Exosomes Derived from Adipose Mesenchymal Stem Cells Carrying miRNA-22-3p Promote Schwann Cells Proliferation and Migration through Downregulation of PTEN

Peripheral nerve injury (PNI) is often resulting from trauma, which leads to severe and permanently disability. Schwann cells are critical for facilitating the regeneration process after PNI. Adipose-derived mesenchymal stem cells (ADSCs) exosomes have been used as a novel treatment for peripheral nerve injury. However, the underlying mechanism remains unclear. In this study, we isolated ADSCs and extracted exosomes, which were verified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blot (WB). Cocultured with Dorsal Root Ganglion (DRG) and Schwann cells (SCs) to evaluate the effect of exosomes on the growth of DRG axons by immunofluorescence, and the proliferation and migration of SCs by CCK8 and Transwell assays, respectively. Through exosomal miRNA sequencing and bioinformatic analysis, the related miRNAs and target gene were predicted and identified by dual luciferase assay. Related miRNAs were overexpressed and inhibited, respectively, to clarify their effects; the downstream pathway through the target gene was determined by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and WB. Results found that ADSC-exosomes could promote the proliferation and migration of SCs and the growth of DRG axons, respectively. Exosomal miRNA-22-3p from ADSCs directly inhibited the expression of Phosphatase and Tensin Homolog deleted on Chromosome 10 (PTEN), activated phosphorylation of the AKT/mTOR axis, and enhanced SCs proliferation and migration. In conclusion, our findings suggest that ADSC-exosomes could promote SCs function through exosomal miRNA-22-3p, which could be used as a therapeutic target for peripheral nerve injury.


Introduction
The peripheral nerve connects the central nerve to the muscles, bones, and visceral organs. Peripheral nerve injuries (PNIs) often result in persistent dysfunction of innervated tissues. PNIs occur secondary to trauma, tumors or inflammation, with an annual incidence of approximately 13-23 per 100,000 individuals [1]. Trauma is the primary cause, and approximately 3% of trauma events are accompanied by PNIs [2]. Because of inflammation and scar formation, recovery from PNIs is often limited by the type and severity of injury. Currently, autologous nerve transplantation is the standard treatment for complete amputation, a type of severe injury. However, this method requires sacrificing the normal donor nerves, and simple end-to-end nerve suturing may result in the wrong direction of sensory and motor nerve regeneration [3]. This results in an unsatisfactory outcome; therefore, new treatments for PNIs are needed.
With the development of regenerative medicine, mesenchymal stem cells (MSCs) have increasingly been used to treat various diseases and injuries. MSCs can promote the repair of nerve injuries [4], which may occur by two mechanisms. First, MSCs can differentiate into Schwann-like cells or promote the massive proliferation of Schwann cells (SCs),

Materials and Methods
2.1. Isolation of ADSCs. Sprague-Dawley rats were purchased from Shanghai SLAC Animal Company. All animal experiments were approved by the Ethics Committee of Shanghai General Hospital and conducted strictly in accordance with the institutional guidelines for the care and use of laboratory animals. Four-week-old SD rats were anesthetized, and adipose biopsies were obtained from inguinal subcutaneous tissue and cut into small pieces using microscissors. Added 0.1% collagenase I (SCR103; Sigma-Aldrich) to digest at a shaker (37°C for 1 hour). Digested adipose tissue was centrifuged at 2000 × rpm for 10 min. After filtering through a 70 μm mesh filter, cells were cultured at 37°C in 5% CO 2 humidified conditions in DMEM medium (11965092; Gibco) with 10% fetal bovine serum (FBS) (10082147; Gibco), and 1% penicillin/streptomycin (15070063; Gibco) [14]. The medium was replaced every two days and passaged when 90% fusion was reached.

