Inhibition of miR-17-5p promotes mesenchymal stem cells to repair spinal cord injury.

Abstract

The aim of this study was to explore the role of microRNA-17-5p (miR-17-5p) in the pathogenesis of spinal cord injury repair by mesenchymal stem cells (MSCs), and to investigate the possible underlying mechanism. MiR-17-5p mimics and negative controls were transfected into MSCs. Dual-luciferase reporter gene assay was used to verify the functional binding between miR-17-5p and its target mRNA. After overexpression or knockdown of miR-17-5p, the expression level of target genes in MSC cells was analyzed by Real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot. The proliferation ability of cells was detected by cell counting kit-8 (CCK-8) assay. The effect of miR-17-5p and VEGF-A on angiogenesis was assessed by HUVEC assay. T8 spinal cord injury model was constructed in nude mice. All mice were divided into the negative control group, the SCI group, the miR-17-5p-NC group, the miR-17-5p-inhibitor group, and the miR-17-5p-inhibitor + sh-VEGF-A group. After injection of different treated MSCs at the lesion site, the proportion of intact tissue as well as reduced lumen volume was measured at 28 d. Meanwhile, the motor function of hind limbs was scored based on the Basso Beattie Bresnahan (BBB) standard scale at 7 d, 14 d, 21 d, and 28 d after transplantation, respectively. A binding site of miR-17-5p was found on the mRNA of VEGF-A. The protein expression of VEGF-A was strikingly altered after overexpression or knockdown of miR-17-5p. Knocking down miR-17-5p expression significantly increased the protein level of VEGF-A and GDNF. Meanwhile, miR-17-5p down-regulation significantly enhanced the viability and the angiogenic ability of MSCs. However, simultaneous knockdown of miR-17-5p and VEGF-A showed the opposite results. After spinal cord injury, the proportion of intact spinal cord tissues in mice was significantly reduced, whereas reduced lumen volume was remarkably increased. After injection of MSCs alone, the proportion of intact tissues was significantly increased. After knocking down miR-17-5p, the proportion was further increased. However, no significant effect was found on the amount of intact tissues after knocking out VEGF-A. Moreover, the reduction in cavity volume appeared to present an opposite trend comparable to the proportion of intact tissues. The BBB scores were significantly decreased in the mice model, while remarkably increased after MSC transplantation. Furthermore, the BBB score was the highest in the miR-17-5p knockout group, while VEGF-A knockout had little effect on it. In addition, no significant difference was found in the mRNA expression GFP in the spinal cord of mice in different groups after MSCs treatment. Inhibition of miR-17-5p up-regulates the expression of VEGF-A and GDNF in MSCs, and promotes the repair of spinal cord injury by MSCs.