Abstract
Schwann cell (SC) transplantation is a promising approach for the treatment of spinal cord injury (SCI); however, SC grafts show a low migratory capacity within the astrocytic environment, which inevitably hampers their therapeutic efficacy. The purpose of this study was to explore mechanisms to modify the characteristics of SCs and astrocytes (ASs), as well as to adjust the SC-AS interface to break the SC-AS boundary, thus improving the benefits of SCI treatment. We observed that the expression levels of miR-124 in SCs and ASs were significantly lower than those in the normal spinal cord. Furthermore, overexpressing miR-124 in SCs (miR-124-SCs) significantly inhibited gene and protein expression levels of SC-specific markers, such as GFAP and Krox20. The expression of neurotrophic factors, Bdnf and Nt-3, was up-regulated in miR-124-SCs without affecting their proliferation. Further, the boundary assay showed an increased number of miR-124-SCs that had actively migrated and entered the astrocytic region to intermingle with ASs, compared with normal SCs. In addition, although Krox20 protein expression was down-regulated in miR-124-SCs, the luciferase assay showed that Krox20 is not a direct target of miR-124. RNA sequencing of miR-124-SCs revealed seven upregulated and eleven downregulated genes involved in cell migration and motility. Based on KEGG pathway and KOG functional analyses, changes in these genes corresponded to the activation of Hippo, FoxO, and TGF-beta signaling pathways, cytokine-cytokine receptor interactions, and the cell cycle. Finally, co-culturing of miR-124-SCs and ASs in a transwell system revealed that GFAP and p-STAT3 protein expression in ASs was significantly reduced. Collectively, these results show that overexpression of miR-124 in SCs promotes SC-AS integration in vitro and may attenuate the capacity of ASs to form glial scars. Thus, this study provides novel insights into modifying SCs by overexpressing miR-124 to improve their therapeutic potential in SCI.
Highlights
Spinal cord injury (SCI) is a traumatic disease with direct and indirect causes
We found that the Krox20 protein expression in Schwann cell (SC) after miR124 overexpression was significantly lower than those in wild type (WT) and negative control (NC) (Figures 4G,H), thereby illustrating that the integration of miR-124-SCs and alternative splicing (AS) might be regulated by the down-regulation of Krox20 (∗∗p < 0.01)
We found that 11 genes were down-regulated (Figure 6E), including matrix metallopeptidase 10 (Mmp10), calcitoninrelated polypeptide alpha (Calca), Neurotrophic receptor tyrosine kinase (Ntrk2), secretogranin 2 (Scg2), c-x-c motif chemokine ligand-2 (Cxcl2), neuropeptide Y (Npy), insulin-like growth factor binding protein 5 (Igfbp5), cyclin D2 (Ccnd2), leukemia inhibitory factor (Lif ), serpin family E member 2 (Serpine 2), and pro-neuropeptide Y-like (LOC100912228)
Summary
Spinal cord injury (SCI) is a traumatic disease with direct and indirect causes. It has received increasing attention in the medical community due to its high potential of leading to disability, the difficulty of spinal cord surgery, and its impaired healing process. The scar interlaces, resulting in steric hindrance and production of inhibitory molecules, such as chondroitin sulfate proteoglycans, which hinder the axonal sprouting and interfere with damage repair (O’Shea et al, 2017). Reducing the formation of glial scar and promoting axon growth to effectively connect distal and terminal axons and fill the cavity, are vital strategies for SCI treatment
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