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Highly-expressed lncRNA FOXD2-AS1 in adipose mesenchymal stem cell derived exosomes affects HaCaT cells via regulating miR-185-5p/ROCK2 axis

Highly-expressed lncRNA FOXD2-AS1 in adipose mesenchymal stem cell derived exosomes affects HaCaT... ADIPOCYTE 2023, VOL. 12, NO. 1, 2173513 https://doi.org/10.1080/21623945.2023.2173513 RESEARCH PAPER Highly-expressed lncRNA FOXD2-AS1 in adipose mesenchymal stem cell derived exosomes affects HaCaT cells via regulating miR-185-5p/ROCK2 axis a b a c a Huanchao Chang , Junliang Chen , Kun Ding , Tianling Cheng , and Shengjian Tang a b Plastic Surgery of Plastic Surgery Hospital, Weifang Medical University, Weifang, China; Vascular surgery department, Affiliated Hospital of Weifang Medical College, Weifang, China; Burn plastic surgery, The First Affiliated Hospital of Xi’an Medical University, Xi’an, China ABSTRACT ARTICLE HISTORY The healing of skin wounds is a highly coordinated multi-step process that occurs after trauma Received 8 November 2022 including surgical incisions, thermal burns, and chronic ulcers. In this study, the authors investi- Revised 3 January 2023 Accepted 24 January 2023 gated lncRNA FOXD2-AS1 function in adipose mesenchymal exosomes from ADMSCs that were successfully extracted. Highly expressed lncRNA FOXD2-AS1 in ADMSCs-exosomes accelerated KEYWORDS HaCaT cell migration and proliferation. LncRNA FOXD2-AS1 negatively targeted miR-185-5p, and Exosomes; FOXD2-AS1; miR-185-5p negatively targeted ROCK2. Highly expressed lncRNA FOXD2-AS1 in ADMSCs- wound healing; miR-185-5p; exosomes promoted HaCaT cell migration and proliferation via down-regulating miR-185-5p ROCK2 and further up-regulating ROCK2. In conclusion, LncRNA FOXD2-AS1 overexpression in ADMSCs derived exosomes might accelerate HaCaT cell migration and proliferation via modulating the miR-185-5p/ROCK2 axis. keratinocyte migration and proliferation [9]. Moreover, Introduction human ADMSCs are an attractive resource for wound The healing of skin wounds is a highly coordinated healing due to their regenerative ability to promote multi-step process that occurs after trauma, including injury repair [10]. A study has confirmed that surgical incisions, thermal burns, and chronic ulcers ADMSCs could accelerate wound healing through opti- [1]. The failure of proceeding through such orderly mizing fibroblasts characteristics [11], which further and timely reparation can induce chronic non- verified their regenerative ability to promote injury healing wounds including diabetic, venous, and decu- repair in wound healing. bitus skin ulcers [2,3]. Refractory wound is a huge Long noncoding RNAs (lncRNAs) have been burden to both the patient and society. Thus, explor- reported to exist in exosomes and modulate gene ing a novel marker for wound healing therapy and expression in host cells through intercellular commu- clarifying the mechanisms of this fatal disease are nication [12]. For example, the high expression of lncRNA H19 in ADMSCs-exosomes can up-regulate imperative. the expression of SOX9 through miR-19b to promote Mesenchymal stem cells (MSCs) are one type of wound healing [13]. A study has proven that exosomal multipotent progenitor cells that are derived from lncRNA FOXD2-AS1 can act as the promising biomar- bone marrow, umbilical cord, and adipose tissue and kers for the diagnostics of colorectal cancer [14]. play a well-known function in tissue regeneration [4]. LncRNA FOXD2-AS1 promotes the progression of In recent years, more and more studies have strongly a variety of tumours [15–18]. Moreover, lncRNA proved that exosomes derived from adipose-derived FOXD2-AS1 promotes the proliferation of many mesenchymal stem cells (ADMSCs) are safe and have kinds of cells, including trophoblast cell [19], fibro- become the hot pot of many researches in many differ- blast-like synoviocytes [20], and chondrocyte [21]. ent fields, such as wound healing [5–7]. A previous However, the evidence regarding the implication of study has demonstrated that ADMSCs-derived exo- exosomal lncRNA FOXD2-AS1 derived from somes can accelerate cell proliferation and migration ADMSCs in wound healing is lacking. through regulating Wnt/β-catenin pathway in cuta- In the current study, we paid attention to evaluate neous wound healing [8]. ADMSCs-derived exosomes the effect of exosomal lncRNA FOXD2-AS1 derived are reported to promote wound healing via accelerating CONTACT Shengjian Tang tsj1950@163.com Plastic Surgery Institute, Weifang Medical University, 4948 Shengli East Street, Kuiwen District, Weifang, 261041, China © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2 H. CHANG ET AL. from ADMSCs in wound healing and its potential Highly expressed lncRNA FOXD2-AS1 in mechanisms. Our findings demonstrated that highly ADMSCs-exosomes accelerated HaCaT cell expressed lncRNA FOXD2-AS1 in ADMSCs derived migration and proliferation exosomes accelerated HaCaT cell migration and prolif- We overexpressed lncRNA FOXD2-AS1 in ADSCs and eration via the regulation of miR-185-5p/rho-associated extracted exosomes from ADSCs. The level of lncRNA coiled-coil-containing protein kinase 2 (ROCK2) axis, FOXD2-AS1 was significantly upregulated following suggesting that exosomal lncRNA FOXD2-AS1 derived lncRNA FOXD2-AS1 overexpression plasmid transfec- from ADMSCs may be a novel therapeutic strategy for tion (Figure 2a). As shown in Figure 2b, treatment with wound healing. exosomes significantly increased lncRNA FOXD2-AS1 expression in HaCaT cells, and lncRNA FOXD2-AS1 overexpression-transfected exosomes further elevated Results lncRNA FOXD2-AS1 expression. CCK-8 and EdU assays data proved that exosomes treatment markedly promoted Identification of ADMSCs and exosomes the proliferation of HaCaT cells (Figure 2c and 2d). Flow cytometric analysis was utilized to examine the Meanwhile, the proliferation was significantly increased cell surface protein expression of isolated ADMSCs. As in FOXD2-AS1 exosome group relative to NC exosome shown in Figure 1a, ADMSCs were positive with CD44 group (Figure 2c and 2d). In addition, we assessed the and CD105, but negative with CD31 and HLA-DR. migrative function of highly expressed lncRNA FOXD2- Then, we further identified ADMSCs-exomoses AS1 in exosomes by wound healing analyses. Figure 2e through TEM (Figure 1b). Furthermore, the data of confirmed that exosomes treatment markedly increased NTA confirmed that the particle size of the extracted HaCaT cell migration, and lncRNA FOXD2-AS1 over- exosomes was mainly distributed at about 100 nm expression-transfected exosomes further elevated HaCaT (Figure 1c). Moreover, the presentation of specific bio- cell migration compared with NC exosomes. Similarly, markers CD9, CD63 and TSG101 indicated that the the results are also demonstrated using western blot by exosomes were successfully extracted (Figurerue 1D). detecting MMP-2 and MMP-9 (figure 2f). Figure 1. Identification of ADMSCs and exosomes. (a) Flow cytometric analysis of cell surface protein expression of isolated ADMSCs. (b) The ultrastructure of ADSCs-exo under TEM. (c) Exosomes size distribution was analysed using NTA. (d) The expression of CD9, CD63 and TSG101 was detected using western blot. ADIPOCYTE 3 Figure 2. Highly expressed lncRNA FOXD2-AS1 in ADMSCs-exosomes promoted the migration and proliferation of HaCaT cells. Following different treatment, the expression of lncRNA FOXD2-AS1 in ADMSCs-exosomes was detected using qRT-PCR (a); the expression of lncRNA FOXD2-AS1 in HaCaT cells was detected using qRT-PCR (b); HaCaT cell variety was evaluated using CCK-8 assay (B); HaCaT cell proliferation was determined with EdU assay (c); HaCaT cell migration at 0, 24, 48 h was analysed with wound healing assay (d); MMP-2 and MMP-9 level were tested utilizing western blot (e). *P < 0.05 vs. exosome or control group; P < 0.05 vs. NC exosome group. 