MiR-124 inhibits spinal neuronal apoptosis through binding to GCH1

S. Yuan, Y.-X. Wang, P.-H. Gong, C.-Y. Meng

Qingdao University, Qingdao, China. txfbhm@163.com

---

• Orthopedics

OBJECTIVE: To explore the effect of micro ribonucleic acid (miR)-124 on the spinal neuronal apoptosis and to explore its related mechanism.

MATERIALS AND METHODS: 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.

RESULTS: 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.

CONCLUSIONS: 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.

Free PDF Download