Exosomal microRNA⁃93⁃3p secreted by bone marrow mesenchymal stem cells downregulates apoptotic peptidase activating factor 1 to promote wound healing.

Wounds are soft tissue injuries, which are difficult to heal and can easily lead to other skin diseases. Bone marrow mesenchymal stem cells (BMSCs) and the secreted exosomes play a key role in skin wound healing. This study aims to clarify the effects and mechanisms of exosomes derived from BMSCs in wound healing. Exosomes were extracted from the supernatant of the BMSCs. The expression of the micro-RNA miR-93-3p was determined by qRT-PCR analysis. HaCaT cells were exposed to hydrogen peroxide (H2O2) to establish a skin lesion model. MTT, flow cytometry, and transwell assays were conducted to determine cellular functions. The binding relationship between miR-93-3p and apoptotic peptidase activating factor 1 (APAF1) was measured using a dual luciferase reporter gene assay. The results showed that BMSC-derived exosomes or BMSC-exos promoted proliferation and migration and suppressed apoptosis in HaCaT cells damaged by H2O2. However, the depletion of miR-93-3p in BMSC-exos antagonized the effects of BMSC-exos on HaCaT cells. In addition, APAF1 was identified as a target of miR-93-3p. Overexpression of APAF1 induced the dysfunction of HaCaT cells. Collectively, the results indicate that BMSC-derived exosomes promote skin wound healing via the miR-93-3p/APAF1 axis. This finding may help establish a new therapeutic strategy for skin wound healing.

Introduction

Wound healing is a complex biological process. There are many factors that affect wound healing, including systemic factors (such as age, nutrition, endocrine function, drugs, etc.) and local factors (such as infection and oxidation). Wound healing is mainly achieved through inflammation, granulation, tissue filling, and reconstruction [1]. The epidermis functions as the first barrier of the skin [2]. The degradation of the epidermis stimulated by ultraviolet rays and oxidative factors is associated with skin aging, inflammatory skin diseases, and epidermal tumors [3,4].

MicroRNAs (miRNAs) are a set of non-coding RNAs with a length of approximately 18–23 nucleotides. Functionally, miRNAs bind to the 3ʹ-UTR of target mRNAs and inhibit gene expression at the post-transcriptional level [5]. miRNAs are key regulators of various biological processes, such as cell differentiation, proliferation, and apoptosis [6]. Recently, increasing evidence has demonstrated that miRNAs are involved in human skin repair and wound healing [7]. miR-93 belongs to the miR-106b-25 family [8]. Aberrant expression of miR-93 is associated with cancer development [9], brain injuries [10], and bone disorders [11]. Moreover, miR-93-3p promotes the proliferation and migration of HaCaT cells [12]. However, the potential role of miR-93-3p in wound healing remains unclear.

Exosomes are secreted by a variety of cells and exist in almost all body fluids [13], and exert similar cellular functions [14,15]. An increasing number of studies have found that the therapeutic effect of bone marrow mesenchymal stem cells (BMSCs) is mainly due to their paracrine mechanism, which has anti-inflammatory and protective effects. BMSC-derived exosomes play an important role in cell-to-cell communication [16]. In addition, compared with bone marrow mesenchymal stem cell therapy, transplantation of BMSC-derived exosomes has more advantages, such as non-immunogenicity, non-tumorigenicity, and convenient storage and transportation [17], suggesting that exosomes derived from BMSCs may be a promising strategy for skin wound treatment [18].

Apoptotic peptidase activating factor 1 (APAF1) is mainly involved in the signal transduction of the mitochondrial apoptosis pathway. Under the combined action of adenine deoxynucleotide triphosphate (dATP) and ATP, cytochrome C activated-APAF1 upregulates caspase-9 and caspase-3, thereby initiating the apoptotic cascade and cell apoptosis [19]. However, the molecular mechanism of BMSC-derived exosomes in wound healing has not been fully elucidated.

This study investigated the effect and mechanism of exosomes isolated from BMSCs on a H2O2-induced skin injury model. We hypothesized that BMSC-exos played a beneficial role in wound healing.