4 H. CHANG ET AL. Knockdown lncRNA FOXD2-AS1 in miR-185-5p mimics group was elevated relative to ADMSCs-exosomes inhibited HaCaT cell migration NC exosome + miR-185-5p mimics group, but was and proliferation reduced relative to FOXD2-AS1 exosome + mimics NC group (Figure 5a and 5b). Moreover, the results We knockdown lncRNA FOXD2-AS1 in ADSCs and of Figure 4c showed that the migration of HaCaT cells extracted exosomes from ADSCs. The level of was notably decreased in NC exosome + miR-185-5p lncRNA FOXD2-AS1 was significantly downregulated mimics group relative to NC exosome + mimics NC following si-lncRNA FOXD2-AS1 transfection group, but increased in FOXD2-AS1 exosome + (Figure 3a). Exosomes significantly increased mimics NC group. The migration of HaCaT cells in lncRNA FOXD2-AS1 expression in HaCaT cells, FOXD2-AS1 exosome + miR-185-5p mimics group and si-lncRNA FOXD2-AS1 transfection exosomes was increased compared with NC exosome + miR- decreased lncRNA FOXD2-AS1 expression 185-5p mimics group but was decreased relative to (Figure 3b). CCK-8 and EdU assays data proved FOXD2-AS1 exosome + mimics NC group that the proliferation was significantly decreased in (Figure 5c). Similarly, the results are also demon- si-FOXD2-AS1 exosome group relative to the NC strated using western blot. MMP-2 and MMP-9 level exosome group (Figure 3c and 3d). si-FOXD2-AS1 was lower in NC exosome + miR-185-5p mimics transfected exosomes decreased HaCaT cell migration group relative to NC exosome + mimics NC group, (Figure 3e). Similarly, the results are also demon- but higher in FOXD2-AS1 exosome + mimics NC strated using western blot by detecting MMP-2 and group (Figure 5d). Meanwhile, the expressions of MMP-9 (figure 3f). MMP-2 and MMP-9 level in FOXD2-AS1 exosome + miR-185-5p mimics group was elevated relative to NC exosome + miR-185-5p mimics group and down- lncRNA FOXD2-AS1 negatively targeted miR-185-5p regulated relative to FOXD2-AS1 exosome + mimics After transfection of lncRNA FOXD2-AS1 overexpres- NC group (Figure 5d). sion vector in HaCaT cells, lncRNA FOXD2-AS1 level was significantly upregulated (Figure 4a), and the expression of miR-185-5p was markedly decreased miR-185-5p negatively targeted ROCK2 (Figure 4b). Additionally, following transfected with miR-185-5p mimics in HaCaT cells, miR-185-5p level As shown in Figure 6a, the expression of ROCK2 was was elevated (Figure 4c) and lncRNA FOXD2-AS1 level upregulated in HaCaT cell followed by transfection of was reduced (Figure 4d). The predicted binding sites ROCK2 overexpression vector. Additionally, the between lncRNA FOXD2-AS1 and miR-185-5p are expression of ROCK2 was downregulated followed shown in Figure 3e. Furthermore, miR-185-5p mimics by transfection of miR-185-5p mimics (Figure 6b). notably inhibited luciferase activities in FOXD2-AS1- Figure 6c is predicted for binding sites between WT group, while had no function on luciferase activ- miR-185-5p and ROCK2. Figure 6d proves miR- ities in FOXD2-AS1-MUT group (Figure 4f). 185-5p mimics decreased luciferase activity in the Moreover, we also found that exosomes treatment ROCK2-WT group, while had no function on luci- only significantly reduced miR-185-5p level, and ferase activities in ROCK2-MUT group. Furthermore, lncRNA FOXD2-AS1 overexpression-transfected exo- Figure 5e and 5f data showed that ROCK2 expression somes further inhibited the expression of miR-185-5p were higher in mimics NC + ROCK2 vector group (Figure 4g). than that in mimics NC + NC vector group, but lower in miR-185-5p mimics + NC vector group. Meanwhile, mRNA and protein expression of Highly expressed lncRNA FOXD2-AS1 in ROCK2 in miR-185-5p mimics + ROCK2 vector ADMSCs-exosomes promoted HaCaT cell migration group was reduced relative to mimics NC + ROCK2 and proliferation via down-regulating miR-185-5p vector group, but higher than that in miR-185-5p mimics + NC vector group (Figure 6e and 6f). The The results of CCK-8 and EdU assays demonstrated above data suggested that miR-185-5p could nega- that in the comparison with NC exosome + mimics tively target ROCK2. Moreover, we also found that NC group, the proliferation of HaCaT cells was nota- exosomes treatment significantly induced ROCK2 bly reduced in NC exosome + miR-185-5p mimics expression, and lncRNA FOXD2-AS1 overexpression- group, but elevated in FOXD2-AS1 exosome + mimics transfected exosomes further promoted the expres- NC group (Figure 5a and 5b). Meanwhile, the prolif- sion of ROCK2 (Figure 6g and 6h). eration of HaCaT cells in FOXD2-AS1 exosome + ADIPOCYTE 5 Figure 3. Knockdown lncRNA FOXD2-AS1 in ADMSCs-exosomes inhibited HaCaT cell migration and proliferation. Following different treatment, the expression of lncRNA FOXD2-AS1 in ADMSCs-exosomes was detected using qRT-PCR (a); the expression of lncRNA FOXD2-AS1 in HaCaT cells was detected using qRT-PCR (b); HaCaT cell variety was evaluated using CCK-8 assay (B); HaCaT cell proliferation was determined with EdU assay (c); HaCaT cell migration at 0, 24, 48 h was analysed with wound healing assay (d); MMP-2 and MMP-9 level were tested utilizing western blot (e). *P < 0.05 vs. exosome or control group; P < 0.05 vs. NC exosome group. 6 H. CHANG ET AL. Figure 4. lncRNA FOXD2-AS1 negatively targeted miR-185-5p. After transfection of lncRNA FOXD2-AS1 overexpression plasmid, lncRNA FOXD2-AS1 (a) and miR-185-5p (b) levels were tested using qRT-PCR. Following transfection of miR-185-5p mimics, the expression of miR-185-5p (c) and lncRNA FOXD2-AS1 (d) was tested using qRT-PCR. (e) The bind sites of lncRNA FOXD2-AS1 and miR- 185-5p. (f) A dual luciferase reporter assay verified the binding relationship between lncRNA FOXD2-AS1 and miR-185-5p. (D) After treatment of exosomes and lncRNA FOXD2-AS1 overexpression-transfected exosomes, qRT-PCR was utilized to assess miR-185-5p level (g). *P < 0.05 vs. FOXD2-AS1 vector, mimics NC, mmics NC + FOXD2-AS1 WT group, and control group, P < 0.05 vs. NC exosome group. ADIPOCYTE 7 Figure 5. Highly expressed lncRNA FOXD2-AS1 in ADMSCs-exosomes promoted HaCaT cell migration and proliferation via down- regulating miR-185-5p. Following different treatment, the HaCaT cell variety was assessed utilizing CCK-8 analyses (a); HaCaT cell proliferation was evaluated applying EdU analyses (b); HaCaT cell migration at 0, 24, 48 h was analysed with wound healing analyses (c); MMP-2 and MMP-9 levels were tested utilizing western blot (d). *P < 0.05 vs. NC exosome + mimics NC group, P < 0.05 vs. FOXD2-AS1 exosome + mimics NC group. miR-185-5p overexpression inhibited HaCaT cell ROCK2 vector group was inhibited relative to mimics migration and proliferation through NC + ROCK2 vector group, but was elevated relative