miR-100-5p Promotes Epidermal Stem Cell Proliferation through Targeting MTMR3 to Activate PIP3/AKT and ERK Signaling Pathways

Skin epidermal stem cells (EpSCs) play a critical role in wound healing and are ideal seed cells for skin tissue engineering. Exosomes from human adipose-derived stem cells (ADSC-Exos) promote human EpSC proliferation, but the underlying mechanism remains unclear. Here, we investigated the effect of miR-100-5p, one of the most abundant miRNAs in ADSC-Exos, on the proliferation of human EpSCs and explored the mechanisms involved. MTT and BrdU incorporation assays showed that miR-100-5p mimic transfection promoted EpSC proliferation in a time-dependent manner. Cell cycle analysis showed that miR-100-5p mimic transfection significantly decreased the percentage of cells in the G1 phase and increased the percentage of cells in the G2/M phase. Myotubularin-related protein 3 (MTMR3), a lipid phosphatase, was identified as a direct target of miR-100-5p. Knockdown of MTMR3 in EpSCs by RNA interference significantly enhanced cell proliferation, decreased the percentage of cells in the G1 phase and increased the percentage of cells in the S phase. Overexpression of MTMR3 reversed the proproliferative effect of miR-100-5p on EpSCs, indicating that miR-100-5p promoted EpSC proliferation by downregulating MTMR3. Mechanistic studies showed that transfection of EpSCs with miR-100-5p mimics elevated the intracellular PIP3 level, induced AKT and ERK phosphorylation, and upregulated cyclin D1, E1, and A2 expression, which could be attenuated by MTMR3 overexpression. Consistently, intradermal injection of ADSC-Exos or miR-100-5p-enriched ADSC-Exos into cultured human skin tissues significantly reduced MTMR3 expression and increased the thickness of the epidermis and the number of EpSCs in the basal layer of the epidermis. The aforementioned effect of miR-100-5p-enriched ADSC-Exos was stronger than that of ADSC-Exos and was reversed by MTMR3 overexpression. Collectively, our findings indicate that miR-100-5p promotes EpSC proliferation through MTMR3-mediated elevation of PIP3 and activation of AKT and ERK. miR-100-5p-enriched ADSC-Exos can be used to treat skin wound and expand EpSCs for generating epidermal autografts and engineered skin equivalents.


Introduction
Epidermal stem cells (EpSCs) residing in the basal layer of skin epidermis play an essential role in skin homeostasis and wound repair [1,2]. After skin injury, the EpSCs around the wound are activated to proliferate, migrate to the wound site, and differentiate to keratinocytes to regenerate epidermis [3,4]. Therapeutic approaches based on EpSCs and EpSC-seeded scaffold are being developed to treat extensive and chronic skin wounds, such as burn and diabetic ulcer [3][4][5]. In addition, EpSCs are ideal seed cells for fabricating engineered skin tissues which provide efficacious therapy in cutaneous wound repair [6,7]. Identifying endogenous factors which can promote EpSC proliferation will be helpful for developing the wound healing agent and expanding EpSCs in vitro for clinical treatment of skin injury or skin tissue engineering.
In the present study, we isolated human ADSC-secreted exosomes and analyzed the miRNA profile by small RNA sequencing. miR-100-5p is one of the most abundant miR-NAs in ADSC-Exos. It has been reported to promote or inhibit the proliferation of cells from different origin by targeting different molecules [22][23][24][25][26]. miR-100-5p promoted the proliferation of human placental microvascular endothelial cells by targeting homeodomain-interacting protein kinase 2 to activate the PI3K/AKT pathway [24]. miR-100-5p inhibited endometrial stromal cell proliferation by targeting HOXA1 to inhibit the PI3K/AKT and ERK pathways [26]. As miR-100-5p from umbilical cord mesenchymal stem cell-derived exosomes can accelerate vaginal epithelial cell proliferation [22], we proposed that miR-100-5p from ADSC-Exos might have proproliferative effect on EpSCs. We investigated the effect of miR-100-5p on human EpSC proliferation and explored the underlying mechanisms and confirmed the effect of miR-100-5p on EpSC proliferation in cultured human skin tissue using miR-100-5p-enriched ADSC-Exos.

