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. 2021 Apr 5;12(1):224.
doi: 10.1186/s13287-021-02282-0.

Exosomes derived from miR-26a-modified MSCs promote axonal regeneration via the PTEN/AKT/mTOR pathway following spinal cord injury

Yuyong Chen #  1   2   3 , Zhenming Tian #  1   2   3 , Lei He #  1   2   3 , Can Liu  1   2   3 , Nangxiang Wang  1   2   3 , Limin Rong  4   5   6 , Bin Liu  7   8   9
Affiliations

Exosomes derived from miR-26a-modified MSCs promote axonal regeneration via the PTEN/AKT/mTOR pathway following spinal cord injury

Yuyong Chen et al. Stem Cell Res Ther. .

Abstract

Background: Exosomes derived from the bone marrow mesenchymal stem cell (MSC) have shown great potential in spinal cord injury (SCI) treatment. This research was designed to investigate the therapeutic effects of miR-26a-modified MSC-derived exosomes (Exos-26a) following SCI.

Methods: Bioinformatics and data mining were performed to explore the role of miR-26a in SCI. Exosomes were isolated from miR-26a-modified MSC culture medium by ultracentrifugation. A series of experiments, including assessment of Basso, Beattie and Bresnahan scale, histological evaluation, motor-evoked potential recording, diffusion tensor imaging, and western blotting, were performed to determine the therapeutic influence and the underlying molecular mechanisms of Exos-26a in SCI rats.

Results: Exos-26a was shown to promote axonal regeneration. Furthermore, we found that exosomes derived from miR-26a-modified MSC could improve neurogenesis and attenuate glial scarring through PTEN/AKT/mTOR signaling cascades.

Conclusions: Exosomes derived from miR-26a-modified MSC could activate the PTEN-AKT-mTOR pathway to promote axonal regeneration and neurogenesis and attenuate glia scarring in SCI and thus present great potential for SCI treatment.

Keywords: Axonal regeneration; Exosomes; Mesenchymal stem cells; Spinal cord injury; miR-26a/PTEN axis.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Identification of miR-26a as a differentially expressed miRNA and its predicted target genes. a Heat map of differentially expressed miRNAs based on the GSE19890 dataset. b miR-26a expression level following spinal cord injury. c, d Enriched GO terms and KEGG pathways for miR-26a target genes. e miR-26a target site in the 3′-UTR of PTEN. **P < 0.01 compared with the sham group by t test. n = 5 for each group
Fig. 2
Fig. 2
Characterization and overexpression of exosomes. a, b Isolation and characterization of MSC. c Schematic diagram of exosome isolation. d Nano measurements of exosomes. e Representative transmission electron micrographs of exosomes showing cup-shaped morphology. f Verification of expression of surface markers, including CD9, CD63, Flotillin-1, and non-exosomal marker Calnexin on exosomes by western blot analysis. g The uptake of green fluorescent dye-expressing exosomes into PC12 cells. h miR-26a expression level in BMSC-derived exosomes following miR-26a mimic transfection (Exos-26a group). ***P < 0.001 compared with the Exos group by t test. n = 3 for each group
Fig. 3
Fig. 3
Exos-26a promoted neurofilament generation via the PTEN-AKT-mTOR pathway. a The ability of Exos-26a to generate neurofilament (red fluorescent dye) in PC12 cells. b, c Representative images of western blots for analysis of the expression level of NF and semiquantification of the data. d, e Representative images of western blots for analysis of the expression of PETN-AKT-mTOR pathway-related proteins and semiquantification of the data. The data are normalized to the control group. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control group by t test or ANOVA test. #P < 0.05 and ##P < 0.01 compared with the Exos group by t test. n = 3 for each group
Fig. 4
Fig. 4
Exos-26a treatment promoted functional behavioral recovery following SCI. a Before and after compression of the spinal cord. b Schematic diagram of the method used to establish a spinal cord injury model and exosome treatment. c Hematoxylin and eosin staining of the PBS group, exosome negative control (Exos) group, and miR-26a-overexpressing exosome (Exos-26a) group on day 28 postinjury. d BBB scores of the three experimental groups. e DTI images of all groups on day 28 postinjury. f, g MEP amplitudes of the three groups on day 28 postinjury and quantification. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control group by t test or ANOVA. #P < 0.05 compared with the Exos group by t test. n = 6 for each group
Fig. 5
Fig. 5
Exos-26a facilitated neuronal regeneration and inhibited reactive astrogliosis. a Representative immunostaining images of NF200 (red) in the injured areas of the spinal cord on day 28 postinjury. b Representative immunostaining images of Tuj-1 (green) and GFAP (red) in the injured areas of the spinal cord. c Western blot analysis of NF, Tuj-1, and GFAP in lesioned spinal cord segments and semiquantification of the data. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control group by t test or ANOVA. #P < 0.05 and ##P < 0.01 compared with the Exos group by t test. n = 3 for each group
Fig. 6
Fig. 6
Exos-26a promoted spinal cord injury repair via the PTEN-AKT-mTOR pathway. a Diagram of the mechanism underlying the effect of Exos-26a via the PTEN-AKT-mTOR pathway. b, c Representative images of western blots used to analyze the expression of PETN-AKT-mTOR pathway-related proteins and semiquantification of the data. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control group by t test or ANOVA. #P < 0.05 compared with the Exos group by t test. n = 3 for each group
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