to down-regulating ROCK2 miR-185-5p mimics + NC vector group (Figure 7c). Moreover, western blot results confirmed that MMP-2 As shown in Figure 7a and 7b, the proliferation of and MMP-9 level was higher in mimics NC + ROCK2 HaCaT cells was notably elevated in mimics NC + vector group than those in mimics NC + NC vector ROCK2 vector group than that in mimics NC + NC group, but lower in miR-185-5p mimics + NC vector vector group, but downregulated in miR-185-5p group (Figure 7d). Meanwhile, MMP-2 and MMP-9 mimics + NC vector group. HaCaT cell proliferation level in miR-185-5p mimics + ROCK2 vector group in miR-185-5p mimics + ROCK2 vector group was was decreased when compared with mimics NC + decreased compared with mimics NC + ROCK2 vector ROCK2 vector group, and increased in miR-185-5p group but was increased compared with miR-185-5p mimics + NC vector group (Figure 7d). mimics + NC vector group (Figure 7a and 7b). In comparison with mimics NC + NC vector group, HaCaT cell migration was promoted in mimics NC + Discussion ROCK2 vector group and decreased in miR-185-5p mimics + NC vector group (Figure 7c). Meanwhile, A basic characteristic of wound healing is the repair of HaCaT cell migration in miR-185-5p mimics + the intact epidermal barrier via reepithelialization [22]. 8 H. CHANG ET AL. Figure 6. miR-185-5p negatively targeted ROCK2. (a) After transfection of ROCK2 overexpression vector, the expression of ROCK2 was detected using qRT-PCR. (b) Following transfection of miR-185-5p mimics, ROCK2 level was detected using qRT-PCR. (c) The bind sites of miR-185-5p and ROCK2. (d) A dual luciferase reporter assay verified the binding relationship between miR-185-5p and ROCK2. After co-transfection with ROCK2 overexpression vector and miR-185-5p mimics, ROCK2 expressions were analysed using qRT-PCR (e) and western blot (f). *P < 0.05 vs. vector NC, mimics NC, mimics NC +ROCK2 WT, and mimics NC+ NC vector group, P < 0.05 vs miR-185-5p mimics + NC vector and NC exosome group. It is reported that keratinocytes proliferation and in wound healing. Our data demonstrate that highly migration are closely related to reepithelialization and expressed lncRNA FOXD2-AS1 in ADMSCs-exosomes closure of the wound gap [23,24]. In the current study, might accelerate HaCaT cell migration and proliferation. highly expressed lncRNA FOXD2-AS1 in ADMSCs Recent research data suggest that miRNAs are promis- derived exosomes promoted HaCaT cell migration ing tools for the treatment and diagnosis of skin wound and proliferation via modulating the miR-185-5p/ healing because miRNAs are important regulators of cel- ROCK2 axis. lular physiology and pathology [29]. miR-200b/c-3p can Currently, increasing evidences have suggested that the modulate epithelial plasticity and repress cutaneous novel role of exosomes as cell communication bodies is wound healing through the regulation of TGF-β- dependent on exosomal cargo, including lncRNAs, mediated RAC1 pathway [30]. It is interesting that microRNAs (miRNAs) and proteins [25,26]. lncRNA interacts with miRNA and the interactions play Furthermore, accumulating evidences relate regulatory critical roles in determining cell fate [31]. Adipose- lncRNAs to human diseases. LncRNA FOXD2-AS1 may derived stem cells-exosomes containing lncRNA accelerate proliferation and migration activities in MALAT1 can promote wound healing via targeting a various of cells [20,27,28]. Interestingly, a study has miR-124 [25]. LncRNA GAS5 is proved to accelerate proven that exosomal lncRNA FOXD2-AS1 can also act diabetic wound healing and promote lymphangiogenesis as the promising biomarkers for the diagnostics of color- by miR-217/Prox1 axis [32]. Through bioinformatics ana- ectal cancer [14]. Therefore, we explored the effects of lyses and Dual-luciferase reporter assay, we verified that exosomal lncRNA FOXD2-AS1 derived from ADMSCs lncRNA FOXD2-AS1 targetly regulates miR-185-5p. In ADIPOCYTE 9 Figure 7. miR-185-5p overexpression inhibited the migration and proliferation of HaCaT cells through down-regulating ROCK2. Following different treatment, the HaCaT cell variety was evaluated utilizing CCK-8 analyses (a); HaCaT cell proliferation was analysed with EdU analyses (b); HaCaT cell migration at 0, 24, 48 h was analysed with wound healing analyses (c); the expressions of MMP-2 and MMP-9 levels were tested utilizing western blot (d). *P < 0.05 vs mimics NC +NC vector group, P < 0.05 vs. miR-185-5p mimics + NC vector group. addition, lncRNA FOXD2-AS1 directly interacts with disturbing MKP1 [41]. Croze et al. have reported that miR-185-5p as miRNA sponge and promotes tumour the suppression of ROCK can promote attachment, progression, including colorectal cancer [33], papillary proliferation, and wound closure in human embryonic thyroid cancer [34], and glioma [35]. In the current stem cell-derived retinal pigmented epithelium [42]. study, lncRNA FOXD2-AS1 overexpression in ADMSCs- Additionally, miR-203 overexpression inhibits the pro- exosomes promoted the migration and proliferation of liferation of epidermal stem cells through the down- HaCaT cells via down-regulating miR-185-5p. regulation of ROCK2 associated with Notch and Wnt MiRNAs are verified to reverse gene expression via pathways, leading to a delayed wound healing [43]. We degrading mRNA and inhibiting translation [36]. verified miR-185-5p can targetly regulate ROCK2 in ROCK2 is widely involved in cell biological activities this study. In addition, Niu et al. have proved that and plays an important role in controlling various cell miR-185-5p may inhibit hepatocellular carcinoma cell phenomena [37]. ROCK2 has been suggested to nega- migration through targeting ROCK2 [44]. In the pre- tively regulate the Parkin-dependent mitophagy path- sent study, we demonstrated that miR-185-5p overex- way [38]. ROCK2 contributes to diabetes-induced pression could inhibit the migration and proliferation 2+ impaired cardiac Ca homoeostasis [39]. ROCK2- of HaCaT cells through down-regulating ROCK2. induced glycolysis and proliferation in osteosarcoma [40]. ROCK2 promotes osteosarcoma growth and Conclusion metastasis by modifying the PFKFB3 ubiquitination and degradation [37]. ROCK2 promotes invasion and In the current study, highly expressed lncRNA FOXD2- metastasis in hepatocellular carcinoma through AS1 in ADMSCs derived exosomes might accelerate 10 H. CHANG ET AL. HaCaT cell migration and proliferation via modulating containing 10% FBS and 100 U/ml penicillin and the miR-185-5p/ROCK2 axis, revealing that exosomal streptomycin at 37°C with 5% CO . HaCaT cells lncRNA FOXD2-AS1 derived from ADMSCs may be (1 × 10 ) were seeded into a 100 mm dish and a novel therapeutic strategy for wound healing. incubated with 20 μg exosomes isolated from the ADMSCs for 24 h, then the culture supernatants were collected for the subsequent experiment. The Materials and Methods lncRNA FOXD2-AS1 overexpression plasmid (FOXD2-AS1 overexpression), miR-185-5p mimics, Isolation and identification of human ADMSCs ROCK2 overexpression plasmid (ROCK2 vector) The adipose tissues were acquired from the discarded and their corresponding negative controls were incu- tissue of a 5-year-old female patient who underwent bated with HaCaT cells for 48 h by Lipofectamine full-layer abdominal skin transplant