Isolation and Culture of Human EpSCs and ADSCs.
Human skin and adipose tissues were obtained from patients (under 50 years old) who underwent the second-stage plastic surgery after treatment of skin injury through anterolateral thigh flap transplantation. EpSCs were isolated form skin tissues of 44 patients (34 males and 10 females), and ADSCs were isolated from adipose tissues of 19 patients (12 males and 7 females). The protocol was approved by the ethics committee of Ruihua Affiliated Hospital of Soochow University, Suzhou, China. EpSCs were isolated from the skin tissues as previously described [27,28]. Briefly, the epidermis was separated from the dermis by incubating the skin tissue in 0.25% dispase II (Sigma, St Louis, USA) overnight at 4°C and digested with 0.05% trypsin for 15 min at 37°C. The cell suspension was filtered and centrifuged. The cell pellet was washed with PBS, resuspended in keratinocyte growth medium-2 (KGM2) (PromoCell, Heidelberg, Germany), and seeded in culture dishes coated with collagen IV. After incubation at 37°C for 10 min, non-adherent cells were rinsed off with PBS. The adherent EpSCs were cultured in KGM2 supplemented with 4 μl/ml bovine pituitary extract, 0.125 ng/ml epidermal growth factor, 0.06 mM CaCl 2 , and 10 μM Y-27632 to promote cell proliferation and inhibit cell differentiation. The second passage of EpSCs cultured in KGM2 was used in the following experiments.
ADSCs were isolated from human adipose tissues as previously described [21]. Briefly, adipose tissues were minced and incubated in 0.1% collagenase I (Sigma, Saint Louis, USA) at 37°C with constant shaking for 1 h. The cell suspension was filtered, centrifuged, and washed with PBS. The cell pellets were resuspended in DMEM/F12 medium supplemented with 10% FBS and incubated overnight at 37°C in a humidified chamber with 5% CO 2 . The adherent ADSCs were cultured in fresh complete DMEM/F12 medium. The following experiments were performed with the second to forth passage of ADSCs.

Characterization of ADSCs.
ADSCs cultured in DMEM/ F12 medium to 90%-100% confluent were further cultured in MesenCult™ Adipogenic Differentiation Medium or Osteogenic Differentiation Medium (STEMCELL Technologies, Vancouver, Canada) for 9-10 days. The cells were fixed with 4% paraformaldehyde and stained with oil red O or alizarin red S to identify adipogenic and osteogenic differentiation, respectively.
To induce chondrogenic differentiation, ADSC pellet was incubated with MesenCult™ ACF Chondrogenic Differentiation Medium (STEMCELL Technologies, Vancouver, Canada) for 21 days. The pellets were fixed in 10% formalin and embedded with paraffin and thereafter stained with Alcian blue.
2.3. Isolation and Characterization of Exosomes. ADSCderived exosomes (ADSC-Exos) were isolated from cell culture medium as previously described [21]. Briefly, after the cultured ADSCs reached 80% confluence, the culture medium was replaced with serum-free medium for The morphology of ADSC-Exos was observed under a transmission electron microscopy. The size distribution of ADSC-Exos was analyzed using ZetaView (Particle Metrix, Germany). Western blotting was performed to determine exosome-specific surface markers CD63 and Alix using primary antibodies from Abcam (Cambridge, UK).
2.4. ADSC-Exo miRNA Sequencing. The exosomes were isolated from cultured ADSCs from a patient who underwent the second-stage plastic surgery aforementioned. Total RNA was extracted from ADSC-Exos using the miRNeasy Serum/Plasma Kit (QIAGEN, California, USA) and qualified and quantified using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher, Massachusetts, USA). The small RNA library was constructed and sequenced by BGI Genomics Co. Ltd. using the BGISEQ-500 platform (BGI, Shenzhen, China).