Multipotent Differentiation of ADSCs.
When the ADSCs reached 70% confluence, the medium was replaced with osteogenic induction medium supplemented with L-glutamine, dexamethasone, ascorbic acid, and β-glycerophosphate. The medium was changed every 3 days according to the growth conditions. After 4 weeks, the cells were stained with Alizarin Red S stain [15]. When ADSCs reached 100% confluence, the medium was replaced with adipogenic induction culture medium consisting of DMEM with insu-lin, dexamethasone, indomethacin, and 3-isobutyl-1-methylxanthine. The medium was replaced every three days for 14 days. Oil Red O staining was performed to detect intracellular lipid vacuoles characteristic of adipocytes [16].
2.4. Extraction of Exosomes and miRNA. ADSCs were cultured in DMEM containing 10% exosomes-free FBS (180625; SBI) for 48 h. The medium was then collected and centrifuged at 3000 × g for 10 min at 4°C. The supernatant was collected, transferred to a new tube, and placed on ice. Exosomes and miRNAs were extracted using an exoRNeasy Midi Kit (77144; QIAGEN), according to the manufacturer's protocol.
2.7. Isolation and Culture of DRG. Three-week-old SD rats were sacrificed by decapitation with guillotine after anesthesia. The body trunk of the rat was isolated between the forelimb and femur, and the bilateral lumbar DRGs were collected in a 35-mm culture dish with 2 ml of iced Leibovitz's L-15 medium (11415064; Gibco) [18]. Added 2 ml of prewarmed digestive system (DNase 0.1 mg/ml; Trypsin 0.4 mg/ml; Collagenase I 1 mg/ml) and digested the DRGs for 40 minutes in an incubator (37°C, 5%CO 2 ). After digestion was stopped, the cell suspension was filtered through a 100 μm membrane filter and plated onto a poly D-lysinecoated confocal dish with DMEM complete medium.

Disease Markers
Approximately 1 d later, the Neurobasal complete medium with the addition of 10 μm cytarabine (Ara-C) was replaced. After 3 days, ADSC-Exos were added to the DRGs. The control group contained no exosomes. 2.11. miRNA Sequencing of Exosomes and Bioinformatic Analysis. Total RNA from ADSC-Exos and SC-Exos was isolated for miRNA analysis using the mirVana™ miRNA Isolation Kit (AM1560; Ambion) according to the manufacturer's protocol. Reverse transcription, library construction, miRNA sequencing, and analysis were conducted by OBIO Biotech Company. Criteria applied to select differentially expressed miRNAs were p < 0:05 and fold-change ≥2 or ≤0.5. The potential binding sites of the miRNAs were predicted using the online software TargetScan (http:// www.Targetscan.org/vert_72/).

2.12.
Dual-Luciferase Reporter Assays. The 3 ′ -UTR of the PTEN fragment containing wild-type or mutant binding sites for miR-22-3p was cloned into the pmirGlO luciferase reporter vector (Asia Vector Biotech) to generate the wildtype or mutant plasmids, respectively. miRNA-NC or miR-22-3p was transfected with luciferase reporter plasmids into Schwann cells. After 72 h, luciferase activity was evaluated using a dual-luciferase assay kit (E1910; Promega). All primers used are presented in Supplementary S1.

Quantitative Real-Time Polymerase Chain Reaction.
Total RNA was isolated using the miRNeasy Mini Kit (217004, QIAGEN), according to the manufacturer's protocol. The HyperScript ® RT Supermix Reagent Kit (R202, NovaBio) and HyperScript™ miRNA 1st Strand cDNA synthesis kit (R601, NovaBio) were used to reverse transcribe mRNA and miRNA into cDNA. Real-time qPCR was per-formed on the StepOnePlus™ platform (Applied Biosystems) using a SYBR ® qPCR Mix kit (Q204, NovaBio). Primers and reaction conditions are provided in Supplementary S2. The relative expression levels of the target genes were calculated using the 2 − ΔΔCt method and normalized to GAPDH or U6.
2.14. Western Blot. RIPA lysis buffer (89900, Thermo Fisher) was used to lyse the samples, which were then centrifuged at 12000 g for 20 min at 4°C. The protein concentration in the supernatant was calculated using a BCA protein assay kit (P0012; Beyotime). The samples were loaded onto an SDS-PAGE gel, electrophoresed, and transferred to the PVDF membrane. The membranes were incubated with primary antibodies (1 : 1000; CST and ABclonal) overnight at 4°C and with the corresponding secondary antibodies (1 : 20000; Proteintech) at room temperature for 1 h. Immunoblots were observed with an enhanced chemiluminescence reagent kit (P2300; NCM).