surgery in our 3000 (Thermo Fisher Scientific, USA). The FOXD2- hospital. The protocol of this research has been AS1 overexpression plasmid and ROCK2 overexpres- approved by the Ethics Committee of Weifang sion plasmid were supplied by FulenGen Medical University. All patients have signed written (Guangzhou, China). AndmiR-185-5p mimic was informed consent. First of all, adipose tissues were cut obtained from GenePharma (Shanghai, China). into cubes of about 1 mm , which were free of fibre or blood vesicles. Following washed using 0.9% NS, adi- pose tissues were digested applying 0.75% type Cell counting kit-8 (CCK-8) assay I collagenase at 37°C for 1 h. Subsequently, the resus- pended cells were transplanted into DMEM medium The cell proliferation was evaluated by a CCK-8 Cell rd (Gibco, USA). The 3 ADMSCs were used for the Proliferation and Cytotoxicity Assay Kit (DOJINDO, subsequent experiment. ADMSCs characterization was 4 Japan). HaCaT cells (1 × 10 cells/well) were cultured evaluated utilizing flow cytometry, as described pre- in a 96-well plate. CCK-8 solution (10 μL) was added viously [45]. The antibodies for CD44 (1:100; into plate, subsequently the cells were further incubated #ab243894), CD105 (1:200; #ab231774), CD31 (1:200; for 48 h at 37°C with 5% CO . Optical density at #ab9498), and HLA-DR (1:100; #ab20181) were 450 nm was analysed applying a microplate reader. obtained from Abcam. LncRNA FOXD2-AS1 transfection 5-ethynyl-20-deoxyuridine (EdU) assay The lncRNA FOXD2-AS1 overexpression plasmid, si- FOXD2-AS1, and negative control (FulenGen, The rates of the proliferating cells were determined Guangzhou, China) were incubated with ADMSCs with an EdU proliferation kit (Beyotime, Shanghai, cells for 48 h by Lipofectamine 3000 (Thermo Fisher China). HaCaT cells (1 × 10 cells/well) were incubated Scientific, USA). in a 96-well plate, and the medium with EdU solution (50 μM) was added. Following 120 min of culture, the 4′, 6-diamidino-2-phenylindole (DAPI, Beyotime) was Isolation and identification of exosomes added into each well of the plates and shielded from At 48 h posttransfection, exosome was collected apply- light for 30 min. Finally, the EdU-positive cells were ing the Ribo™ Exosome Isolation Reagent (Ribobio, visualized using a fluorescent microscope and quanti- Guangzhou, China). The extracted exosome morphol- fied applying ImageJ software. ogy was determined using transmission electron micro- scopy (TEM; Leica, Germany). Exosome size distribution was analysed using the nanoparticle track- Wound healing assay ing analysis (NTA) as previously described [46]. Finally, the expression of CD9, CD63 and TSG101 HaCaT cells (4 × 10 cells/well) were plated into a 96- was evaluated by western blotting. well plate and cultured till reaching 90% confluence. After that, sterile pipette tips were applied to scrape cell confluent monolayer in a standardized manner, creat- Cell culture and treatment ing a cell-free zone in each well. After the culture of HaCaT cells for 48 h at 37°C, the wound’s distance at 0, Human keratinocyte cells (HaCaT cells) were sup- 24, and 48 h was photographed under the light plied by NanJing Cobioer Biosciences Co., Ltd microscope. (China). Cells were cultured in a DMEM medium ADIPOCYTE 11 Dual luciferase reporter assay Abcam, UK) were conducted with membranes over- night at 4°C. Subsequently, the secondary antibody Bioinformatics website StarBase (http://starbase.sysu. was utilized to incubate membranes for 1 h. The blots edu.cn/index.php) and TargetScanHuman (http:// were detected using enhanced chemiluminescence www.targetscan.org/vert_72/) were used to identify (Thermo Fisher Scientific, USA). potential binding sites for lncRNA FOXD2-AS1, miR- 185-5p, and ROCK2. ROCK2 3’-UTRs including wild- type (WT) and mutant (MUT) miR-185-5p binding site Statistical analysis or FOXD2-AS1 3’-UTRs including WT and MUT miR- Data obtained in the study were represented as the 185-5p binding site were inserted into pmir-reporter mean ± SD of three independent experimental repeats vector to construct ROCK2-WT and ROCK2-MUT and measured utilizing GraphPad Prism 8.0. Statistical reporter vectors or FOXD2-AS1-WT and FOXD2-AS1- differences were determined by Student's t-test or one- MUT reporter vectors. After that, HaCaT cells trans- way ANOVA. A p-value <0.05 was considered to be fected these reporter vectors, miR-185-5p mimics and significant in all experiments. mimics NC for 2 d utilizing Lipofectamine 2000 (Thermo Fisher Scientific, USA). Luciferase activity was determined utilizing Dual-Luciferase Reporter Kit List of abbreviations (Vazyme, Nanjing, China). Adipose mesenchymal stem cells (ADMSCs), Mesenchymal stem cells (MSCs) qRT-PCR Total RNA was extracted with TRIzol (Beyotime, Disclosure statement Beijing, China). The complementary DNA was synthe- sized using the EasyScript® First-Strand cDNA No potential conflict of interest was reported by the author(s). Synthesis SuperMix (Transgen, Beijing, China) and TransScript® miRNA First-Strand cDNA Synthesis Funding SuperMix (Transgen) based on the instructions of the manufacturer. Afterwards, RT-PCR was carried out The author(s) reported that there is no funding associated with the work featured in this article. with the TransScript®Green Two-Step qRT-PCR SuperMix (Transgen) or TransScript® Green miRNA Two-Step qRT-PCR SuperMix (Transgen). The primer Ethics approval and consent to participate sequences were as follows: FOXD2-AS1-sence: 5′- TGGACCTAGCTGCAGCTCCA-3′, antisense: 5′- The protocol of this research has been approved by the Ethics AGTTGAAGGTGCACACACTG-3′; miR-185-5p-sense Committee of Weifang Medical University. (WFYXY20201003) All patients have signed written : 5′-GCGGCGGTGGAGAGAAAGGCAG-3′, antisense: informed consent. The experiments were carried out accord- 5′-ATCCAGTGCAGGGTCCGAGG-3′; ROCK2-sence: ing to the principles of the Declaration of Helsinki. 5′-AACGTCAGGATGCAGATGGG-3′, antisense: 5′- CAGCCAAAGAGTCCCGTTCA-3′; GAPDH-sence: 5′-GTTGCAACCGGGAAGGAAAT-3′, antisense: 5′- Consent for publication GCCCAATACGACCAAATCAGA-3′ and U6-sence: All authors have read and approved the manuscript. 5′-CAGCACATATACTAAAATTGGAACG-3′, anti- sence: 5′-ACGAATTTGCGTGTCATCC-3′. Data Availability statement Western blot The datasets used and analysed during the current study are available from the corresponding author on reasonable request. Total protein was extracted by Radio Immunoprecipitation Assay buffer (Thermo Fisher Scientific, USA). Equal amounts of protein (50 μg per Author contributions lane) were detached by 10% SDS-PAGE and transferred Conception and design: Huanchao Chang; Perform research: onto nitrocellulose membranes. After that, the primary Huanchao Chang, Junliang Chen, and Tianling Cheng; Data antibodies (CD9, #ab236630; CD63, #ab134045; analysis and interpretation: Kun Ding and Shengjian Tang; TSG101, #ab125011; MMP-2, #ab92536; MMP-9, Manuscript writing: All authors; Final approval of manuscript: #ab137867; ROCK2, #ab125025; GAPDH, #ab181602, All authors 12 H. CHANG ET AL. Regulating MiR-185/AKT Axis. Med Sci Monit. References 2019;25:9618–9629. [1] Du H, Zhou Y, Suo Y, et al. CCN1 accelerates [16] Gao J, Liu F, Zhao X, et al. 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Highly-expressed lncRNA FOXD2-AS1 in adipose mesenchymal stem cell derived exosomes affects HaCaT cells via regulating miR-185-5p/ROCK2 axis