Flow
Cytometry. The expression of biomarkers for EpSCs and ADSCs was examined by flow cytometry. Human EpSCs or ADSCs were suspended in FACS buffer (PBS containing 1% goat serum and 5% FBS) and kept at room temperature for 1 h. EpSCs were incubated with FITC-conjugated rat anti-human α6 integrin antibody and PE-conjugated mouse anti-human CD71 antibody (BD Biosciences, San Jose, USA), and ADSCs were incubated with PE-labeled mouse anti-human CD34 or CD45 antibody, APC-labeled mouse anti-human CD73 antibody, or FITC-labeled mouse anti-human CD90 antibody (BD Biosciences, San Jose, USA) at room temperature for half an hour in the dark. Meanwhile, isotype-matched FITClabeled rat IgG and PE-, FITC-, and APC-labeled mouse IgGs were served as isotype controls. The cells were washed and resuspended in FACS buffer. FACS analyses were performed on a flow cytometry (Beckman Coulter, California, USA).
Cell cycle analyses were carried out by flow cytometry. Briefly, EpSCs were transfected with miR-100-5p mimics or control miRNA (RiboBio, Guangzhou, China), MTMR3 siRNA, or control siRNA (GENEWIZ, Suzhou, China) using Lipofectamine 3000 reagents (Invitrogen, Massachusetts, USA). After 24 h, the cells were collected and fixed in 75% ethanol at 4°C overnight. The cells were then centrifuged to remove ethanol and incubated with PI/RNase Staining Buffer (BD Biosciences, San Jose, USA) for 30 min in the dark. The DNA content was analyzed using a flow cytometer (Beckman Coulter, California, USA). The sequences of miR-100-5p mimics, control miRNA, MTMR3 siRNA, and control siRNA are listed in Supplementary Table 1. 2.6. Cell Proliferation Assays. Human EpSCs seeded on 6 cm dishes were transfected with miR-100-5p mimics or control miRNA, MTMR3 siRNA or control siRNA, and MTMR3 expression plasmids or control vector. After 24 h, the cells were harvested and transferred into 96-well plates. Cell proliferation at the indicated time points was determined by MTT assay or BrdU incorporation assays as previously described [27]. The OD values were measured using the Multiskan™ Spectrum Microplate Reader (Thermo Fisher, Massachusetts, USA) at 490 and 450 nm, respectively. 2.7. Bioinformatics Analysis. The targets of miR-100-5p were predicted using miRanda, TargetScan, RNA22, and miR-Walk. We further used DAVID software to perform gene ontology (GO) analysis on the overlapped target genes, and specific biological process categories were enriched.
2.8. Luciferase Reporter Assay. A MTMR3 3 ′ UTR fragment containing the wild-type or mutant miR-100-5p binding site was synthesized and inserted into a pGL3-control vector (Promega, Wisconsin, USA) to construct luciferase reporter plasmid. Human HEK-293T cells were transfected with luciferase reporter plasmid together with miR-100-5p mimics or control miRNA using Lipofectamine 3000 (Invitrogen, Massachusetts, USA). A reporter plasmid encoding Renilla luciferase was cotransfected for normalization purposes. Cells were harvested 24 h after transfection, and the luciferase signals of firefly and Renilla were measured using the Dual-Luciferase® Reporter Assay System (Promega, Wisconsin, USA).
2.9. Immunofluorescence Staining. The expression of biomarkers of EpSCs was detected by immunofluorescence staining. Briefly, EpSCs were fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton-X. After incubation with 10% goat serum for 30 min, the cells were washed and incubated with primary antibody against CK19 or β1 integrin (Abcam, Cambridge, UK) overnight at 4°C and then washed with PBS and incubated with fluorescence-conjugated secondary antibody for 1 h. The nuclei were stained with DAPI, and the fluorescence signals were detected under a fluorescence microscope (Olympus, Tokyo, Japan).

