miR-15b-5p通过下调BDNF/TrkB/PSD95表达参与PD中抑郁样行为发生

Involvement of miR-15b-5p in depression-like behavior in Parkinson's disease by down-regulating BDNF/TrkB/PSD95 expressions

目的探讨miR-15b-5p参与PD中抑郁样行为发生的机制。方法(1)将18只C57BL/6N小鼠按随机数字表法分为PD组、干预组及对照组,每组6只。PD组小鼠通过脑立体定位仪将0.25 mg/kg鱼藤酮注射至右侧纹状体以制备PD模型,干预组小鼠接受0.25 mg/kg鱼藤酮及miR-15b-5p抑制物慢病毒注射,对照组小鼠注射等体积PBS。4周后进行旷场实验及转棒实验评价小鼠的运动能力,进行糖水偏好实验及强迫游泳实验评价小鼠的抑郁样行为表现,然后提取小鼠黑质及纹状体中相关蛋白及miRNAs,进行Western blotting实验、实时荧光定量逆转录聚合酶链反应(qRT-PCR)实验以及免疫荧光染色实验检测相关指标[miR-15b-5p、酪氨酸羟化酶(TH)、脑源性神经营养因子(BDNF)、原肌球蛋白受体激酶B(TrkB)、突触后密度蛋白95(PSD95)]。(2)将100 nmol/L miR-15b-5p模拟物/抑制物序列及各自阴性对照序列分别转染至6孔板上SH-SY5Y细胞中(分别命名为miR-15b-5p模拟物组、miR-15b-5p模拟物对照组及miR-15b-5p抑制物组、miR-15b-5p抑制物对照组),48 h后进行Western blotting实验及qRT-PCR实验,以检测BDNF、TrkB、PSD95蛋白及miR-15b-5p表达改变。(3)将100 ng BDNF 3'端非编码区(3'-UTR)野生型或突变型荧光素酶报告载体质粒以及100 nmol/L miR-15b-5p模拟物/抑制物序列及各自阴性对照序列共转染至24孔板上SH-SY5Y细胞中,得到相应8组细胞,48 h后进行荧光素酶报告载体活性检测实验,以检测各组细胞的荧光素酶活性。结果(1)与对照组相比,PD组小鼠的运动速度明显减慢、落棒潜伏期明显缩短、糖水偏好百分比明显降低、不动时间明显增加;与PD组相比,干预组小鼠的运动速度明显提高、落棒潜伏期明显延长、糖水偏好百分比明显升高,不动时间明显减少;差异均有统计学意义(P<0.05)。(2)与miR-15b-5p模拟物对照组比较,miR-15b-5p模拟物组细胞中miR-15b-5p表达明显升高,BDNF、TrkB、PSD95表达明显降低(100.00±5.75 vs.66.79±5.90;100.00±5.95 vs.84.46±5.77;100.00±7.02 vs.80.43±3.25);与miR-15b-5p抑制物对照组比较,miR-15b-5p抑制物组细胞中miR-15b-5p表达明显降低,BDNF、TrkB、PSD95表达明显升高(100.00±6.81 vs.119.90±5.66;100.00±2.88 vs.110.10±4.15;100.00±2.19 vs.124.60±11.69);差异均有统计学意义(P<0.05)。与对照组相比,PD组小鼠黑质中miR-15b-5p表达明显增加,TH、BDNF、TrkB、PSD95表达明显降低(100.00±9.20 vs.63.60±12.80;100.00±9.88 vs.71.95±10.00;100.00±5.16 vs.70.37±8.43;100.00±7.01 vs.68.12±10.22),BDNF荧光强度明显降低(100.00±12.99 vs.48.23±12.58);与PD组相比,干预组小鼠黑质中miR-15b-5p表达明显降低,TH、BDNF、TrkB、PSD95表达明显升高(63.60±12.80 vs.90.69±9.84;71.95±10.00 vs.93.31±4.50;70.37±8.43 vs.88.11±4.10;68.12±10.22 vs.89.59±5.93),BDNF荧光强度明显升高(48.23±12.58 vs.83.65±10.52);差异均有统计学意义(P<0.05)。(3)与BDNF 3'-UTR野生型+miR-15b-5p模拟物对照组比较,BDNF 3'-UTR野生型+miR-15b-5p模拟物组细胞中荧光素酶活性明显降低(100.00±5.07 vs.90.59±1.75);与BDNF 3'-UTR野生型+miR-15b-5p抑制物对照组比较,BDNF 3'-UTR野生型+miR-15b-5p抑制物组细胞中荧光素酶活性明显升高(100.00±5.08 vs.152.20±31.87);差异均有统计学意义(P<0.05)。结论miR-15b-5p通过下调BDNF/TrkB/PSD95表达参与PD中抑郁样行为发生。