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Abstract

ADIPOCYTE 2023, VOL. 12, NO. 1, 2173513 https://doi.org/10.1080/21623945.2023.2173513 RESEARCH PAPER Highly-expressed lncRNA FOXD2-AS1 in adipose mesenchymal stem cell derived exosomes affects HaCaT cells via regulating miR-185-5p/ROCK2 axis a b a c a Huanchao Chang , Junliang Chen , Kun Ding , Tianling Cheng , and Shengjian Tang a b Plastic Surgery of Plastic Surgery Hospital, Weifang Medical University, Weifang, China; Vascular surgery department, Affiliated Hospital of Weifang Medical College, Weifang, China; Burn plastic surgery, The First Affiliated Hospital of Xi’an Medical University, Xi’an, China ABSTRACT ARTICLE HISTORY The healing of skin wounds is a highly coordinated multi-step process that occurs after trauma Received 8 November 2022 including surgical incisions, thermal burns, and chronic ulcers. In this study, the authors investi- Revised 3 January 2023 Accepted 24 January 2023 gated lncRNA FOXD2-AS1 function in adipose mesenchymal exosomes from ADMSCs that were successfully extracted. Highly expressed lncRNA FOXD2-AS1 in ADMSCs-exosomes accelerated KEYWORDS HaCaT cell migration and proliferation. LncRNA FOXD2-AS1 negatively targeted miR-185-5p, and Exosomes; FOXD2-AS1; miR-185-5p negatively targeted ROCK2. Highly expressed lncRNA FOXD2-AS1 in ADMSCs- wound healing; miR-185-5p; exosomes promoted HaCaT cell migration and proliferation via down-regulating miR-185-5p ROCK2 and further up-regulating ROCK2. In conclusion, LncRNA FOXD2-AS1 overexpression in ADMSCs derived exosomes might accelerate HaCaT cell migration and proliferation via modulating the miR-185-5p/ROCK2 axis. keratinocyte migration and proliferation [9]. Moreover, Introduction human ADMSCs are an attractive resource for wound The healing of skin wounds is a highly coordinated healing due to their regenerative ability to promote multi-step process that occurs after trauma, including injury repair [10]. A study has confirmed that surgical incisions, thermal burns, and chronic ulcers ADMSCs could accelerate wound healing through opti- [1]. The failure of proceeding through such orderly mizing fibroblasts characteristics [11], which further and timely reparation can induce chronic non- verified their regenerative ability to promote injury healing wounds including diabetic, venous, and decu- repair in wound healing. bitus skin ulcers [2,3]. Refractory wound is a huge Long noncoding RNAs (lncRNAs) have been burden to both the patient and society. Thus, explor- reported to exist in exosomes and modulate gene ing a novel marker for wound healing therapy and expression in host cells through intercellular commu- clarifying the mechanisms of this fatal disease are nication [12]. For example, the high expression of lncRNA H19 in ADMSCs-exosomes can up-regulate imperative. the expression of SOX9 through miR-19b to promote Mesenchymal stem cells (MSCs) are one type of wound healing [13]. A study has proven that exosomal multipotent progenitor cells that are derived from lncRNA FOXD2-AS1 can act as the promising biomar- bone marrow, umbilical cord, and adipose tissue and kers for the diagnostics of colorectal cancer [14]. play a well-known function in tissue regeneration [4]. LncRNA FOXD2-AS1 promotes the progression of In recent years, more and more studies have strongly a variety of tumours [15–18]. Moreover, lncRNA proved that exosomes derived from adipose-derived FOXD2-AS1 promotes the proliferation of many mesenchymal stem cells (ADMSCs) are safe and have kinds of cells, including trophoblast cell [19], fibro- become the hot pot of many researches in many differ- blast-like synoviocytes [20], and chondrocyte [21]. ent fields, such as wound healing [5–7]. A previous However, the evidence regarding the implication of study has demonstrated that ADMSCs-derived exo- exosomal lncRNA FOXD2-AS1 derived from somes can accelerate cell proliferation and migration ADMSCs in wound healing is lacking. through regulating Wnt/β-catenin pathway in cuta- In the current study, we paid attention to evaluate neous wound healing [8]. ADMSCs-derived exosomes the effect of exosomal lncRNA FOXD2-AS1 derived are reported to promote wound healing via accelerating CONTACT Shengjian Tang tsj1950@163.com Plastic Surgery Institute, Weifang Medical University, 4948 Shengli East Street, Kuiwen District, Weifang, 261041, China © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2 H. CHANG ET AL. from ADMSCs in wound healing and its potential Highly expressed lncRNA FOXD2-AS1 in mechanisms. Our findings demonstrated that highly ADMSCs-exosomes accelerated HaCaT cell expressed lncRNA FOXD2-AS1 in ADMSCs derived migration and proliferation exosomes accelerated HaCaT cell migration and prolif- We overexpressed lncRNA FOXD2-AS1 in ADSCs and eration via the regulation of miR-185-5p/rho-associated extracted exosomes from ADSCs. The level of lncRNA coiled-coil-containing protein kinase 2 (ROCK2) axis, FOXD2-AS1 was significantly upregulated following suggesting that exosomal lncRNA FOXD2-AS1 derived lncRNA FOXD2-AS1 overexpression plasmid transfec- from ADMSCs may be a novel therapeutic strategy for tion (Figure 2a). As shown in Figure 2b, treatment with wound healing. exosomes significantly increased lncRNA FOXD2-AS1 expression in HaCaT cells, and lncRNA FOXD2-AS1 overexpression-transfected exosomes further elevated Results lncRNA FOXD2-AS1 expression. CCK-8 and EdU assays data proved that exosomes treatment markedly promoted Identification of ADMSCs and exosomes the proliferation of HaCaT cells (Figure 2c and 2d). Flow cytometric analysis was utilized to examine the Meanwhile, the proliferation was significantly increased cell surface protein expression of isolated ADMSCs. As in FOXD2-AS1 exosome group relative to NC exosome shown in Figure 1a, ADMSCs were positive with CD44 group (Figure 2c and 2d). In addition, we assessed the and CD105, but negative with CD31 and HLA-DR. migrative function of highly expressed lncRNA FOXD2- Then, we further identified ADMSCs-exomoses AS1 in exosomes by wound healing analyses. Figure 2e through TEM (Figure 1b). Furthermore, the data of confirmed that exosomes treatment markedly increased NTA confirmed that the particle size of the extracted HaCaT cell migration, and lncRNA FOXD2-AS1 over- exosomes was mainly distributed at about 100 nm expression-transfected exosomes further elevated HaCaT (Figure 1c). Moreover, the presentation of specific bio- cell migration compared with NC exosomes. Similarly, markers CD9, CD63 and TSG101 indicated that the the results are also demonstrated using western blot by exosomes were successfully extracted (Figurerue 1D). detecting MMP-2 and MMP-9 (figure 2f). Figure 1. Identification of ADMSCs and exosomes. (a) Flow cytometric analysis of cell surface protein expression of isolated ADMSCs. (b) The ultrastructure of ADSCs-exo under TEM. (c) Exosomes size distribution was analysed using NTA. (d) The expression of CD9, CD63 and TSG101 was detected using western blot. ADIPOCYTE 3 Figure 2. Highly expressed lncRNA FOXD2-AS1 in ADMSCs-exosomes promoted the migration and proliferation of HaCaT cells. Following different treatment, the expression of lncRNA FOXD2-AS1 in ADMSCs-exosomes was detected using qRT-PCR (a); the expression of lncRNA FOXD2-AS1 in HaCaT cells was detected using qRT-PCR (b); HaCaT cell variety was evaluated using CCK-8 assay (B); HaCaT cell proliferation was determined with EdU assay (c); HaCaT cell migration at 0, 24, 48 h was analysed with wound healing assay (d); MMP-2 and MMP-9 level were tested utilizing western blot (e). *P < 0.05 vs. exosome or control group; P < 0.05 vs. NC exosome group. 