Adipose‐derived mesenchymal stem cells‐sourced exosomal microRNA‐7846‐3p suppresses proliferation and pro‐angiogenic role of keloid fibroblasts by suppressing neuropilin 2

Exosomes (Exos) and their contained microRNAs (miRNAs) have been emergingly recognized as key regulators in spanning biological processes, including proliferation and angiogenesis.


| INTRODUC TI ON
Skin wounds are frequent during human life time. Healthy cutaneous wound healing is a complex and well-orchestrated process involving hemostasis, inflammation, angiogenesis, granulation tissue formation, reepithelialization, and extracellular matrix (ECM) remodeling. 1,2 However, abnormal proliferation and differentiation of fibroblasts can lead to redundant deposition of ECM and pathological scarring, including hypertrophic scars and keloid. 3,4 The two pathological scars have several adverse consequences such as pain, itching, disfiguring, and motion restriction, though, they have major distinctions in prognosis: hypertrophic scars usually develop within the original wound sites and may regress over time, whereas keloids grow without limitation and barely regress. 5 Nowadays, keloid formation continues to evade the normal wound healing process and remains recalcitrant despite several medical and surgical therapies, including intralesional steroid injection and surgical excision. 6 Owing to their pluripotency and abilities in self-renewal and secreting regenerative cytokines, mesenchymal stem cells (MSCs) have emerged as an attractive source of stem cells for tissue repair and wound healing. 7,8 Exosomes (Exos) represent one of the major components of paracrine and the major contributor to MSC efficacy. 7 They are nano-sized membrane vesicles responsible for intercellular communication by transporting proteins, nucleic acid, and drugs to target specific cells. 9 Adipose-derived MSCs (adMSCs) are among the most frequently studied MSC types due to their high frequency in liposuctions and convenience in extraction. 10 The adMSCs and their derived Exos (adMSC-Exos) have critical functions in augmenting cutaneous wound healing. 7 However, their roles in excessive scarring are less concerned.
MicroRNAs (miRNAs) are ~22 nt noncoding RNAs which direct posttranscriptional degradation of mRNA targets by binding to complementary sites in the 3′untranslated region (3′UTR). 11 They are the major component of Exos, and their delivery by Exos protects them from degradation, enabling them to affect gene expression in target cells. 12 Dysregulation of miRNAs has been implicated in spanning cellular behaviors such as proliferation, fibroblast activation, apoptosis, and collagen production during keloid formation. 13 This study aims to explore the function of adMSC-Exos in keloid and the major miRNA involved.

| Extraction and authentication of adMSC-Exos
To rule out the potential impact of inherent Exos in FBS, the FBScontaining culture medium for adMSC cell culture was centrifugated at 100 000 g at 4°C for 16 h, and then filtered at 0.22 μm to discard the Exos. The ad-MSCs were cultured in the Exos-deprived medium for another 24 h at 37°C with 5% CO 2 . The supernatant was collected and successively centrifuged at 300 g for 10 min,

| Uptake of adMSC-Exos by KFs
The extracted Exos were labeled using PKH67 (green fluorescence) membrane labeling kit (HR8659, Bio-Lab Technologies). In short, 10 μg (1 mL) Exos were mixed with 4 μL PKH67 for 15 min, followed by ultracentrifugation at 110 000 g at 4°C for 1 h with the supernatant discarded. The Exos were resuspended in PBS and cocultured with KFs for 24 h. Thereafter, the KFs were fixed with 4% paraformaldehyde. Nuclei of cells were stained with 10 μg/mL DAPI (C1002, Beyotime Biotechnology Co., Ltd.) for 10 min. The fluorescence labeling in cells was observed under fluorescence microscopy.

| RNA extraction and quantification
Total RNA from cells was extracted using the TRIzol reagent  Table 1.

| Cell counting kit-8 (CCK-8) assay
Cells in each group were cultured into 96-well plates (1 × 10 3 cells per well). After incubation for 0, 24, 48, and 72 h, each well was filled with 10 μL CCK8 reagent (C0038, Beyotime), followed by 1 h of incubation. The optical density at 450 nm was read by microplate reader to analyze the viability of cells.