Methods and Materials

Cell culture and transfection

Human bone marrow mesenchymal stem cells (hBMSCs) and benign epidermal keratinocyte cell line HaCaT were purchased from ATCC (Manassas, VA, USA). DMEM/F12 medium (Hyclone, Logan, UT, USA) supplemented with 10% FBS (Hyclone) was used for the cultivation of hBMSCs or HaCaTs in a humid atmosphere at 37°C with 5% CO2. HaCaTs were incubated with 500 μM H2O2 for 4 h to establish the cell models.

miR-93-3p mimics, Anti-miR-93-3p and relevant negative control (NC mimic and Anti-NC; GenePharma, Shanghai, China), pcDNA 3.1-APAF1 or pcDNA 3.1-NC adenoviral vectors (HanBio Technology Co. Ltd., Shanghai, China) were used for HaCaT transfection using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer’s protocols. After 48 h, the cells were co-cultured with BMSCs-Exos for 48 h.

MTT assay

HaCaT cells were exposed to 0 μM, 100 μM, 500 μM, and 1000 μM H2O2. For the MTT assay, target cells were cultured in 96-well plates at a density of 2 × 103 cells/mL and cultured for 48 h. Then cells were incubated with 10 μL of MTT solution (0.5 mg/mL; Beyotime, Shanghai, China) for 4 h. A spectrophotometer (BioTek, Winooski, VT, USA) was used to measure absorbance at 490 nm.

Flow cytometry

An Annexin V PE/7-AAD tool kit (Solarbio, Beijing, China) was used to detect apoptosis. The cells were resuspended at a density of 1 × 105/ml. After incubation with Annexin V PE and 7-AAD, according to the manufacturer’s protocols, the cells were detected using a flow cytometer (Verse, BD, USA).

RT-qPCR assay

Total RNA was isolated from cells using TRIzol reagent (Invitrogen). cDNA synthesis was performed using the GoScript™ Reverse Transcription System (Promega, WI, USA). PCR was conducted using SYBR Premix EX Taq (Takara, Dalian, China) on a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The primers were purchased from the Sangon Biological Engineering Technology Company (Shanghai, China). The results were calculated using the 2−ΔΔCt method. GAPDH and U6 were used as internal controls for mRNAs and miRNAs.

Western blotting

The cells were harvested, lysed with RIPA lysis buffer, and centrifuged. Protein concentration was determined using the BCA method. The total protein (20 μg) was separated by 10% SDS-PAGE gel electrophoresis (90 V for 30 min, 120 V for 1 h) and then transferred to a PVDF membrane (400 mA for 2 h). The membranes were blocked with 5% BSA for 1 h and incubated with primary antibodies, such as CD63, CD9, TSG101, and GAPDH at 4°C overnight, and then with a horseradish peroxidase-labeled secondary antibody at room temperature for 1 h. The membrane was then washed with TBST buffer for 30 min, and the SuperSignalTM chemiluminescence kit was used to visualize the target protein expression of each group of cells.

Isolation and identification of exosomes

A Total Exosome Isolation tool kit (Invitrogen, Carlsbad, USA) was used to isolate exosomes from the hBMSC supernatant according to the manufacturer’s protocol. Cells, at 80% confluence, were added with serum-free culture medium. The supernatants were collected after culturing for 24 h. Then the exosomes were resuspended in 0.01 M PBS and centrifuged at 100,000 × g for 70 min for purification.

Transmission electron microscopy (TEM) was performed using H-7650 (Hitachi Corp., Tokyo, Japan). Subsequently, a nanoparticle tracking analyzer (Particle Metrix, Germany) was used to detect the size, distribution, and number of the isolated particles. Total RNA and protein of the exosomes were extracted using a Total Exosome RNA and Protein Isolation Kit (Invitrogen).

Dual-luciferase reporter assay

Putative miR-93-3p binding sites of APAF1 were first predicted using TargetScan (http://www.targetscan.org/vert_72/). The wild type (WT)-APAF1 3ʹ-UTR and mutant (MUT)-APAF1 3ʹ-UTR were cloned into the pmirGLO luciferase reporter vector (Promega, WI, USA). The cells were then co-transfected with miR-93-3p mimics (GenePharma, Shanghai, China) or NC mimic (GenePharma, Shanghai, China) pmirGLO vectors. A Dual-Luciferase® Reporter Assay Kit (Solarbio, Beijing, China) was used to detect luciferase activity.

