荧光素酶报告基因法检测miR-137-3p与KDM4A的靶向关系。应用GraphPad Prism 8.2.1软件,对数据进行配对t检验、ANOVA单因素方差分析及双因素方差分析等方法分析。结果(1)与Sham组比较,miR-137-3p在CCI大鼠DRG和SC中的表达水平(0.363±0.130、0.490±0.099)显著低于Sham组(1.013±0.164、0.950±0.120),且差异有统计学意义(t=8.772、8.425,P<0.05),但KDM4A在CCI大鼠DRG和SC中的表达水平(3.113±0.083、3.613±0.136)高于Sham组(1.025±0.158、0.975±0.198),差异有统计学意义(t=33.020、31.060,P<0.01)。(2)与Sham组大鼠机械撤足阈值(1 d,23.220±5.262;3 d,22.790±5.764;7 d,23.220±5.262)和热撤足潜伏期(1 d,11.617±0.518;3 d,11.345±0.867;7 d,11.645±0.588)比较,CCI组机械撤足阈值(1 d,10.269±2.422;3 d,6.484±2.314;7 d,4.434±2.273)热撤足潜伏期(1 d,8.061±0.123;3 d,6.661±0.298;7 d,5.067±0.335)显著下降(F=7.841,P<0.05;F=162.900,P<0.01);而与CCI组比较,鞘内注射miR-137-3p mimic可显著促进大鼠机械撤足阈值(1 d,15.302±8.044;3 d,13.891±5.894;7 d,11.006±2.284)和热撤足潜伏期(1 d,10.995±0.139;3 d,9.972±0.336;7 d,8.522±0.322)的上升(F=12.420,P<0.01;F=80.300,P<0.01),抑制KDM4A的表达(DRG中,1.554±0.071比2.698±0.160,t=15.672,P<0.05;SC中,1.684±0.059比3.006±0.088,t=5.223,P<0.05)。(3)与Sham组(153.600±15.600;150.900±6.302)比较,CCI组大鼠DRG和SC中BDNF表达水平(354.400±13.020;350.700±26.850)显著上升(t=4.673,P<0.05;t=1.981,P<0.05);而与CCI组比较,鞘内注射miR-137-3p mimic可显著抑制BDNF的产生(188.000±5.685,t=5.767,P<0.05;182.200±4.983,t=20.034,P<0.05)。(4)与miR-scramble组相比,miR-137-3p mimic转染可显著降低KDM4A野生型载体的荧光素酶报告基因的荧光强度(1.002±0.080比0.486±0.063,t=11.290,P<0.05)。结论miR-137-3p通过调控大鼠背根神经节和脊髓背角中KDM4A的表达参与神经病理性痛。

Objective To observe the role of microRNA(miR)-137-3p regulation of the lysine specific demethylase 4A gene(KDM4A)in neuropathic pain caused by chronic constriction injury(CCI)of the sciatic nerve.Methods Rats were randomly divided into sham-operated group(Sham),CCI model group,2 mg/kg miR-137-3p mimic(mimic)intrathecal injection group,and 4 mg/kg miR-137-3p mimic intrathecal injection group.The expression of miR-137-3p and KDM4A in the dorsal root ganglion(DRG)and spinal cord(SC)of rats was measured by real-time quantitative polymerase chain reaction(Real-time PCR)on the 14th day after surgery.The expression of miR-137-3p and KDM4A in the DRG and SC was measured by von Frey before and at d 1,3,7 and 14 after injection.Western blotting was used to detect the changes of KDM4A expression in DRG and SC.The enzyme linked immunosorbent assay(ELISA)was used to detect the content of brain-derived neurotrophic factor(BDNF)in L4-L5 tissue of SC.The dual luciferase reporter gene method was used to detect the targeting relationship between miR-137-3p and KDM4A.Using GraphPad Prism 8.2.1 software,the data were analyzed by paired t-test,one-way Anova and two-way Anova.Results(1)Compared with the Sham group,the expression levels of miR-137-3p in DRG and SC of CCI rats(0.363±0.130,0.490±0.099)were significantly reduced(1.013±0.164,0.950±0.120,t=8.772,8.425,P<0.05),and the expression levels of KDM4A in DRG and SC of CCI rats(3.113±0.083,3.613±0.136)significantly increased(1.025±0.158,0.975±0.198,t=33.020,31.060,P<0.01).(2)Compared with the Sham group for mechanical withdrawal threshold(1 d:23.220±5.262;3 d:22.790±5.764;7 d:23.220±5.262)and heat withdrawal latency(1 d:11.617±0.518;3 d:11.345±0.867;7 d:11.645±0.588),the mechanical withdrawal threshold(1 d:10.269±2.422;3 d:6.484±2.314;7 d:4.434±2.273)and thermal withdrawal latency(1 d:8.061±0.123;3 d:6.661±0.298;7 d:5.067±0.335)were significantly lowered(F=7.841,162.900,P<0.01)in the CCI group,while intrathecal injection of miR-137-3p mimic significantly promoted the increase of mechanical withdrawal threshold(1 d:15.302±8.044;3 d:13.891±5.894;7 d:11.006±2.284)and heat withdrawal latency(1 d:10.995±0.139;3 d:9.972±0.336;7 d:8.522±0.322)as compared with the CCI group(F=12.420,80.300,P<0.01),and inhibited KDM4A expression(DRG:1.554±0.071 vs.2.698±0.160,t=15.672,P<0.05;SC:1.684±0.059 vs.3.006±0.088,t=5.223,P<0.05).(3)Compared with the Sham group(153.600±15.600,150.900±6.302),BDNF expression levels in DRG and SC of rats in the CCI group(354.400±13.020,350.700±26.850)were significantly increased(t=4.673,1.981,P<0.05);and compared with the CCI group,intrathecal injection of miR-137-3p mimic could significantly inhibit the production of BDNF(188.000±5.685,t=5.767,P<0.05;182.200±4.983,t=20.034,P<0.05);and compared with the CCI group,intrathecal injection of miR-137-3p mimic could significantly inhibit the production of BDNF.(4)Transfection of miR-137-3p mimic significantly reduced the fluorescence intensity of the luciferase reporter gene of the KDM4A wild-type vector compared with the miR-scramble group(1.002±0.080 vs.0.486±0.063,t=11.290,P<0.05).Conclusion MiR-137-3p is involved in neuropathic pain by regulating the expression of KDM4A in the dorsal root ganglion and the dorsal horn of the SC in rats.