4 H. CHANG ET AL. Knockdown lncRNA FOXD2-AS1 in miR-185-5p mimics group was elevated relative to ADMSCs-exosomes inhibited HaCaT cell migration NC exosome + miR-185-5p mimics group, but was and proliferation reduced relative to FOXD2-AS1 exosome + mimics NC group (Figure 5a and 5b). Moreover, the results We knockdown lncRNA FOXD2-AS1 in ADSCs and of Figure 4c showed that the migration of HaCaT cells extracted exosomes from ADSCs. The level of was notably decreased in NC exosome + miR-185-5p lncRNA FOXD2-AS1 was significantly downregulated mimics group relative to NC exosome + mimics NC following si-lncRNA FOXD2-AS1 transfection group, but increased in FOXD2-AS1 exosome + (Figure 3a). Exosomes significantly increased mimics NC group. The migration of HaCaT cells in lncRNA FOXD2-AS1 expression in HaCaT cells, FOXD2-AS1 exosome + miR-185-5p mimics group and si-lncRNA FOXD2-AS1 transfection exosomes was increased compared with NC exosome + miR- decreased lncRNA FOXD2-AS1 expression 185-5p mimics group but was decreased relative to (Figure 3b). CCK-8 and EdU assays data proved FOXD2-AS1 exosome + mimics NC group that the proliferation was significantly decreased in (Figure 5c). Similarly, the results are also demon- si-FOXD2-AS1 exosome group relative to the NC strated using western blot. MMP-2 and MMP-9 level exosome group (Figure 3c and 3d). si-FOXD2-AS1 was lower in NC exosome + miR-185-5p mimics transfected exosomes decreased HaCaT cell migration group relative to NC exosome + mimics NC group, (Figure 3e). Similarly, the results are also demon- but higher in FOXD2-AS1 exosome + mimics NC strated using western blot by detecting MMP-2 and group (Figure 5d). Meanwhile, the expressions of MMP-9 (figure 3f). MMP-2 and MMP-9 level in FOXD2-AS1 exosome + miR-185-5p mimics group was elevated relative to NC exosome + miR-185-5p mimics group and down- lncRNA FOXD2-AS1 negatively targeted miR-185-5p regulated relative to FOXD2-AS1 exosome + mimics After transfection of lncRNA FOXD2-AS1 overexpres- NC group (Figure 5d). sion vector in HaCaT cells, lncRNA FOXD2-AS1 level was significantly upregulated (Figure 4a), and the expression of miR-185-5p was markedly decreased miR-185-5p negatively targeted ROCK2 (Figure 4b). Additionally, following transfected with miR-185-5p mimics in HaCaT cells, miR-185-5p level As shown in Figure 6a, the expression of ROCK2 was was elevated (Figure 4c) and lncRNA FOXD2-AS1 level upregulated in HaCaT cell followed by transfection of was reduced (Figure 4d). The predicted binding sites ROCK2 overexpression vector. Additionally, the between lncRNA FOXD2-AS1 and miR-185-5p are expression of ROCK2 was downregulated followed shown in Figure 3e. Furthermore, miR-185-5p mimics by transfection of miR-185-5p mimics (Figure 6b). notably inhibited luciferase activities in FOXD2-AS1- Figure 6c is predicted for binding sites between WT group, while had no function on luciferase activ- miR-185-5p and ROCK2. Figure 6d proves miR- ities in FOXD2-AS1-MUT group (Figure 4f). 185-5p mimics decreased luciferase activity in the Moreover, we also found that exosomes treatment ROCK2-WT group, while had no function on luci- only significantly reduced miR-185-5p level, and ferase activities in ROCK2-MUT group. Furthermore, lncRNA FOXD2-AS1 overexpression-transfected exo- Figure 5e and 5f data showed that ROCK2 expression somes further inhibited the expression of miR-185-5p were higher in mimics NC + ROCK2 vector group (Figure 4g). than that in mimics NC + NC vector group, but lower in miR-185-5p mimics + NC vector group. Meanwhile, mRNA and protein expression of Highly expressed lncRNA FOXD2-AS1 in ROCK2 in miR-185-5p mimics + ROCK2 vector ADMSCs-exosomes promoted HaCaT cell migration group was reduced relative to mimics NC + ROCK2 and proliferation via down-regulating miR-185-5p vector group, but higher than that in miR-185-5p mimics + NC vector group (Figure 6e and 6f). The The results of CCK-8 and EdU assays demonstrated above data suggested that miR-185-5p could nega- that in the comparison with NC exosome + mimics tively target ROCK2. Moreover, we also found that NC group, the proliferation of HaCaT cells was nota- exosomes treatment significantly induced ROCK2 bly reduced in NC exosome + miR-185-5p mimics expression, and lncRNA FOXD2-AS1 overexpression- group, but elevated in FOXD2-AS1 exosome + mimics transfected exosomes further promoted the expres- NC group (Figure 5a and 5b). Meanwhile, the prolif- sion of ROCK2 (Figure 6g and 6h). eration of HaCaT cells in FOXD2-AS1 exosome + ADIPOCYTE 5 Figure 3. Knockdown lncRNA FOXD2-AS1 in ADMSCs-exosomes inhibited HaCaT cell migration and proliferation. Following different treatment, the expression of lncRNA FOXD2-AS1 in ADMSCs-exosomes was detected using qRT-PCR (a); the expression of lncRNA FOXD2-AS1 in HaCaT cells was detected using qRT-PCR (b); HaCaT cell variety was evaluated using CCK-8 assay (B); HaCaT cell proliferation was determined with EdU assay (c); HaCaT cell migration at 0, 24, 48 h was analysed with wound healing assay (d); MMP-2 and MMP-9 level were tested utilizing western blot (e). *P < 0.05 vs. exosome or control group; P < 0.05 vs. NC exosome group. 6 H. CHANG ET AL. Figure 4. lncRNA FOXD2-AS1 negatively targeted miR-185-5p. After transfection of lncRNA FOXD2-AS1 overexpression plasmid, lncRNA FOXD2-AS1 (a) and miR-185-5p (b) levels were tested using qRT-PCR. Following transfection of miR-185-5p mimics, the expression of miR-185-5p (c) and lncRNA FOXD2-AS1 (d) was tested using qRT-PCR. (e) The bind sites of lncRNA FOXD2-AS1 and miR- 185-5p. (f) A dual luciferase reporter assay verified the binding relationship between lncRNA FOXD2-AS1 and miR-185-5p. (D) After treatment of exosomes and lncRNA FOXD2-AS1 overexpression-transfected exosomes, qRT-PCR was utilized to assess miR-185-5p level (g). *P < 0.05 vs. FOXD2-AS1 vector, mimics NC, mmics NC + FOXD2-AS1 WT group, and control group, P < 0.05 vs. NC exosome group. ADIPOCYTE 7 Figure 5. Highly expressed lncRNA FOXD2-AS1 in ADMSCs-exosomes promoted HaCaT cell migration and proliferation via down- regulating miR-185-5p. Following different treatment, the HaCaT cell variety was assessed utilizing CCK-8 analyses (a); HaCaT cell proliferation was evaluated applying EdU analyses (b); HaCaT cell migration at 0, 24, 48 h was analysed with wound healing analyses (c); MMP-2 and MMP-9 levels were tested utilizing western blot (d). *P < 0.05 vs. NC exosome + mimics NC group, P < 0.05 vs. FOXD2-AS1 exosome + mimics NC group. miR-185-5p overexpression inhibited HaCaT cell ROCK2 vector group was inhibited relative to mimics migration and proliferation through NC + ROCK2 vector group, but was elevated relative to down-regulating ROCK2 miR-185-5p mimics + NC vector group (Figure 7c). Moreover, western blot results confirmed that MMP-2 As shown