Migration assay

Transwell was performed previous described [20]. Briefly, after transfection, cells were collected and resuspended in 200 μL of serum-free culture medium and then placed in the upper chamber of the culture system, whereas the lower chamber was filled with normal medium. The migrated cells were fixed with 4% formaldehyde after 24 h of incubation, and 0.1% crystal violet solution was used to stain the target cells. The cells that migrated into the lower chamber were captured using a microscope and calculated.

RNA pull-down

RNA pull-down was performed as previously described [21]. Briefly, cells were seeded on a 10 cm petri dish and cultured overnight. Cells were transfected with biotin-labeled miR-93-3p mimic (probe customized by RiboBio) at a final concentration of 100 nmol/L. The control group was transfected with biotin-labeled NC sequence dye. Cells were collected after 48 h of transfection. The cell lysate was added and incubated at 4°C for 4 h; 50 μL streptavidin magnetic beads were added to the cell lysate, and rotated at 4°C for 30 min. After centrifugation, the magnetic beads were collected. The results were determined using RT-PCR.

Statistical analysis

Data were analyzed using SPSS 18.0 and presented as the means ± standard deviation (SD). Student’s t-test or one-way ANOVA was used to evaluate the statistical differences. Statistical significance was set at p < 0.05.

Results

This study investigated the potentials of BMSC-exos in wound healing and the underlying molecular mechanisms. BMSC-exos derived miR-93-3p promoted the migration and suppressed the apoptosis of HaCaT cells via targeting APAF1.

Isolation and characterization of BMSCs and BMSC-exos

We collected and isolated exosomes from BMSCs. Transmission electron microscopy and nanoparticle tracking analysis showed that the exosomes were cup-shaped or circular membrane-bound vesicles with a diameter of 100–300 nm (Figure 1a,b). The expression of exosomal markers CD63, CD9, and TSG101 was decreased in the BMSC-derived exosome (BMSC-exos) group (Figure 1c). In addition, miR-93-3p expression was significantly upregulated in the BMSC-exos group (P < 0.001) (Figure 1d).

Figure 1.

Isolation and characterization of BMSCs. (a) The morphology of exosomes (200 nm) was observed under TEM. (b) The particle size distribution and concentration in exosomes was analyzed by nanoparticle tracking analysis. (c) The protein expression of CD63, CD9, and TSG101 was measured by Western blot assay. (d) The qRT-PCR assay measures the expression of miR-93-3p. *** P < 0.001 vs. BMSCs

Construction of skin lesion model

HaCaT cells were treated with 0, 100, 500, and 1000 µM concentrations of H2O2, to construct a skin injury model. The results of the MTT assay showed that H2O2 significantly inhibited the viability of HaCaT cells, indicating that H2O2 can induce skin damage (Figure 2a). The results from flow cytometry showed that compared with the control, H2O2 exposure significantly promoted the apoptosis of HaCaT cells (Figure 2b).

Figure 2.

Construction of skin lesion model in vitro. (a) HaCaT cell viability was determined by MTT assay. (b) The apoptosis rates of HaCaT cells were detected by flow cytometry. ** P < 0.01, *** P < 0.001 vs. 0 μm group

BMSC-exos inhibited cell proliferation and migration and suppressed the apoptosis of HaCaT cells

As shown in Figure 3a, BMSC-exos administration increased the viability of HaCaT cells in a dose-dependent manner. Moreover, BMSC-exos significantly reduced the apoptosis rates of HaCaT cells induced by H2O2 (Figure 3b). The inhibition of the migratory ability of HaCaT cells induced by H2O2 was also significantly alleviated by BMSC-exos (Figure 3c). The above findings suggest the protective effects of BMSC-exos on skin wound healing.

Figure 3.