RT-PCR and Quantitative
Real-Time PCR. Target gene expression was measured through RT-PCR or RTquantitative real-time PCR (qPCR). Target miRNA expression was examined by qPCR. Briefly, total RNA was extracted from exosomes, cells, or tissues using TRIzol reagent (Invitrogen, Massachusetts, USA). Reverse transcription of mRNA and miRNA were performed using the PrimeScript™ RT Reagent Kit with the gDNA Eraser and Mir-X miRNA First-Strand Synthesis Kit (TaKaRa, Dalian, China), respectively. PCR products were separated by agarose gel electrophoresis. GAPDH was applied as endogenous control. The expression levels of target genes were semiquantified using ImageJ software (NIH Image, Bethesda, Maryland, USA). qPCR was performed using SYBR® Premix Ex Taq TM II Kit (TaKaRa, Dalian, China) on a StepOne-Plus™ Real-Time PCR System (Applied Biosystems, Massachusetts, USA). Transcriptional levels for target mRNA 3 Stem Cells International and miRNA were normalized to GAPDH and U6, respectively. The relative expression levels were calculated using the 2 −ΔΔCT method. The primer sequences used for RT-PCR and RT-qPCR are listed in Supplementary Table 2. 2.11. Measurement of Phosphatidylinositol-3,4,5-Trisphophate. Human EpSCs were lysed via repeated cycles of freeze-thaw. After centrifugation, the supernatant was collected and the phosphatidylinositol-3,4,5-trisphophate (PIP3) level was measured using the Human PIP3 ELISA Kit (J&L Biological, Shanghai, China) according to the manufacturer's instruction. The absorbance was measured at 450 nm with the Multiskan™ Spectrum Microplate Reader (Thermo Fisher, Massachusetts, USA).

Skin Explant Culture.
Human skin tissues were cultured in a 24-well transwell system as previously described [27]. Briefly, the skin tissues were cut into 0:5 cm × 0:5 cm pieces. Each piece of the skin was injected intradermally into four spots with 20 μg ADSC-Exos, 20 μg exosomes isolated from miR-100-5p expression plasmid-transfected ADSCs (miR-100-Exos), 20 μg miR-100-Exos plus 5 μg MTMR3 plasmids, or same volume of PBS and cultured in Ham's F-12 medium (Gibco, California, USA) supplemented with 10% FBS and antibiotics. The epidermal side of the skin was kept at the air-liquid interface. The medium was changed every other day. After 2 and 5 days, the skin tissues were lysed in TRIzol reagent for extracting RNA to detect miR-100-5p and MTMR3 mRNA, or fixed in paraformaldehyde and embedded in paraffin for histological and immunohistochemical assays.
2.14. Histology and Immunohistochemistry. Paraffin-embedded skin tissues were sectioned at 4 μm using a rotary microtome (Leica, Wetzlar, Germany) and stained with hematoxylin and eosin (HE). The thickness of the epidermis was measured using ImageJ software (NIH Image, Bethesda, Maryland, USA). For immunohistochemical assay, the sections were heated in an oven at 60°C for 2 h, deparaffinized in xylene, rehydrated in an ethanol gradient (100%-80%), incubated in antigen retrieval solution containing 0.4 g/l citric acid and 3.0 g/l trisodium citrate for 20 min, and rinsed with PBS. The sections were incubated with the endogenous peroxidase blocker (MXB, Fuzhou, China) for 10 min, rinsed with PBS, and blocked with 10% goat serum for 1 h. Then, the slides were incubated with the primary antibody against α6 integrin, β1 integrin, or PCNA (Abcam, Cambridge, UK) at 4°C overnight, rinsed with PBS, and incubated with the secondary antibody from the MaxVision™ HRP-Polymer anti-Mouse IHC Kit or anti-Rabbit IHC Kit (MXB, Fuzhou, China) for 1 h. After rinsing with PBS, the sections were incubated with diaminobenzidine (MXB, Fuzhou, China), counterstained with hematoxylin, and imaged under a microscope. The positive signals of immunohistochemical staining were analyzed using ImageJ software (NIH Image, Bethesda, Maryland, USA).