Statistical Analysis.
All data are expressed as the mean ± SD. Comparisons between two groups were evaluated using an unpaired Student's t-test. One-way ANOVA with a Bonferroni posthoc test was used for groups ≥3. Statistical significance was set at p < 0:05. Statistical analysis was performed using the GraphPad Prism 8.0.2 software.

Identification of ADSCs.
ADSCs were isolated from SD rats and passaged to the 3rd generation. Flow cytometry analysis revealed that the ADSCs were positive for MSC markers, including CD29 (99.71%) and CD90 (98.64%), and negative for CD45 (0.43%) and CD11b/c (0.37%) (Figure 1(a)). To verify the multipotent differentiation ability, we conducted adipogenic and osteogenic experiments, and the results indicated that they exhibited significant differentiation potential (Figure 1(b)).

Characterization and Internalization of ADSC-Exos.
Next, we characterized ADSC-Exos by isolating exosomes from the conditioned medium of the ADSCs. First, exosomes were resuspended in PBS and characterized using transmission electron microscopy (TEM). ADSC-Exos exhibited the characteristic structure of exosomes ( Figure 2(a)). Second, we used NTA to confirm the size and concentration of released exosomes. The mean particle diameter was 110.2 nm, with a concentration of 1:5 × 10 10 particles/ml (Figure 2(b)). Finally, western blot analysis of the purified exosomes revealed the expression of exosomal markers CD9 and ALIX, as well as the negative marker GAPDH (Figure 2(c)). Exosomes were labeled with PKH67 to determine whether ADSC-Exos were taken up by Schwann cells. We cocultured different concentrations (5, 10, and 20 μg/ml) of ADSC-Exos with Schwann cells. After 24 h, confocal imaging revealed the presence of PKH67 spots in the recipient cells, indicating that the labeled exosomes released by ADSCs could be delivered to Schwann cells, and the uptake efficiency was concentration-dependent ( Figure 2(d)).

Effects of ADSC-Exos on DRG Neurite Growth and SCs
Proliferation and Migration. We isolated rat primary DRGs to determine the effects of ADSC-Exos on neurite growth. After coculturing with ADSC-Exos for 3 days, immunofluorescence staining was performed to evaluate the effect of ADSC-Exos on DRG neurite growth compared with the PBS-treated control group (Figure 3(a)). The DRGs clearly developed neurites in the ADSC-Exos group, and the length of the longest neurite was significantly increased compared with that of the control group (Figure 3(b)). Next, we determined whether ADSC-Exos affected the migration and proliferation of Schwann cells. The migration assay revealed that ADSC-Exos markedly enhanced cells migration at 48 h (Figures 3(c) and 3(d)). In addition, the CCK-8 assay indicated that ADSC-Exos promoted the proliferation of Schwann cells, and there was a statistical difference between the two groups at 72 h (Figure 3(e) and 3(f)).
3.4. Exosomal miRNA Sequencing. miRNA sequencing results revealed that 124 miRNAs were upregulated in ADSC-Exos compared to SC-Exos (p < 0:05) (Supplementary S3). The distribution of the differentially expressed miR-NAs of ADSC-Exos compared to SC-Exos is shown in a volcano plot, as shown in Figure 4(a). Using the KEGG database, the target genes were found to be enriched in several signaling pathways, in particular, 143 miRNAs were enriched in the mTOR signaling pathway (Figure 4(b)). We selected ten miRNAs with high different expression (p < 0:05) in sequencing and high score in the TargetScan software as candidates (Figure 4(c)). The results indicated that miR-22-3p has significant potential, and Pten (tensin homolog deleted on chromosome 10) was predicted to be its target gene (Figure 4(d)).