The effects of BMSC-exos on the proliferation, apoptosis and migration of HaCaT cells damaged by H2O2. (a) The cell viability of HaCaT cells was measured by MTT assay. (b) Cell apoptosis of HaCaT was determined by flow cytometry. (c) The cell migration ability of HaCaT cells was analyzed by transwell assay. ***P < 0.001 vs. control, ##P < 0.01 vs. H2O2 + PBS group

miR-93-3p knocked-down BMSC-exos lose their protective effect on H2O2-damaged HaCaT cells

We further verified whether BMSC-exos containing miR-93-3p played a role in mitigating skin lesions. miR-93-3p expression was significantly decreased in the anti-miR-93-3p group and increased in the miR-93-3p mimic group, suggesting successful transfection (Figure 4a). MTT test results showed that BMSC-exos greatly increased the viability of H2O2-damaged HaCaT cells, which was significantly inhibited after exposure to anti-miR-93-3p (Figure 4b). Similarly, flow cytometry showed that after BMSC-exos administration, the apoptosis of HaCaT cells was significantly reduced, and this was significantly antagonized by miR-93-3p knockdown (Figure 4c). Additionally, the increase in the migratory ability of HaCaT cells induced by BMSC-exos was alleviated by anti-miR-93-3p (Figure 4d).

Figure 4.

miR-93-3p-silenced BMSC-exos lose their protective effect on HaCaT cells damaged by H2O2. (a) The expression of miR-93-3p was evaluated by qRT-PCR method. (b) The cell viability of HaCaT cells was detected by MTT assay. (c) Flow cytometry was used to detect cell apoptotic rates. (d) The migration ability of cells was assessed by Transwell assay. **P < 0.01, ***P < 0.001 vs. Anti-NC or control group; ## P < 0.01 vs. H2O2 group; & P < 0.05, && P < 0.01, &&& P < 0.001 vs. H2O2 + BMSC-exos group

APAF1 was target for miR-93-3p in HaCaT cells

Figure 5a shows the binding site between apoptotic peptidase activating factor 1 (APAF1) and miR-93-3p. Luciferase assay revealed that luciferase activity was significantly decreased in cells co-transfected with APAF1 3ʹ-UTR WT and miR-93-3p mimic (Figure 5b). RNA pull-down further verified the interaction between APAF1 and miR-93-3p (Figure 5c). Moreover, downregulation of miR-93-3p significantly increased the expression of APAF1 (Figure 5d-e).

Figure 5.

APAF1 was a target of miR-93-3p. (a) The binding sites between miR-93-3p and APAF1. (b) Relative luciferase activity of HaCaT cells. (c) The interaction between APAF1 and miR93-3p was analyzed by RNA pull-down. (d) Relative expression of APAF1 was detected by RT-qPCR. (e) The protein expression of APAF1 was determined by Western blot. *** P < 0.001 vs. NC mimic, Biotin-NC or Anti-NC group

APAF1 overexpression induced dysfunction of HaCaT cells

Rescue assays were performed to further verify the potential of APAF1 in wound healing. As shown in Figure 6a, the expression of APAF1 was significantly increased by APAF1 overexpression plasmids. Such overexpression of APAF1 significantly decreased the viability and migration ability of HaCaT cells (Figure 6e and e). Additionally, APAF1 overexpression significantly augmented the apoptosis rate of HaCaT cells (Figure 6c and d).

Figure 6.

Exosomes secreted by BMSCs exerted their roles via regulating miR-93-3p/APAF1 axis. (a) The expression of APAF1 was analyzed by RT-qPCR. (b) Cell viability was detected by MTT assay. (c and d) The apoptosis rates were detected by flow cytometry. (e) The migration of HaCaT cells was examined by a Transwell assay. ***P < 0.001 vs. H2O2 or control; ##P < 0.01 vs. H2O2; &P < 0.05 vs. H2O2 + BMSC-exos + NC mimic + vector; $$P < 0.01 vs. H2O2 + BMSC-exos + miR-93-3p mimic + APAF1

Discussion

In this study, BMSC-derived exosomes restored the cellular functions of HaCaT cells degraded by H2O2. Moreover, BMSC-exos induced the upregulation of miR-93-3p and exerted its protective role by regulating the miR-93-3p/APAF1 axis. Therefore, BMSC-derived exosomes may be a promising therapeutic agent for wound healing.