Statistical Analysis.
Each experiment was repeated at least three times and the results are expressed as mean ± SD. Statistical analysis was performed with GraphPad Prism 8.0 (GraphPad Software Inc). Unpaired two-tailed Student's t-test was used to assess the statistical difference between two groups. Comparisons between more than two groups were performed using one-way ANOVA. P < 0:05 was considered significantly different.

Characterization of ADSC-Exos and the Profile of miRNAs in ADSC-Exos.
ADSCs were isolated from human adipose tissues and cultured. Flow cytometry assay showed that these cells were positive for mesenchymal stem cell markers CD73 and CD90 and negative for vascular endothelial marker CD34 and leukocyte marker CD45 (Supplementary Figure 1(a)). These cells could differentiate to osteocytes, adipocytes, and chondrocytes, as shown by alizarin red S staining, oil red O staining, and Alcian blue staining, respectively (Supplementary Figure 1(b)). These results demonstrated the successful isolation and culture of ADSCs.
ADSC-Exos were isolated from the serum-free culture supernatant of ADSCs. ADSC-Exos displayed a saucershaped morphology under transmission electron microscopy (Figure 1(a)). Nanoparticle tracking analysis showed the size of ADSC-Exos in the 100 nm range (Figure 1(b)). Western blot assay showed that ADSC-Exos expressed exosome marker proteins Alix and CD63, but not GAPDH (Figure 1(c)). These results demonstrated the successful isolation of ADSC-Exos.

miR-100-5p Promotes Human EpSC Proliferation.
To examine the effect of miRNA on the proliferation of EpSCs, we isolated EpSCs from human skin tissues. Flow cytometry assay showed that these cells were positive for EpSC markers α6 integrin, β1 integrin, and CK19 and negative for CD71 (Supplementary Figure 2), which was consistent with the previous studies [21,27,28]. 4 Stem Cells International To determine the miRNA(s) from ADSC-Exos which can stimulate EpSC proliferation, we searched the literature to find out which miRNA among the top 5 miRNAs in ADSC-Exos could promote cell proliferation. Among these miRNAs, miR-127 and Let-7b have inhibitory effect on cell proliferation [29][30][31]; miR-92a, miR-222-3p, and miR-100-5p have been reported to stimulate or inhibit cell proliferation [22][23][24][25][26][32][33][34][35]. We then examined the effect of miR-92a-3p, miR-222-3p, and miR-100-5p on the proliferation of human EpSCs. While miR-92a-3p and miR-222-3p had no effect (data not shown), miR-100-5p had stimulatory effect on EpSC proliferation. As shown in Figure 2(a), transfection of human EpSCs with miR-100-5p mimics significantly elevated the intracellular miR-100-5p level and stimulated cell proliferation in a time-dependent manner as examined by MTT assay. BrdU incorporation assay confirmed the proproliferative effect of miR-100-5p on EpSCs (Figure 2(b)). Furthermore, flow cytometry assay showed that transfection of EpSCs with miR-100-5p mimics significantly decreased the percentage of cells in the G1 phase and increased the percentage of cells in the G2 phase. The cells in the S phase had a tendency to increase (Figure 2(c)). All together, these results demonstrated that miR-100-5p promotes the proliferation of EpSCs.
3.4. miR-100-5p Promotes EpSC Proliferation through MTMR3. To investigate whether miR-100-5p promotes EpSC proliferation by inhibiting the expression of MTMR3, we first examined the effect of MTMR3 on EpSC proliferation by knockdown of MTMR3 in human EpSCs through RNA interference. RT-qPCR showed that the mRNA level of MTMR3 was significantly decreased after transfecting the cells with MTMR3 siRNA (Figure 4(a)). MTT assay showed that MTMR3 knockdown remarkably promoted the proliferation of EpSCs (Figure 4(a)). Meanwhile, knockdown of MTMR3 in EpSCs significantly reduced the percentage of cells in the G1 phase and increased the percentage of cells in the S phase (Figure 4(b)). These results indicate that knockdown of MTMR3 promotes EpSC proliferation by facilitating G1/S phase transition.
Then, we checked if overexpression of MTMR3 could reverse the proproliferative effect of miR-100-5p on EpSC proliferation. As shown in Figure 5, compared with EpSCs transfected with negative control miRNA and control vector, cotransfection of miR-100-5p mimics and control vector significantly downregulated the expression of MTMR3 in EpSCs and promoted cell proliferation, cotransfection of miR-100-5p mimics and MTMR3 plasmid reversed the effect of miR-100-5p on MTMR3 expression and cell proliferation, and transfection of EpSCs with MTMR3 expression plasmid significantly elevated the MTMR3 mRNA level and inhibited cell proliferation. These results confirmed that MTMR3 negatively regulated EpSC proliferation and demonstrated that miR-100-5p promotes EpSC proliferation through downregulating MTMR3.