Objective To explore the mechanism of miR-15b-5p involving in depression-like behavior in Parkinson's disease(PD).Methods(1)Eighteen C57BL/6N mice were randomly divided into PD group,intervention group and control group(n=6).PD models in PD group were established by stereotaxically injecting 0.25 mg/kg rotenone into the right striatum;mice in the intervention group were injected with 0.25 mg/kg rotenone and miR-15b-5p inhibitor lentivirus,while mice in the control group were injected with equal volume of PBS.Four weeks after that,open field test and rotarod test were performed to evaluate the motor ability,and sucrose preference and forced swimming tests were performed to evaluate the depression-like behaviors.And then,proteins and miRNAs in the substantia nigra were extracted;real-time fluorescent quantitative reverse transcription PCR(qRT-PCR)was used to detect the miR-15b-5p expression,and Western blotting and immunofluorescent staining were used to detect the tyrosine hydroxylase(TH),brain-derived neurotrophic factor(BDNF),tropomyosin receptor kinase B(TrkB),and postsynaptic density protein 95(PSD95)protein expressions.(2)The 100 nmol/L miR-15b-5p mimic/inhibitor and their negative control sequences were transfected into SH-SY5Y cells on 6-well plates(named miR-15b-5p mimic group,miR-15b-5p mimic control group,miR-15b-5p inhibitor group and miR-15b-5p inhibitor control group,respectively);48 h after that,BDNF,TrkB and PSD95 protein expressions were detected by Western blotting and miR-15b-5p expression by qRT-PCR.(3)The 100 ng BDNF 3'-UTR wild-type or mutant luciferase reporter vector plasmids and 100 nmol/L miR-15b-5p mimic/inhibitor or their negative control sequences were co-transfected into SH-SY5Y cells on 24-well plates,and luciferase reporter activity assay was performed 48 h after co-transfection to detect the luciferase activity.Results(1)Compared with the control group,the PD group had significantly reduced movement speed,shortened rotarod drop latency,decreased percentage of sucrose preference,and prolonged immobility time(P<0.05);compared with the PD group,the intervention group had significantly increased movement speed,prolonged rotarod drop latency,increased percentage of sucrose preference,and shortened immobility time(P<0.05).(2)Compared with the miR-15b-5p mimic control group,the miR-15b-5p mimic group had significantly increased miR-15b-5p expression,and decreased BDNF,TrkB and PSD95 protein expressions(100.00±5.75 vs.66.79±5.90;100.00±5.95 vs.84.46±5.77;100.00±7.02 vs.80.43±3.25,P<0.05).Compared with the miR-15b-5p inhibitor control group,the miR-15b-5p inhibitor group had significantly decreased miR-15b-5p expression,and increased BDNF,TrkB and PSD95 expressions(100.00±6.81 vs.119.90±5.66;100.00±2.88 vs.110.10±4.15;100.00±2.19 vs.124.60±11.69,P<0.05).Compared with the control group,PD group had significantly increased miR-15b-5p expression,and significantly decreased TH,BDNF,TrkB and PSD95 expressions and BDNF fluorescent intensity(100.00±9.20 vs.63.60±12.80;100.00±9.88 vs.71.95±10.00;100.00±5.16 vs.70.37±8.43;100.00±7.01 vs.68.12±10.22;100.00±12.99 vs.48.23±12.58)in the substantia nigra(P<0.05);compared with the PD group,the intervention group had significantly lower miR-15b-5p expression and increased TH,BDNF,TrkB and PSD95 expressions and BDNF fluorescent intensity(63.60±12.80 vs.90.69±9.84;71.95±10.00 vs.93.31±4.50;70.37±8.43 vs.88.11±4.10;68.12±10.22 vs.89.59±5.93;48.23±12.58 vs.83.65±10.52)in the substantia nigra(P<0.05).(3)Compared with the BDNF 3'-UTR wild-type+miR-15b-5p mimic control group,the BDNF 3'-UTR wild-type+miR-15b-5p mimic group had significantly decreased luciferase activity(100.00±5.07 vs.90.59±1.75,P<0.05);compared with the BDNF 3'-UTR wild-type+miR-15b-5p inhibitor control group,the BDNF 3'-UTR wild-type+miR-15b-5p inhibitor group had significantly increased luciferase activity(100.00±5.08 vs.152.20±31.87,P<0.05).Conclusion MiR-15b-5p is involved in depression-like behavior in PD by down-regulating the BDNF/TrkB/PSD95 expressions.