Adipose stem cell‐derived exosomes promote wound healing by regulating the let‐7i‐5p/GAS7 axis

Injury to skin tissue is devastating for human health, making it imperative to devise strategies for hastening wound healing. Normal wound healing is a complex process comprising overlapping steps, including hemostasis, inflammatory response, proliferation, and matrix remodeling. This study investigated the effects of adipose stem cell‐derived exosomes (ADSC‐exos) on wound healing and the underlying mechanisms.

response, proliferation, and matrix remodeling. 3,4Clinically chronic non-healing wounds have been reported to have higher incidence and recurrence rates, bringing a substantial economic burden.Skin regeneration following a wound encompasses a variety of cells, where keratinocytes have been found to play a pivotal role in wound healing.Therefore, investigating the pathological mechanism of keratinocytes in chronic non-healing wounds is envisaged to provide novel wound-healing treatment methods. 5ipose-derived stem cells (ADSCs) are a group of cells within the adipose matrix 6 that have multi-directional differentiation potential, including osteoblasts, chondrocytes, and adipocytes in vitro.They are primarily derived from adipocytes but can also be obtained through liposuction or excised fat. 7Compared to other mesenchymal stem cells (MSCs), ADSCs have easy access, large quantities, and unique immune regulatory functions. 8They promote skin regeneration by upregulating the secretion levels of epidermal and vascular endothelial growth factors and play roles in repairing damaged bone tissue and cartilage. 9It has been reported recently that ADSCs facilitate tissue repair through their paracrine action, where exosomes are the major paracrine function products.
Exosomes are extracellular vesicles secreted into intercellular spaces through the endosome pathway, having a diameter of 30-100 nm and a density of 1.13-1.19g/mL. 10,11Exosomes can be secreted by different cells, including T and B cells, platelets, and MSCs, and body fluids rich in exosomes, including blood, urine, and milk. 12Increasing evidence has demonstrated that adipose mesenchymal stem cell-derived exosomes (ADSCs-exos) promote wound healing, 13 but the underlying mechanism remains to be investigated.
This study investigated the effects of ADSCs-exos on migration and inflammation of human keratinocyte (HaCaT) cell lines, followed by evaluating regulation growth-arrest-specific-7 (GAS7) levels elicited by ADSCs-exos-derived microRNA-let-7i-5p (let-7i-5p).The findings of this study are envisaged to open novel theoretical horizons in applying the let-7i-5p/GAS7 axis in skin wound healing.

| Collection of ADSCs
Subcutaneous adipose tissues were harvested from patients who underwent liposuction at the hospital after obtaining informed consent from all subjects and institutional ethical committee approval.
The collected tissues were washed with phosphate-buffered saline (PBS) and trimmed into 1 mm 3 sections.The tissues were digested with 0.1% collagenase I (Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 0.5 h, followed by centrifugation.The sedimented ADSCs were resuspended using PBS and maintained in Dulbecco's Modified Eagle Medium F12 (DMEM/F12; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 100 U penicillin/100 mg/mL streptomycin (Gibco) under humidified 5% CO 2 at 37°C.The ADSCs were passaged several times, and cells at 3-5 passages were used in the subsequent experiments.

| Characterizations of ADSCs
The ADSCs were identified following isolation by culturing in an osteogenic induction medium for 14 days, while their osteogenic differentiation ability was evaluated using alkaline phosphatase and alizarin red staining.The stem cell surface markers were investigated using flow cytometry with FITC-conjugated anti-CD34, anti-CD44, anti-CD90, and anti-CD105, as previously reported. 14

| Exosomes isolation
Exosomes were isolated from ADSCs using the exosome isolation kit (Yessen, Shanghai, China) according to the manufacturer's protocol.The cell culture medium was collected and centrifugated at 3000 g for 10 min to remove cell debris and impurities.The supernatant (10 mL) was vortexed with 2.5 mL extraction reagent for 1 min and allowed to stand for 2 h.Following centrifuging at 10000 g for 60 min, the sediment was collected and resuspended using PBS.The collected ADSC-exos were stored at −80°C until further use.The exosome authentication was done using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blot.