miR-22-3p Directly Targets Pten in SCs.
To explore the mechanism of miR-22-3p regulation, a bioinformatic analysis was performed, which revealed that the target site was located at 674-681 bp in the 3 ′ UTR of Pten. Therefore, we designed a luciferase vector containing a wild-type or mutant Pten 3 ′ -UTR ( Figure 5(a)). To verify whether miR-22-3p directly targets the 3 ′ UTR of Pten directly, a dual luciferase reporter assay was conducted. The results showed that the luciferase activity of the Pten WT 3 ′ UTR was significantly reduced after cotransfection with miR-22-3p compared to that in the NC group. In contrast, no significant direct interaction was observed between miR-22-3p and the vector containing the Pten MUT 3 ′ UTR ( Figure 5(b)).    (Figure 5(d)). Accordingly, we performed transwell and CCK-8 assays. Upregulation of miR-22-3p significantly increased cell migration, whereas the miR-22-3p inhibitor decreased cell motility in Schwann cells (Figure 6(a) and 6(b)). The CCK-8 assay revealed that the mimic also promoted the proliferation of Schwann cells, whereas the inhibitor had a negative effect (Figure 6(c)). Overall, these results indicate that exosomal miR-22-3p promotes proliferation and migration of Schwann cells by activating the AKT/mTOR signaling pathway.