| Tube formation assay
The 96-well plates were pre-coated with Matrigel until the mixture solidification. The HUVECs were then seeded into the plates containing conditioned medium of KFs for 12 h of incubation at 37°C, 5% The "Average Vessels Length" outcomes were collected for statistical quantification.

| Dual luciferase reporter gene assay
The

| Statistical analysis
All data were analyzed by SPSS21.0 (IBM Corp.). Measurement data were expressed as the mean ± standard deviation. Differences between two groups were compared by the unpaired t test. When over two groups were involved, one-or two-way analysis of variance (ANOVA) followed by Tukey's post hoc test was applied for difference analysis. The cutoff value for significant difference was set at p < 0.05.

| adMSC-Exos can be taken up by KFs
The adMSC-Exos were extracted, which showed round or ellipse shape in different sizes under TEM, most of which were approximately 100 nm in diameter ( Figure 2A). The NTA showed that the the successful extraction of adMSC-Exos and the uptake of Exos by KFs.

| AdMSC-Exos suppress proliferation and proangiogenetic ability of KFs by delivering miR-7846-3p
To examine if the extracted adMSC-Exos affect keloid development, we had KFs co-incubated with PBS, Exos, Exo-inhibitor NC, or Exo-miR-7846-3p inhibitor. After 24 h, the KFs were harvested. The qPCR analysis showed that the KFs showed significantly elevated expression of miR-7846-3p after coculture with Exos; however, the miR-7846-3p expression was reduced when the KFs were coincubated with Exo-miR-7846-3p inhibitor ( Figure 3A). CCK-8 and EdU labeling assays revealed that the viability of the KFs and the EdU positive rate in cells were substantially decreased by Exo treatment. However, the decrease in cell viability and proliferation was blocked when miR-7846-3p was inhibited in the Exos ( Figure 3B,C).
According to the TUNEL assay, apoptosis of the KFs was increased by Exos, and the proapoptotic role of the Exos was suppressed upon miR-7846-3p inhibition ( Figure 3D).
The conditioned medium of KFs in each group was collected and used for HUVEC incubation to analyze the pro-angiogenic ability of KFs. It was found that the Exos-treated KFs showed weakened pro-angiogenic ability. However, the pro-angiogenic role was restored when the KFs were treated with Exo-miR-7846-3p inhibitor ( Figure 3E).

| miR-7846-3p targets NRP2 mRNA to inactivate the Hedgehog pathway
To explore the downstream targets of miR-7846-3p responsible for the inhibition of KF activity, we predicted the target transcripts of   Figure 4A). Among them, NRP2 has been reported to show specific pro-angiogenic roles during tumor development. 14 Meanwhile, it has been found to accelerate wound healing by activating the Hedgehog signaling pathway. 15 We therefore selected NRP2 mRNA as a candidate target of miR-7846-3p for further research. The putative binding sequence between miR-7846-3p and NRP2 3′UTR was predicted from TargetScan ( Figure 4B) for luciferase assay. In KFs, transfection of miR-7846-3p mimic significantly reduced the luciferase activity of the NRP2-WT reporter vector, but it had no impact on the NRP2-MUT vector ( Figure 4C). Moreover, we transfected miR-7846-3p mimic or miR-7846-3p inhibitor directly into the KFs. The miR-7846-3p mimic successfully led to an increase in miR-7846-3p expression in cells along with a decline in the mRNA levels of NRP2, SHH, SMO, and GLI1, whereas the miR-7846-3p inhibitor resulted in inverse trends ( Figure 4D). Indeed, the WB analysis also found that the miR-7846-3p mimic decreased, whereas the miR-7846-3p inhibitor increased the protein levels of NRP2, SHH, SMO, and GLI1 ( Figure 4E).