| Transmission electron microscopy (TEM)
Exosome morphology was determined using TEM.The exosomes were added to carbon-pre-coated copper mesh.After fixing with 1% glutaraldehyde, the copper mesh was stained with 10 μL uranyl acetate for 30 s in the dark.After drying, the images of exosomes were photographed under a TEM at 120 kV.

| Nanoparticle tracking analysis (NTA)
The exosome size was evaluated using NTA. 13Briefly, the exosomes were diluted using PBS until 20-100 objects per frame, followed by analysis using the Nanosight LM10 system (Nanosight technology, Malvern) with the NTA analytical software (version 2.3).

| Cell treatment
HaCaT cells at the logarithmic phase were seeded into 96-well plates, treated with 20 μg exosomes premixed with cell culture medium, and incubated under conditions detailed earlier.The in vitro model was developed by treating HaCaT cells with 1 mM H 2 O 2 , while exosome release was inhibited using a 10 mM exosome inhibitor (GW4869, Sigma-Aldrich, USA) added to the culture medium of the transwell system.

| Western blot
The surface markers of exosomes Calnexin, CD9, and CD63 were analyzed using western blot.Total protein was extracted from ADSC-exos using WB lysis buffer (Yeasen) and resolved using 10% SDS-PAGE, followed by transfer to PVDF membranes (Beyotime).

| Transwell assay
Cell migration was assessed with a 24-well transwell system containing 8 μm pore-sized filters without Matrigel.The HaCaT cells suspended in serum-free medium were added to the upper chambers, and the cell culture medium was filled into the lower chambers.
The filters were immobilized using 4% paraformaldehyde under ambient conditions after 24 h of incubation.The migrated cells were stained with 0.1% crystal violet and observed under an inverted light microscope (Olympus, Tokyo, Japan).The wound healing rate was then calculated using the following equation.

| Wound healing assay
The HaCaT cells were seeded into 6-well plates and incubated until they reached 100% confluence, followed by wound creation in each plate using a 1 mL sterile pipette tip.The cells were gently washed with PBS, and floating cells were decanted.The cells were then incubated in the serum-free medium at 37°C for 24 h.
Respective sections on each plate were photographed at 0 h and 24 h intervals.The percentage of wound healing was analyzed using ImageJ software (version 1.52a; Media Cybernetics, Silver Springs, MD, USA).

| Alizarin red S (ARS) staining and activity
ADSCs at passage 3 were cultured in growth medium for 24 h, then transferred to osteogenic induction medium (OM) consisting of growth medium +50 μg/mL ascorbic acid, 5 mM β-glycerophosphoric acid, and 100 nM dexamethasone, and placed in a high glucose and proinflammatory environment.The medium was changed every 2 days.After osteogenic induction for 21 days, Alizarin Red staining (ARS) solution (Beyotime, Shanghai, China) was used to detect intracellular calcium deposition.The staining was observed and photographed using an inverted phase contrast microscope (Leica, Germany).

| Quantitative real-time PCR
Total RNA was extracted from HaCaTs using the TRIzol reagent (Invitrogen).Following detection of RNA purity at 260/280 nm and integrity using agarose gel electrophoresis, reverse transcription was performed using the Quant cDNA first-strand synthesis kit (TIANGEN, Beijing, China).Subsequently, the SuperReal premix plus (SYBR Green) (TIANGEN) was used for qPCR.The quantification of mRNA expression was calculated using the 2 −ΔΔC t method.GAPDH was used as an internal reference.

| Statistical analysis
All experiments were conducted three-time repetition.The data were accessed using the GraphPad Prism software (version 8.0, La Jolla, CA, USA) and are expressed by mean ± SD.Comparisons were analyzed using the unpaired Student's t-test (two groups) or one-way ANOVA (multiple groups).p < 0.05 means a statistically significant difference.

