脊神经结扎术后脊髓背角小胶质细胞中miR-195的表达及其对自噬水平的影响

Effect of miR-195 on microglia autophagy after spinal nerve ligation in rats

中文关键词:  微核糖酸-195  小胶质细胞  自噬相关基因14  自噬  神经结扎术  大鼠

英文关键词:MicroRNA-195  Microglia  Autophagy associated gene 14  Autophagy  Nerve operation  Rat

中文摘要:

  【摘要】 目的：探讨大鼠脊神经结扎术后脊髓背角小胶质细胞中微核糖核酸-195 （microRNA-195，miR-195）的表达及其对自噬水平的影响与调节机制。方法：32只成年雄性SD大鼠随机分为脊神经结扎组（SNL组）和假手术组（S组），每组16只，SNL组结扎L5段神经制备脊神经结扎模型，造模后1d、3d、5d、10d两组各处死4只大鼠，分离脊髓腰膨大，采用Percoll梯度离心法分离脊髓背角小胶质细胞，Real-time PCR检测两组各时间点脊髓背角小胶质细胞的miR-195表达水平。取12只成年雄性SD大鼠，处死后取脊髓腰膨大，分离脊髓背角小胶质细胞，分为两组，一组采用脂质体法转染miR-195模拟物，另一组加入转染试剂，检测两组小胶质细胞自噬体膜标记蛋白轻链3（light chain 3，LC3）的表达水平；再取小胶质细胞分为三组，分别将含有野生型及突变型自噬相关基因14（ATG14）3′非编码区（3′UTR）的报告基因质粒、pRL-TK质粒miR-195模拟物和对照转入HEK 293细胞培养48h，荧光显微镜下观察miR-195的直接作用靶点，并检测三组ATG14蛋白的表达。取24只健康SD大鼠脊髓腰膨大，分离脊髓背角小胶质细胞，分为三组分别转染miR-195模拟物、抑制物和转染试剂，培养48h，采用免疫蛋白印记（Western blot）法检测三组ATG14蛋白的表达水平；再取小胶质细胞分为三组，分别转染pEGFP-LC3、pEGFP-LC3+miR-195模拟物及pEGFP-LC3+miR-195模拟物+pCMV-ATG14培养48h，染色后荧光显微下观察自噬斑点数量。结果：SNL组各时间点大鼠脊髓背角小胶质细胞中miR-195表达均明显上调，与同时间点S组比较均有显著性差异（P<0.05）。转染miR-195模拟物后，LC3的表达水平（0.61±0.07）较对照组（1.21±0.08）显著性降低（P<0.05）。转入野生型ATG14 3′UTR的报告基因质粒的HEK 293细胞的荧光强度较对照显著性减弱（0.65±0.04 vs 1.01±0.01，P<0.05），突变型与对照无显著性变化（0.99±0.03 vs 1.00±0.02，P>0.05）。转染miR-195模拟物后小胶质细胞中ATG14的表达显著性降低（P<0.05）；转染miR-195抑制物后ATG14的表达显著性升高（P<0.05）。转染pEGFP-LC3与pEGFP-LC3+miR-195模拟物较转染pEGFP-LC3+miR-195模拟物+pCMV-ATG14时自噬数量降低（P<0.05）。结论：miR-195在大鼠脊神经结扎模型脊髓背角小胶质细胞中高表达，降低自噬蛋白LC3表达；ATG14蛋白受miR-195负调控，miR-195可能通过降低ATG14的表达抑制小胶质细胞自噬。

英文摘要:

  【Abstract】 Objectives: To explore the effect of microRNA-195(miR-95) on microglia autophagy after spinal nerve ligation and its regulatory mechanism in rats. Methods: Thirty-two adult male SD rats were randomly divided into the spinal nerve ligation model group(SNL group) and the sham operation group(S group), with 16 rats in each group. In SNL group, L5 nerve was ligatured in each rat to prepare spinal nerve ligation model. At each time point of 1d, 3d, 5d, 10d after modeling, 4 rats in each group were executed. Microglia in spinal cord dorsal horn was separated by Percoll gradients centrifugation, real-time PCR was used to detect miR-195 expression. 12 adult male SD rats were sacrificed, microglia in spinal dorsal horn of lumbar enlargements was isolated and divided into two groups, one group with liposome transfection of miR-195 mimics and the other group with transfection of reagent. In the two groups, the expression of autophagic membrane marker protein light chain 3(LC3) was detected. Then microglial cells were divided into three groups, each group contained wild type and mutant autophagy related gene 14(ATG14) 3′ untranslation region(3′UTR) reporter plasmid. HEK293 were transfected by pRL-TK plasmid miR-195 mimics and controls and cultured for 48h. The direct effect of miR-195 target was observed under fluorescent microscope, and the expression of ATG14 protein was detected in three groups. 24 healthy SD rats were sacrificed, microglia in spinal dorsal horn of lumbar enlargements was isolated and divided into three groups, which were transfected with miR-195 mimics, inhibitor and transfection reagent respectively, then cultured for 48h. The expression of ATG14 protein was detected by Western blot in the three groups. Microglial cells were divided into three groups, which were transfected with pEGFP-LC3, pEGFP-LC+miR-195 mimics and pEGFP-LC+miR-195 mimics +pCMV-ATG14, then cultured for 48h. The dot staining of autophagy was observed under fluorescence microscope. Results: The miR-195 expression in microglia of spinal cord dorsal horn increased obviously in the spinal nerve ligation model when compared with S group at the same time, there was significant difference between the two groups(P<0.05). After transfection with miR-195 analog, the expression level of LC-3(0.61±0.07) was significantly lower than that in the control group(1.21±0.08). The fluorescence intensity of wild-type ATG14 3′ UTR plasmid HEK293 cells was significantly lower than that in the control group(0.65±0.04 vs 1.01±0.01), and there was no significant change between the mutant and the control group(0.99±0.03 vs 1.01±0.02). The transfection of miR-195 simulant reduced autophagy level in normal microglia. Luciferase experiments showed ATG14 was the direct target of miR-195, the expression of ATG14 declined after transfection of miR-195 simulant, and rised after transfection of inhibition of miR-19. Conclusions: The miR-195 is highly expressed and inhibited autophagy in microglia of spinal cord dorsal horn in the spinal nerve ligation model.