Long-term unhealed wounds and hyperplasia of scar tissue not only affect the daily life of patients, but also lead to adverse effects on patients’ psychology [22], and bring a huge economic burden to society [23]. Therefore, finding a way to accelerate wound healing, reduce scar formation, and promote wound repair seem to play a vital role in wound management [24]. Bone marrow mesenchymal stem cells (BMSCs) are an important type of non-hematopoietic stem cells in the bone marrow. BMSCs are easy to obtain, and possess strong self-replication ability and multi-directional differentiation properties. BMSCs exert protective functions by increasing the release of cytokines and growth factors, promoting collagen synthesis and granulation tissue growth, and inducing immune reconstitution [25-28]. The paracrine function of BMSCs can mediate their beneficial therapeutic effects on a variety of diseases. Paracrine substances include a variety of cytokines, exosomes, and nucleic acids [29]. Exosomes derived from bone marrow mesenchymal stem cells have therapeutic effects in a variety of diseases [30]. In this study, BMSC-derived exosomes restored HaCaT cells, as manifested by the increase in cell viability and migration ability, and decrease in apoptosis rates of the latter. These results indicate that BMSC-secreted exosomes may play a protective role in wound healing, which is consistent with the results of previous studies. Moreover, compared with other MSC, such as umbilical cord MSC, umbilical cord blood MSC, amniotic fluid MSC, epidermal MSC, bone marrow MSCs (BMSCs) have shown good results in repairing damaged tissues in various degenerative diseases, in animal models and human clinical trials [31-35]. Therefore, it is of vital importance to investigate the underlying mechanisms that BMSC-secreted exosomes promotes wound healing.

Exosome-derived miRNAs play anti-tumor, anti-inflammatory, cardiac-protective and wound healing roles [36-39]. miRNAs can affect eukaryotic cell proliferation, differentiation, apoptosis, and other processes. Dysregulated miRNAs are closely associated with the occurrence of various diseases [40]. For instance, miR-93, belonging to the miR-106b-25 family, functions as a tumor suppressor and inhibits the invasion and metastasis of tumor cells [40]. Moreover, miR-93-3p has a beneficial role in wound healing [12]. In this study, miR-93-3p was upregulated in BMSC-exos-treated cells. However, downregulation of miR-93-3p antagonized the effects of BMSC-exos on the proliferation and migration of HaCaT cells. Therefore, BMSC-exos are thought to exert a protective role by upregulating miR-93-3p. Moreover, miR-93-3p plays a beneficial role in wound healing, which is consistent with the results of a previous study.

Apoptotic peptidase activating factor 1 (APAF1) is a core pro-apoptotic factor in the cytochrome C-dependent apoptosis signaling pathway [41]. Its expression is significantly downregulated in many tumors, suggesting that it is a tumor suppressor gene [42]. Induction of apoptosis is an important goal in tumor therapy. Apoptosis is a process of programmed death that occurs in multicellular organisms. It is regulated by multiple genes [43,44]. In the present study, APAF1 was predicted to be a target of miR-93-3p. Overexpression of APAF1 reversed the effects of miR-93-3p, suppressed the proliferation and migration ability, and promoted the apoptosis of HaCaT cells. However, the roles of APAF1 in tumors and wound healing are contradictory; the activation of APAF1 protects against tumorigenesis and the upregulation of APAF1 deteriorates wound injuries. This may be due to the fact that the roles of APAF1 vary with the cell type and that the apoptosis of epithelial cells is a crucial factor in wound healing. Therefore, the apoptosis of epithelial cells in wound injuries should not be inhibited.

Conclusion

In short, BMSC-exos play a protective role in wound healing. BMSC-exos-derived miR-93-3p was observed to restore cellular functions and inhibit apoptosis of epithelial HaCaT cells by inactivating APAF1. Therefore, the BMSC-exos/miR-93-3p/APAF1 axis represents a promising therapeutic strategy for wound healing.