Discussion
Although the vast majority of PNIs do not threaten the lives of patients, persistent damage brings not only long-term 7 Disease Markers physical and mental harm to patients but also a heavy burden on families and society [19]. Following PNI, massive proliferation of SCs is triggered to form Bungner bands, which provide a large number of nutritional substrates for axon growth, including a considerable amount of extracellular matrix proteins and a variety of neurotrophic factors [20]. Additionally, SCs form a "myelin scaffold" to support axons extending distally at an average speed of 1 mm/day [21,22]. These findings demonstrate that changes in the proliferative and migratory abilities of SCs during the repair of PNIs can influence the regeneration of nerve axons.
In the present study, we selected ADSCs as subjects because they have the advantage of minimal tissue damage after sampling, mass availability, repeatability, and high clinical value [23]. We successfully isolated primary ADSCs, identified the surface antigens using flow cytometry, and confirmed their adipogenic and osteogenic abilities through direction-induced differentiation. ADSCs promote the repair of PNIs; however, it has been reported that a paracrine mechanism is involved in MSC-mediated tissue repair, whereas direct differentiation of stem cells is weak [24,25]. Considering the limitations of stem cell applications and the fact that the paracrine function of stem cells can be mediated by exosomes, the use of exosomes to replace stem cells for direct treatment has become a feasible alternative method.
By coculturing rat ADSC-Exos with SCs, we found that ADSC-Exos not only promoted SC proliferation but also enhanced their migratory ability, which was demonstrated for the first time. As mentioned above, SC proliferation results in the formation of new myelin channels that promote axonal regeneration. The results of our DRGs' experiment confirmed this hypothesis. Some researchers cocultured conditioned medium from mouse ADSCs with SCs and DRGs and found that the proliferation of SCs were enhanced as well as the growth of DRG axons [26]; the underlying reason may be that exosomes in the conditioned medium are responsible for the above effects. Another study reported that human ADSC-Exos significantly promoted SC proliferation, migration, and myelination [13], which are consistent with the results of this study.
However, the mechanism by which ADSC-Exos promote SC proliferation and migration remains unclear. Exosomes can carry a variety of bioactive substances and mediate intercellular signal transduction in MSC-derived exosomes mediate intercellular signal transduction [12,27]. In the 8 Disease Markers peripheral nervous system, exosomes can transport miRNAs to axons [28]. Therefore, exosomal miRNAs may be key elements in the effects of exosomes, and identifying the relevant miRNAs in exosomes is a prerequisite to clarify the role of exosomes in promoting peripheral nerve regeneration and postinjury repair. We speculated that miRNAs in rat ADSC-Exos may play an important role in promoting the proliferation and migration of SCs. To further explore the role of miRNAs, we performed exosomal miRNA sequencing and subsequent bioinformatic analysis. Sequencing results revealed that 124 miRNAs were highly expressed in ADSCs. Based on KEGG pathway analysis, 143 miRNAs were enriched in the mTOR pathway. To identify the miRNAs associated with SC proliferation and migration, miR-181a-3p, miR-365-3p, miR-22-3p, miR-22-5p, miR-181a-5p, miR-181b-5p, miR-129-5p, miR-431, miR-212-5p, and miR-365-3p were selected for further bioinformatic analysis. TargetScan database search revealed that miR-22-3p is very likely to bind directly to phosphatase and tensin homolog deleted on chromosome 10 (PTEN). In addition, overexpression of miR-22-3p in HK-2 cells can directly inhibit PTEN [29]. Consequently, we focused on miR-22-3p and examined the target gene, Pten, of miR-22-3p in the Akt/mTOR pathway. PTEN is an inhibitor of the Akt/mTOR signaling pathway. Following PTEN downregulation, Akt is activated and phosphorylated to promote the activation of downstream mTOR. As an important signal transduction pathway, the PI3K/Akt/mTOR pathway plays an important biological role in cell growth, survival, proliferation, apoptosis, angiogenesis, and autophagy [30]. Conditional knockout of PTEN gene can promote regeneration of corticospinal tract (CST) axons and recovery of motor function in mice [31]. Another study also showed that PTEN knockout promotes CST regeneration, and the mechanism may involve increased mTOR activity, thus enhancing the regeneration ability of cortical neurons [32]. This indicates that repair of the central nervous system can be promoted by enhancing Akt/mTOR signaling activity. In our previous study, we found that negative regulation of PTEN activates the PI3K/Akt signaling pathway and promotes repair of the recurrent laryngeal nerve [33]. Moreover, the results of this study further confirmed that miR-22-3p directly binds to PTEN and promotes the phosphorylation of the downstream Akt/mTOR axis by inhibiting PTEN expression, thus promoting the proliferation and migration of SCs. Regarding future clinical translation for the treatment of PNIs, ADSCs may be extracted from the adipose tissue of patients and exosomes overexpressing miR-22-3p may be obtained after rapid amplification. Alternatively, exosomes may be obtained by overexpression of miR-22-3p and transplanted into the site of PNIs combined with nerve conduits. independent experiments with duplicates. Data are expressed as means ± SD. Statistical significance was obtained with unpaired Student's t test. * means p < 0:05; * * means p < 0:01; * * * means p < 0:001. ADSC-Exos, the exosomes extracted from Adipose-derived mesenchymal stem cells; SCs, Schwann cells; miR, miRNA.

Conclusion
In this study, we confirmed that ADSC-Exos promoted the proliferation and migration of SCs and the growth of DRG axons. Through exosomal sequencing and bioinformatic analysis, miR-22-3p was identified and its role in activating the Akt/mTOR axis by inhibiting PTEN was confirmed. These results provide a new strategy for the treatment of PNIs with stem cell-derived exosomes as a novel treatment method for PNIs.

Data Availability
All data used to support the findings of this study are available from the corresponding author upon request.

Ethical Approval
Animal experiments were approved by the Experimental Animal Care and Ethics Committee of the Institute of Shanghai General Hospital, Shanghai Jiao Tong University, School of Medicine.