miR-100-5p from ADSC-Exos Promotes EpSC
Proliferation in Human Skin Tissue through MTMR3. To investigate if miR-100-5p could promote EpSC proliferation in the human skin, we used miR-100-5p-enriched ADSC-Exos (miR-100-Exos) to deliver miR-100-5p. We first examined the effect of miR-100-Exos on the proliferation of EpSCs in vitro. As shown in Figure 9(a), both ADSC-Exos  , and A2 at mRNA (c) and protein levels (e). Data are shown as mean ± SD, n = 3. * P < 0:05, * * P < 0:01, and * * * P < 0:001, compared with cells transfected with (a, d) NC, (b) control siRNA, or (c, e) NC and Vector; # P < 0:05, ## P < 0:01, and ### P < 0:001, compared with cells transfected with (c, e) miR-100-5p and Vector or (d) control siRNA. 9 Stem Cells International (Exos) and miR-100-Exos significantly stimulated EpSC proliferation; the proproliferative effect of miR-100-Exos was greater than that of Exos. We then examined the effect of miR-100-Exos on the proliferation of EpSCs in cultured skin tissues. Human skin tissue explants were intradermally injected with PBS, Exos, or miR-100-Exos and cultured for 2 and 5 days. HE staining showed that the thickness of epidermis was significantly increased at 2 and 5 days after the injection with Exos or miR-100-Exos (Figure 9(b)). Immunohistochemical staining of cell proliferation marker PCNA or EpSC markers β1 integrin and α6 integrin showed that the cells expressing PCNA, α6 integrin, or β1 integrin were located in the basal layer of the epidermis in PBSinjected skin tissue. Five days after the injection of Exos, or 2 and 5 days after the injection of miR-100-Exos, the number of PCNA-, β1 integrin-, and α6 integrin-positive cells was significantly increased in the basal layer of epidermis (Figure 9(b)). The thickness of epidermis and numbers of PCNA-, β1 integrin-, and α6 integrin-positive cells was greater and increased earlier in miR-100-Exos-treated skin tissues than in Exos-treated skin tissues. These results demonstrated that exosomal miR-100-5p promotes the proliferation of EpSCs in human skin tissue. Furthermore, intradermal injection of miR-100-Exos together with MTMR3 expression plasmid completely abolished miR-100-Exos-induced increase of epidermis thickness and numbers of PCNA-, β1 integrin-, and α6 integrin-positive cells in the basal layer (Figure 9(b)). RT-qPCR assays showed that both Exos-treated and miR-100-Exos-treated skin tissues had higher levels of miR-100-5p and lower levels of MTMR3 than PBS-treated skin tissues; miR-100-Exos-treated skin tissues had higher levels of miR-100-5p and lower levels of MTMR3 than Exos-treated skin tissues (Figures 9(c) and 9(d)). Intradermal injection of miR-100-Exos together with MTMR3 expression plasmid reversed miR-100-Exos-induced decrease of MTMR3 (Figure 9(d)). Taken together, these results indicate that miR-100-5p promotes EpSC proliferation in human skin tissues through inhibiting MTMR3 expression.