| Identification of ADSCs
The flow cytometry results (as shown in Figure 1A) showed that CD44, CD90, and CD105 were expressed but barely CD34 by ADSCs.The ARS and oil red O staining results showed that ADSCs could differentiate into osteoblasts (Figures 1B,C).These results cemented the identification of isolated ADSCs.

| Characterizations of ADSC-exos
The ADSC-exos characterization results showed bilayer membranecupped vesicular geometry, as shown in Figure 2A, where most of the exosomes had sizes of 100-200 nm (Figure 2B).Moreover, the levels of exosome markers, including CD9 and CD63, were higher in ADSC-exos compared to ADSCs, while the Calnexin expression in ADSC-exos was significantly lower than in ADSCs (Figure 2C).
These results demonstrated the successful isolation of exosomes from ADSCs.

| ADSC-exos promoted the cell viability and migration of HaCaT under H 2 O 2 treatment
The HaCaT cells were treated with

| ADSC-exos exerts its roles via transporting let-7i-5p
The underlying mechanism of ADSC-exos was investigated by detecting the expression of several previously reported mRNAs that play critical roles in the biological function of exosomes.The qRT-PCR results showed that after exosome treatment, the level of miR-let-7i-5p was significantly elevated (Figure 4A).The miR-let-7i-5p-mediated effect of ADSC-exos was verified by knocking down miR-let-7i-5p in ADSCs followed by exosome extraction.The qPCR results showed that anti-let-7i-5p reduced the let-7i-5p level in the ADSC-exos (Figure 4B).

| GAS7 expression was targeted and inhibited by let-7i-5p
miRNAs play their roles in physiological processes via binding with various targets; thus, the potential target of let-7i-5p was further predicted.Results showed that among various candidates, the GAS7 was found to exert its effect on cell growth.The potential binding between let-7i-5p and GAS7 is demonstrated in Figure 5A, where luciferase assays showed that let-7i-5p could directly bind with GAS7 (Figure 5B).

| GAS7 overexpression reversed the effects of let-7i-5p on the viability and migration of HaCaT
Rescue experiments were performed to verify the interaction between let-7i-5p and GAS7 further.For this purpose, ago-let-7i-5p and GAS7 overexpressing vectors were co-transfected into HaCaT, and results showed that Ago-let-7i-5p and GAS7 overexpressing vectors significantly elevated the levels of let-7i-5p and GAS7, respectively (Figures 6A,B).Moreover, the ago-let- that ADSC-exos exerted their roles via let-7i-5p.Furthermore, GAS7 overexpression significantly reversed the effects of let-7i-5p (Figures 6C-E).These results indicated that ago-let-7i-5p promoted the viability and migration of HaCaT by targeting GAS7.