| Overexpression of NRP2 in KFs negates the effects of adMSC-Exos
To investigate the implication of NRP2 downregulation in the events mediated by adMSC-Exos, the Exo-treated KFs were further transfected with oe-NRP2. The oe-NRP2 transfection had no effect on the expression of miR-7846-3p, though, it significantly elevated the mRNA and protein levels of NRP2, SHH, SMO, and GLI1 ( Figure 5A,B). When it comes to the activity of cells, the NRP2 overexpression restored the viability ( Figure 5C) and proliferation ( Figure 5D) of KFs but reduced cell apoptosis ( Figure 5E). The conditioned medium of oe-NRP2-transfected KFs was collected as well for HUVEC incubation. It was observed that the tube formation ability of the HUVECs was restored when they were cultured in the NRP2-overexpressed condition ( Figure 5F). Therefore, it can be F I G U R E 4 miR-7846-3p targets NRP2 mRNA to inactivate the Hedgehog pathway. (A) A Venn diagram for common target mRNAs of miR-7846-3p predicted in three bioinformatics systems; (B) Putative binding sequence between miR-7846-3p and NRP2 3′UTR predicted from TargetScan; (C) Luciferase assay to validate the binding between miR-7846-3p and NRP2 mRNA; (D) qPCR analysis to detect miR-7846-3p expression and mRNA levels of NRP2, SHH, SMO, and GLI1 in KFs; (E) WB analysis to detect protein levels of NRP2, SHH, SMO, and GLI1 in KFs. Differences were compared by two-way ANOVA (C-E). *p < 0.05 versus mimic NC; # p < 0.05 versus inhibitor.
opined that NRP2 activates the Hedgehog signaling and enhances proliferation of KFs and angiogenesis.

| DISCUSS ION
In addition to their known effects in accelerating wound healing, 17 MSCs also showed alleviating roles in excessive scarring due to their anti-inflammatory and anti-fibrotic paracrine effects through the secretory microvesicles and chemokines. 18 In this study, we of adMSCs has been found to reduce the collagen deposition and scar formation. 21,22 Collectively, we report that the adMSC-Exos can suppress proliferation of KFs and block angiogenesis to limit keloid development.
MiRNAs have shown robust functions in controlling cellular processes linked to excessive scarring. Aberrantly expressed miRNAs in keloid tissue has reportedly been closely linked to TGFβ, MAPK, apoptosis, and cell cycle signaling pathways. 23 For instance, miR-152-3p has been found to promote proliferation and invasion of KFs as well as ECM deposition by targeting forkhead box F1 mRNA. 24 Upregulation of miR-21 in human keloid tissues and KFs increased proliferation and collagen production by targeting SMAD family member 7. 25 Conversely, miR-148b-3p-mediated downregulation of insulin-like growth factor binding protein 5 suppressed keloid formation. 26 The discrepancy of the function of distinct miRNAs might be ascribed to the different targets they affected. As a type of major cargos in Exos, specific miR-NAs can also affect the function of the carrier Exos. For instance, miR-29a-modified adMSC-Exos significantly blocked excessive scar formation by suppressing the TGF-β2/Smad3 signaling. 27 Here, by analyzing aberrantly expressed miRNAs in keloid in the GSE113620 dataset and querying adMSC-Exos-contained miRNAs in bioinformatics system, we obtained miR-7846-3p as an exosomal miRNA that showed aberrant expression in keloids. The role of miR-7846-3p in human diseases has hardly been investigated.
Of note, we observed that specific inhibition of miR-7846-3p in the adMSC-Exos blocked their roles in suppressing proliferation and angiogenesis. This attracted our attention to define the downstream molecules implicated.
By querying four bioinformatics systems, we obtained NRP2 as a target of miR-7846-3p. The previous work by Luo et al. revealed that NRP2 promoted migration of HUVECs to induce angiogenesis. 14 Indeed, NRP2 has reportedly been linked to survival and proliferation of endothelial cells, therefore representing an angiogenesis marker. 28 Meanwhile, NRP2 has been reported as important positive regulators of signal transduction of the Hedgehog pathway. 29 The NRP2-Hedgehog axis was linked to M2 polarization of macrophages and proliferation of human dermal fibroblasts. 15 Activation of the Hedgehog pathway-related molecules has reportedly been linked to cell growth and proliferation for keloid development. 16 We found that the adMSC-Exos treatment

| CON CLUS IONS
In summary, this work demonstrates that adMSCs-sourced exoso-

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare that they have no conflict of interest related to this study including any commercial or associated interests that may represent a conflict of interest in connection with the submitted work.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the first author upon reasonable request.

E TH I C A L A PPROVA L
Not applicable.