Discussion
In the present study, we found that human ADSC-derived exosomes contain high levels of miR-100-5p. miR-100-5p promoted the proliferation of human EpSCs in culture and in human skin tissue explants. Mechanistic studies revealed that miR-100-5p promoted EpSC proliferation through MTMR3-mediated elevation of PIP3 and activation of AKT and ERK. It has been reported that ADSC-Exos promoted the proliferation and migration of keratinocytes by activating the AKT signaling pathway [56,57]. Our previous study showed that ADSC-Exos promoted human EpSC proliferation partly through upregulating the expression of βcatenin, c-Myc, and cyclins D1, E1, and A2 [21] but the exosomal molecules that mediate the proproliferative effect of ADSC-Exos on EpSCs remain unclear. Human ADSC-Exos are rich in miRNAs which represent approximately 44% of all small noncoding RNA detected in ADSC-Exos [17]. By small RNA sequencing, we found that human ADSC-Exos contained multiple miRNAs. miR-100-5p was one of the most abundant miRNAs in ADSC-Exos ( Figure 1). In vitro study showed that miR-100-5p promoted human EpSC proliferation in a time-dependent manner (Figures 2(a) and 2(b)). We found that transfection of EpSCs with miR-100-5p mimics decreased the percentage of cells in the G1 phase and increased the percentage of cells in the S phase and G2 phase (Figure 2(c)), which confirmed the proproliferative effect of miR-100-5p on EpSCs. Cyclins are a group of proteins that regulate genes essential for cell cycle progression by binding with various cyclin-dependent kinases (CDK). In mammalian cell division cycle, cyclin D-CDK4/6 complexes control entry into the cell cycle from quiescence and progression throughout the G1 phase. Cyclin E-CDK2 complex controls entry into the S phase. Cyclin A-CDK2/1 complexes control DNA replication and progression through the G2 phase [58]. Our study demonstrated that miR-100-5p promoted EpSC proliferation by upregulating the expression of cyclins D1, E1, and A2 (Figures 6(a) and 6(d)), which was consistent with the effect of ADSC-Exos on EpSC proliferation [21].
Exosomes have recently emerged as a promising drug carrier with low immunogenicity, high biocompatibility, and high efficacy of delivery. We isolated exosomes form ADSCs with or without overexpression of miR-100-5p and compared their effect on cultured EpSCs and EpSCs in the cultured human skin. miR-100-5p-enriched ADSC-Exos had more potent proproliferative effect on EpSCs than that of ADSC-Exos. Intradermal injection of ADSC-Exos or miR-100-5p-enriched ADSC-Exos both significantly elevated the miR-100-5p level and reduced MTMR3 expression in skin tissues and increased the number of EpSCs in the basal layer of epidermis and the thickness of the epidermis. Injection of miR-100-5penriched ADSC-Exos together with MTMR3 expression plasmid increased MTMR3 expression and reversed the effect of miR-100-5p-enriched ADSC-Exos on EpSC proliferation ( Figure 9). These data demonstrated that exosomal miR-100-5p promotes skin EpSC proliferation through MTMR3. Compared with ADSC-Exos, miR-100-5p-enriched ADSC-Exos showed stronger proproliferative effect on EPSCs in the skin. Therefore, miR-100-5penriched ADSC-Exos can be used to effectively expand EpSCs for basic research and skin tissue engineering. It is also a potential therapeutic reagent to promote wound healing.
In the present study, we demonstrated that both miR-100-5p and miR-100-5p-enriched ADSC-Exos could promote EpSC proliferation in vitro. The limitation is that we have not performed in vivo experiment to verify the beneficial effect of miR-100-5p on skin wound healing through stimulating EpSC proliferation. We tried to enrich miR-100-5p in ADSC-Exo by transfecting ADSCs with miR-100-5p expression plasmid, but the level of miR-100-5p in the exosomes only increased to some extent. To enhance the efficacy of ADSC-Exo-mediated delivery of miR-100-5p, it is crucial to develop a convenient and efficient method to enrich miR-100-5p in isolated exosomes.

Conclusions
miR-100-5p promotes EpSCs proliferation through MTMR3mediated activation of PIP3-AKT and ERK signaling ( Figure 10). miR-100-5p-enriched ADSC-Exos is a useful reagent for the expansion of EpSCs for skin tissue engineering and for skin wound treatment.

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