| DISCUSS ION
An increasing number of studies have demonstrated the great potential of ADSC-exos in wound healing.Human adipose MSCderived exosomes have been reported to attenuate hypertrophic scar fibrosis via the miR-192-5p/IL-17RA/SMAD axis. 15Similarly, exosomes derived from adipose MSCs was found to promote diabetic chronic wound healing through SIRT3/SOD2, 16 while exosomal microRNA-125a-3p from human adipose-derived MSCs were found to promote angiogenesis of wound healing by inhibiting PTEN. 17Our results revealed that ADSC-exos facilitated the viability and migration of H 2 O 2 -treated HaCaT cells.
Mechanistically, ADSC-exos are responsible for transporting various biomacromolecules such as DNA, RNA, and proteins, while increasing evidence has demonstrated that ADSC-exos transport miRNAs to exert their biological functions.For instance, adiposederived stem cell-derived exosomes carried miR-138-5p 18 and miR-192-5p 15 to promote wound healing.Our results showed that ADSC-exos were responsible for carrying let-7i-5p.
The let-7i-5p has been extensively studied in cancer and has been found to play the roles of oncogene in clear cell renal cell carcinoma, 19 nasopharyngeal carcinoma, 20 glioblastoma, 21 while as an anti-cancer gene in colon 22 and bladder cancer. 23Moreover, let-7i-5p is contained in exosomes, where exosomal let-7i-5p from threedimensional cultured human umbilical cord MSCs has been reported to inhibit fibroblast activation in silicosis by targeting TGFBR1. 24milarly, let-7i-5p had been found to mediate the therapeutic effects of exosomes from human placenta choriodecidual membranederived MSCs on mitigating endotoxin-induced mortality and hepatic injury in high-fat diet-induced obese mice. 25The MSCs were also found to attenuate renal fibrosis via exosome-mediated delivery of microRNA let-7i-5p antagomir. 26However, it was still not reported in ADSC-derived exosomes, nor the let-7i-5p role in wound healing.
Our study demonstrated that let-7i-5p accelerated the HaCaT cell's viability and migration.
H 2 O 2 to generate an in vitro wound model.Results showed that H 2 O 2 treatment significantly inhibited cell viability and migration, which were reversed considerably by co-culturing HaCaT cells with ADSCs.To confirm these effects were exerted by ADSCs via exosomes, an exosome release inhibitor (GW4869) was added, and results showed that GW4689 treatment impeded the viability and migration of ADSCexos-treated HaCaT cells (Figures 3A-C), cementing the results that ADSC-exos facilitated the HaCaT cells viability and migration through exosomes.

F I G U R E 1
It was then proceeded by conducting functional studies, and results showed that let-7i-5p-silenced ADSC-exos partially lost their cell viability and migration of function in HaCaT cells (Figure 4C-E), cementing the postulation that the ADSC-exos effect was driven via let-7i-5p transportation into HaCaT.Characterization of ADSC.(A) The expression of ADSC surface markers CD34, CD44, CD90, and CD105 were detected using flow cytometry.(B, C) The osteogenic differentiation ability of ADSCs was evaluated using ARS and oil red O staining.

F I G U R E 2
Characterization of ADSC-exos.(A) The morphology of ADSC-exos was observed by TEM.(B) Exosome size was analyzed by NTA.(C) The expression of exosome surface markers CD9, CD63, and Calnexin were detected using a western blot.F I G U R E 3 ADSC-exos promoted the cell viability and migration of HaCaT cells following H 2 O 2 treatment.(A) The viability of HaCaT was evaluated by CCK-8 assay.(B) A Transwell assay was performed to detect the migration of HaCaT.(C) A wound healing assay was performed to detect the migration of HaCaT.*p < 0.05, **p < 0.01, ***p < 0.001.

F I G U R E 5
7i-5p promoted the viability and migration of HaCaT, cementing F I G U R E 4 ADSC-exo exerted its roles via transporting let-7i-5p.(A, B) The relative expression after different treatments was evaluated by qPCR.(C) The viability of HaCaT was evaluated by CCK-8 assay.(D) A Transwell assay was performed to detect the migration of HaCaTs.(E) A wound healing assay was performed to detect the migration of HaCaTs.**p < 0.01, ***p < 0.001.The let-7i-5p directly targets GAS7.(A) The potential binding site between let-7i-5p and GAS7 was predicted by targetscan8.0.(B) Luciferase assay was performed to detect the binding between let-7i-5p and GAS7.(C, D) The expression of GAS7 was evaluated under different treatments.

F I G U R E 6
GAS7 overexpression reversed the effects of let-7i-5p on the viability and migration of HaCaT.(A, B) The relative RNA expression was evaluated by qPCR.(C) The viability of HaCaT was evaluated by CCK-8 assay.(D) A Transwell assay was performed to detect the migration of HaCaT.(E) A wound healing assay was performed to detect the migration of HaCaT.**p < 0.01, ***p < 0.001.