Yuan S, Wang Y-X, Gong P-H, Meng C-Y
Qingdao University, Qingdao, China.
Eur Rev Med Pharmacol Sci. 2019 Jun;23(11):4564-4574. doi: 10.26355/eurrev_201906_18032.
To explore the effect of micro ribonucleic acid (miR)-124 on the spinal neuronal apoptosis and to explore its related mechanism.
The rat model of spinal cord injury (SCI) was established, agomir-124 was injected intrathecally and the effect of agomir-124 on motor function recovery of rats was evaluated using the Basso-Beattie-Bresnahan (BBB) score. The gene expression levels of miR-124 and GTP-cyclohydrolase 1 (GCH1) in spinal cord tissues were detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR), and the correlation between them was detected using the Pearson correlation coefficient. Then, the direct interaction between miR-124 and GCH1 mRNA was detected using the TargetScan software and luciferase reporter assay. The changes in apoptosis in each group were examined via flow cytometry and Western blotting. Moreover, the changes in the tetrahydrobiopterin (BH4) content in each group were detected via high-performance liquid chromatography, and the changes in the nitrite level in the supernatant in each group were detected using the Griess reagent. Finally, the changes in the activity of the inducible nitric oxide synthase (iNOS) protein were detected using the iNOS kit.
Compared with that in the model group, the BBB score was significantly increased in agomir-124 group at 21, 28, 35 and 42 d. In the agomir-124 group, the relative expression level of miR 124 in spinal cord tissues was significantly increased at 7-28 d and reached the peak at 21 d, while the mRNA level of GCH1 in spinal cord tissues declined and touched the bottom at 21 d. According to the Pearson correlation coefficient, there was a significant negative correlation between the expression of miR-124 and mRNA expression of GCH1 (r =- 0.87, p = 1.5e-6). It was found in the prediction using TargetScan software that GCH1 might be a potential target for miR-124, which was further verified by the luciferase reporter assay. The results of flow cytometry and Western blotting showed that miR-124 significantly reduced the LPS-induced primary spinal neuronal apoptosis, while the miR-124 inhibitor remarkably increased the primary spinal neuronal apoptosis. Moreover, it was also found that the knockout of GCH1 reduced the LPS-induced spinal neuronal apoptosis. In addition, the GCH1 overexpression assay revealed that miR-124 inhibited spinal neuronal apoptosis by suppressing the GCH1 expression. LPS + miR-124 remarkably decreased the BH4 content, nitrite level, and iNOS activity while LPS + miR-124 + GCH1 remarkably increased the BH4 content, nitrite level, and iNOS activity.
MiR-124 inhibits neuronal apoptosis in SCI by binding to GCH1. The results in the present study may provide a new mechanism for the therapeutic effect of miR-124, and miR-124 may have a potential therapeutic value in the treatment of SCI in the future.
探讨微小核糖核酸（miR）-124对脊髓神经元凋亡的影响并探究其相关机制。
建立大鼠脊髓损伤（SCI）模型，鞘内注射agomir-124，采用Basso-Beattie-Bresnahan（BBB）评分评估agomir-124对大鼠运动功能恢复的影响。通过定量逆转录-聚合酶链反应（qRT-PCR）检测脊髓组织中miR-124和鸟苷三磷酸环化水解酶1（GCH1）的基因表达水平，并使用Pearson相关系数检测它们之间的相关性。然后，使用TargetScan软件和荧光素酶报告基因检测法检测miR-124与GCH1 mRNA之间的直接相互作用。通过流式细胞术和蛋白质印迹法检测各组细胞凋亡的变化。此外，采用高效液相色谱法检测各组四氢生物蝶呤（BH4）含量的变化，使用Griess试剂检测各组上清液中亚硝酸盐水平的变化。最后，使用诱导型一氧化氮合酶（iNOS）试剂盒检测iNOS蛋白活性的变化。
与模型组相比，agomir-124组在第21、28、35和42天时BBB评分显著升高。在agomir-124组中，脊髓组织中miR-124的相对表达水平在7-28天显著升高，并在第21天达到峰值，而脊髓组织中GCH1的mRNA水平下降，并在第21天降至最低。根据Pearson相关系数，miR-124的表达与GCH1的mRNA表达之间存在显著负相关（r = -0.87，p = 1.5e-6）。使用TargetScan软件预测发现GCH1可能是miR-124的潜在靶点，荧光素酶报告基因检测法进一步验证了这一结果。流式细胞术和蛋白质印迹法的结果表明，miR-124显著降低了脂多糖（LPS）诱导的原代脊髓神经元凋亡，而miR-124抑制剂显著增加了原代脊髓神经元凋亡。此外，还发现敲除GCH1可减少LPS诱导的脊髓神经元凋亡。另外，GCH1过表达实验表明，miR-124通过抑制GCH1表达来抑制脊髓神经元凋亡。LPS + miR-124显著降低了BH4含量、亚硝酸盐水平和iNOS活性，而LPS + miR-124 + GCH1显著增加了BH4含量、亚硝酸盐水平和iNOS活性。
MiR-124通过与GCH1结合抑制SCI中的神经元凋亡。本研究结果可能为miR-124的治疗作用提供新的机制，miR-124未来可能在SCI治疗中具有潜在的治疗价值。
