| Objective The regeneration of injured peripheral nerves is an extremely complex process, and there are many genes and proteins changed in the nerves during sciatic nerve regeneration. microRNAs(miRNAs) are short, non-coding RNA molecules that play an important role in the post-transcriptional level of gene expression,and its discovery provides a new mode of the molecular mechanisms of regeneration. Schwann cells(SCs), the principal glial cells in the peripheral nerve system, can improve the microenvironment for peripheral nerve and have a very important role in the degeneration and regeneration after peripheral nerve injury. In this study, therefore, we aimed to explore the possible involvement of miR-9 in regulating responses of SCs in peripheral nerve repair and regeneration.Methods Thirty adult, male Sprague-Dawley(SD) rats(180±20 g) were provided by the Experimental Animal Center of Nantong University and randomly divided into five groups of six rats each. The 1-cm-long segment of left sciatic nerve of all animals was transected and the proximal stumps of sciatic nerve(0.5 cm) were collected at 0, 1, 4, 7 and 14 days after injury. We extracted total RNA for miRNA microarray, and used qRT-PCR and ISH to determine the alterations of miR-9 between serial time points. Then, the cell proliferation, apotosis and migration of SCs were examined by using EdU, Annexin V-PE,Transwell and Wound healing assay, respectively. Collagen triple helix repeat-containing protein 1(CTHRC1) might be the target of miR-9, and its 3’-UTR was subcloned into the region downstream of the stop codon in the luciferase gene to generate different p-Luc-UTR luciferase reporter vectors. The activity of luciferases was measured to determine whether miR-9 directly target the 3’-UTR of CTHRC1. We performed qRT-PCR and Western blotting to verify the expression of CTHRC1 in proximal sciatic nerve tissue and conducted immunohistochemistry to detect the location of CTHRC1 in cells. The role of CTHRC1 in regulating migration of SCs was assayed by using CTHRC1 siRNA. In addition, the correlation among miR-9, CTHRC1 and Rac1 GTPase in SCs was tested by Rac1 inhibitor treatment. In vivo, we used an animal model of peripheral nerve transection to investigate the effect of miR-9 overexpression on SCs.Results We detected that there were many miRNAs changed obviously in injured sciatic nerves, and miR-9 was decreased from 1 d post injury, and the expression was significantly lower at 1, 4, 7 and 14 d post injury than at 0 d post injury. miR-9 inhibited the migration of primary SCs, but had no influence on the proliferation and apotosis of SCs. miR-9 could downregulated the expression of CTHRC1 by directly targeting the 3’-UTR of CTHRC1. In addition, miR-9 inhibited the migration of SCs by inactivating Rac1 GTPase which was a downstream regulator of CTHRC1. Besides that, the number and the distance of SC migration were lower by miR-9 agomir injection than by negative control injection in the injuried nerves, suggesting that miR-9 could also inhibit the migration of SCs in vivo.Conclusions In this study, using a microarray screen, we found there were many important miRNAs which showed dynamic alteration in the proximal sciatic nerves. miR-9 showed a significantly lower expression in the injured nerve, and it could inhibit SC migration in vitro and in vivo. The role of miR-9 in regulating SC migration was played by inhibiting CTHRC1 expression at the post-transcriptional level, and further inactivating Rac1 GTPase which was a downstream regulator of CTHRC1. We discussed the effect of miR-9 on SC migration in peripheral nerve injury and regeneration, and imply that mi RNA may be an important component of the regulatory network responsible for the activation and the phenotypic modulation of SCs. Our findings demonstrated that the miRNA signal was important for understanding the molecular mechanisms responsible for peripheral nerve regeneration, and offered a new approach to peripheral nerve injury and repair. |
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