in Figure 7a and 7b, the proliferation of and MMP-9 level was higher in mimics NC + ROCK2 HaCaT cells was notably elevated in mimics NC + vector group than those in mimics NC + NC vector ROCK2 vector group than that in mimics NC + NC group, but lower in miR-185-5p mimics + NC vector vector group, but downregulated in miR-185-5p group (Figure 7d). Meanwhile, MMP-2 and MMP-9 mimics + NC vector group. HaCaT cell proliferation level in miR-185-5p mimics + ROCK2 vector group in miR-185-5p mimics + ROCK2 vector group was was decreased when compared with mimics NC + decreased compared with mimics NC + ROCK2 vector ROCK2 vector group, and increased in miR-185-5p group but was increased compared with miR-185-5p mimics + NC vector group (Figure 7d). mimics + NC vector group (Figure 7a and 7b). In comparison with mimics NC + NC vector group, HaCaT cell migration was promoted in mimics NC + Discussion ROCK2 vector group and decreased in miR-185-5p mimics + NC vector group (Figure 7c). Meanwhile, A basic characteristic of wound healing is the repair of HaCaT cell migration in miR-185-5p mimics + the intact epidermal barrier via reepithelialization [22]. 8 H. CHANG ET AL. Figure 6. miR-185-5p negatively targeted ROCK2. (a) After transfection of ROCK2 overexpression vector, the expression of ROCK2 was detected using qRT-PCR. (b) Following transfection of miR-185-5p mimics, ROCK2 level was detected using qRT-PCR. (c) The bind sites of miR-185-5p and ROCK2. (d) A dual luciferase reporter assay verified the binding relationship between miR-185-5p and ROCK2. After co-transfection with ROCK2 overexpression vector and miR-185-5p mimics, ROCK2 expressions were analysed using qRT-PCR (e) and western blot (f). *P < 0.05 vs. vector NC, mimics NC, mimics NC +ROCK2 WT, and mimics NC+ NC vector group, P < 0.05 vs miR-185-5p mimics + NC vector and NC exosome group. It is reported that keratinocytes proliferation and in wound healing. Our data demonstrate that highly migration are closely related to reepithelialization and expressed lncRNA FOXD2-AS1 in ADMSCs-exosomes closure of the wound gap [23,24]. In the current study, might accelerate HaCaT cell migration and proliferation. highly expressed lncRNA FOXD2-AS1 in ADMSCs Recent research data suggest that miRNAs are promis- derived exosomes promoted HaCaT cell migration ing tools for the treatment and diagnosis of skin wound and proliferation via modulating the miR-185-5p/ healing because miRNAs are important regulators of cel- ROCK2 axis. lular physiology and pathology [29]. miR-200b/c-3p can Currently, increasing evidences have suggested that the modulate epithelial plasticity and repress cutaneous novel role of exosomes as cell communication bodies is wound healing through the regulation of TGF-β- dependent on exosomal cargo, including lncRNAs, mediated RAC1 pathway [30]. It is interesting that microRNAs (miRNAs) and proteins [25,26]. lncRNA interacts with miRNA and the interactions play Furthermore, accumulating evidences relate regulatory critical roles in determining cell fate [31]. Adipose- lncRNAs to human diseases. LncRNA FOXD2-AS1 may derived stem cells-exosomes containing lncRNA accelerate proliferation and migration activities in MALAT1 can promote wound healing via targeting a various of cells [20,27,28]. Interestingly, a study has miR-124 [25]. LncRNA GAS5 is proved to accelerate proven that exosomal lncRNA FOXD2-AS1 can also act diabetic wound healing and promote lymphangiogenesis as the promising biomarkers for the diagnostics of color- by miR-217/Prox1 axis [32]. Through bioinformatics ana- ectal cancer [14]. Therefore, we explored the effects of lyses and Dual-luciferase reporter assay, we verified that exosomal lncRNA FOXD2-AS1 derived from ADMSCs lncRNA FOXD2-AS1 targetly regulates miR-185-5p. In ADIPOCYTE 9 Figure 7. miR-185-5p overexpression inhibited the migration and proliferation of HaCaT cells through down-regulating ROCK2. Following different treatment, the HaCaT cell variety was evaluated utilizing CCK-8 analyses (a); HaCaT cell proliferation was analysed with EdU analyses (b); HaCaT cell migration at 0, 24, 48 h was analysed with wound healing analyses (c); the expressions of MMP-2 and MMP-9 levels were tested utilizing western blot (d). *P < 0.05 vs mimics NC +NC vector group, P < 0.05 vs. miR-185-5p mimics + NC vector group. addition, lncRNA FOXD2-AS1 directly interacts with disturbing MKP1 [41]. Croze et al. have reported that miR-185-5p as miRNA sponge and promotes tumour the suppression of ROCK can promote attachment, progression, including colorectal cancer [33], papillary proliferation, and wound closure in human embryonic thyroid cancer [34], and glioma [35]. In the current stem cell-derived retinal pigmented epithelium [42]. study, lncRNA FOXD2-AS1 overexpression in ADMSCs- Additionally, miR-203 overexpression inhibits the pro- exosomes promoted the migration and proliferation of liferation of epidermal stem cells through the down- HaCaT cells via down-regulating miR-185-5p. regulation of ROCK2 associated with Notch and Wnt MiRNAs are verified to reverse gene expression via pathways, leading to a delayed wound healing [43]. We degrading mRNA and inhibiting translation [36]. verified miR-185-5p can targetly regulate ROCK2 in ROCK2 is widely involved in cell biological activities this study. In addition, Niu et al. have proved that and plays an important role in controlling various cell miR-185-5p may inhibit hepatocellular carcinoma cell phenomena [37]. ROCK2 has been suggested to nega- migration through targeting ROCK2 [44]. In the pre- tively regulate the Parkin-dependent mitophagy path- sent study, we demonstrated that miR-185-5p overex- way [38]. ROCK2 contributes to diabetes-induced pression could inhibit the migration and proliferation 2+ impaired cardiac Ca homoeostasis [39]. ROCK2- of HaCaT cells through down-regulating ROCK2. induced glycolysis and proliferation in osteosarcoma [40]. ROCK2 promotes osteosarcoma growth and Conclusion metastasis by modifying the PFKFB3 ubiquitination and degradation [37]. ROCK2 promotes invasion and In the current study, highly expressed lncRNA FOXD2- metastasis in hepatocellular carcinoma through AS1 in ADMSCs derived exosomes might accelerate 10 H. CHANG ET AL. HaCaT cell migration and proliferation via modulating containing 10% FBS and 100 U/ml penicillin and the miR-185-5p/ROCK2 axis, revealing that exosomal streptomycin at 37°C with 5% CO . HaCaT cells lncRNA FOXD2-AS1 derived from ADMSCs may be (1 × 10 ) were seeded into a 100 mm dish and a novel therapeutic strategy for wound healing. incubated with 20 μg exosomes isolated from the ADMSCs for 24 h, then the culture supernatants were collected for the subsequent experiment. The Materials and Methods lncRNA FOXD2-AS1 overexpression plasmid (FOXD2-AS1 overexpression), miR-185-5p mimics, Isolation and identification of human ADMSCs ROCK2 overexpression plasmid (ROCK2 vector) The adipose tissues were acquired from the discarded and their corresponding negative controls were incu- tissue of a 5-year-old female patient who underwent bated with HaCaT cells for 48 h by Lipofectamine full-layer abdominal skin transplant surgery in our 3000 (Thermo Fisher Scientific, USA). The FOXD2- hospital. The protocol of this research has been AS1 overexpression plasmid and ROCK2 overexpres- approved by the Ethics Committee of Weifang sion plasmid were supplied by FulenGen Medical University. All patients have signed written (Guangzhou, China). AndmiR-185-5p mimic was informed consent. First of all, adipose tissues were cut obtained from GenePharma (Shanghai, China). into cubes of about 1 mm , which were free of fibre or blood vesicles. Following washed using 0.9% NS, adi- pose tissues were digested applying 0.75% type Cell counting kit-8 (CCK-8) assay I collagenase at 37°C for 1 h. Subsequently, the resus- pended cells were transplanted into DMEM medium The cell proliferation was evaluated by a CCK-8 Cell rd (Gibco, USA). The 3 ADMSCs were used for the Proliferation and Cytotoxicity Assay Kit (DOJINDO, subsequent experiment. ADMSCs characterization was 4 Japan). HaCaT cells (1 × 10 cells/well) were cultured evaluated utilizing flow cytometry, as described pre- in a 96-well plate. CCK-8 solution (10 μL) was added viously [45]. The antibodies for CD44 (1:100; into plate, subsequently the cells were further incubated #ab243894), CD105 (1:200; #ab231774), CD31 (1:200; for 48 h at 37°C with 5% CO . Optical density at #ab9498), and HLA-DR (1:100; #ab20181) were 450 nm was analysed applying a microplate reader. obtained from Abcam. LncRNA FOXD2-AS1 transfection 5-ethynyl-20-deoxyuridine (EdU) assay The lncRNA FOXD2-AS1 overexpression plasmid, si- FOXD2-AS1, and negative control (FulenGen, The rates of the proliferating cells were determined Guangzhou, China) were incubated with ADMSCs with an EdU proliferation kit (Beyotime, Shanghai, cells for 48 h by Lipofectamine 3000 (Thermo Fisher China). HaCaT cells (1 × 10 cells/well) were incubated Scientific, USA). in a 96-well plate, and the medium with EdU solution (50 μM) was added. Following 120 min of culture, the 4′, 6-diamidino-2-phenylindole (DAPI, Beyotime) was Isolation and identification of exosomes added into each well of the plates and shielded from At 48 h posttransfection, exosome was collected apply- light for 30 min. Finally, the EdU-positive cells were ing the Ribo™ Exosome Isolation Reagent (Ribobio, visualized using a fluorescent microscope and quanti- Guangzhou, China). The extracted exosome morphol- fied applying ImageJ software. ogy was determined using transmission electron micro- scopy (TEM; Leica, Germany). Exosome size distribution was analysed using the nanoparticle track- Wound healing assay ing analysis (NTA) as previously described [46]. Finally, the expression of CD9, CD63 and TSG101 HaCaT cells (4 × 10 cells/well) were plated into a 96- was evaluated by western blotting. well plate and cultured till reaching 90% confluence. After that, sterile pipette tips were applied to scrape cell confluent monolayer in a standardized manner, creat- Cell culture and treatment ing a cell-free zone in each well. After the culture of HaCaT cells for 48 h at 37°C, the wound’s distance at 0, Human keratinocyte cells (HaCaT cells) were sup- 24, and 48 h was photographed under the light plied by NanJing Cobioer Biosciences Co., Ltd microscope. (China). Cells were cultured in a DMEM medium ADIPOCYTE 11 Dual luciferase reporter assay Abcam, UK) were conducted with membranes over- night at 4°C. Subsequently, the secondary antibody Bioinformatics website StarBase (http://starbase.sysu. was utilized to incubate membranes for 1 h. The blots edu.cn/index.php) and TargetScanHuman (http:// were detected using enhanced chemiluminescence www.targetscan.org/vert_72/) were used to identify (Thermo Fisher Scientific, USA). potential binding sites for lncRNA FOXD2-AS1, miR- 185-5p, and ROCK2. ROCK2 3’-UTRs including wild- type (WT) and mutant (MUT) miR-185-5p binding site Statistical analysis or FOXD2-AS1 3’-UTRs including WT and MUT miR- Data obtained in the study were represented as the 185-5p binding site were inserted into pmir-reporter mean ± SD of three independent experimental repeats vector to construct ROCK2-WT and ROCK2-MUT and measured utilizing GraphPad Prism 8.0. Statistical reporter vectors or FOXD2-AS1-WT and FOXD2-AS1- differences were determined by Student's t-test or one- MUT reporter vectors. After that, HaCaT cells trans- way ANOVA. A p-value <0.05 was considered to be fected these reporter vectors, miR-185-5p mimics and significant in all experiments. mimics NC for 2 d utilizing Lipofectamine 2000 (Thermo Fisher Scientific, USA). Luciferase activity was determined utilizing Dual-Luciferase Reporter Kit List of abbreviations (Vazyme, Nanjing, China). Adipose mesenchymal stem cells (ADMSCs), Mesenchymal stem cells (MSCs) qRT-PCR Total RNA was extracted with TRIzol (Beyotime, Disclosure statement Beijing, China). The complementary DNA was synthe- sized using the EasyScript® First-Strand cDNA No potential conflict of interest was reported by the author(s). Synthesis SuperMix (Transgen, Beijing, China) and TransScript® miRNA First-Strand cDNA Synthesis Funding SuperMix (Transgen) based on the instructions of the manufacturer. Afterwards, RT-PCR was carried out The author(s) reported that there is no funding associated with the work featured in this article. with the TransScript®Green Two-Step qRT-PCR SuperMix (Transgen) or TransScript® Green miRNA Two-Step qRT-PCR SuperMix (Transgen). The primer Ethics approval and consent to participate sequences were as follows: FOXD2-AS1-sence: 5′- TGGACCTAGCTGCAGCTCCA-3′, antisense: 5′- The protocol of this research has been approved by the Ethics AGTTGAAGGTGCACACACTG-3′; miR-185-5p-sense Committee of Weifang Medical University. (WFYXY20201003) All patients have signed written : 5′-GCGGCGGTGGAGAGAAAGGCAG-3′, antisense: informed consent. The experiments were carried out accord- 5′-ATCCAGTGCAGGGTCCGAGG-3′; ROCK2-sence: ing to the principles of the Declaration of Helsinki. 5′-AACGTCAGGATGCAGATGGG-3′, antisense: 5′- CAGCCAAAGAGTCCCGTTCA-3′; GAPDH-sence: 5′-GTTGCAACCGGGAAGGAAAT-3′, antisense: 5′- Consent for publication GCCCAATACGACCAAATCAGA-3′ and U6-sence: All authors have read and approved the manuscript. 5′-CAGCACATATACTAAAATTGGAACG-3′, anti- sence: 5′-ACGAATTTGCGTGTCATCC-3′. Data Availability statement Western blot The datasets used and analysed during the current study are available from the corresponding author on reasonable request. Total protein was extracted by Radio Immunoprecipitation Assay buffer (Thermo Fisher Scientific, USA). Equal amounts of protein (50 μg per Author contributions lane) were detached by 10% SDS-PAGE and transferred Conception and design: Huanchao Chang; Perform research: onto nitrocellulose membranes. After that, the primary Huanchao Chang, Junliang Chen, and Tianling Cheng; Data antibodies (CD9, #ab236630; CD63, #ab134045; analysis and interpretation: Kun Ding and Shengjian Tang; TSG101, #ab125011; MMP-2, #ab92536; MMP-9, Manuscript writing: All authors; Final approval of manuscript: #ab137867; ROCK2, #ab125025; GAPDH, #ab181602, All authors 12 H. CHANG ET AL. Regulating MiR-185/AKT Axis. Med Sci Monit. References 2019;25:9618–9629. [1] Du H, Zhou Y, Suo Y, et al. CCN1 accelerates [16] Gao J, Liu F, Zhao X, et al. 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Journal

AdipocyteTaylor & Francis

Published: Dec 31, 2023

Keywords: Exosomes; FOXD2-AS1; wound healing; miR-185-5p; ROCK2

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