Down‐regulation of exosomal miR‐200c derived from keratinocytes in vitiligo lesions suppresses melanogenesis

Abstract Vitiligo is a refractory disfiguring skin disease. However, the aetiology and pathogenesis of vitiligo have not been fully defined. Previous studies have shown that exosomes from normal human keratinocytes improve melanogenesis by up‐regulating the expression of melanogenesis‐related proteins. Several microRNAs (miRNAs) have been demonstrated to be effective in modulating melanogenesis via exosomes. In the present study, it was found that the effect of exosomes derived from keratinocytes in vitiligo lesions in regulating melanin synthesis is weakened. Furthermore, miR‐200c was detected to be significantly down‐regulated in exosomes from keratinocytes in vitiligo lesions. In addition, miR‐200c enhanced the expression of melanogenesis‐related genes via suppressing SOX1 to activate β‐catenin. In conclusion, our study revealed that the effect of exosomes secreted by keratinocytes in vitiligo lesions exhibited a weaker capacity in promoting melanogenesis of melanocytes. Moreover, the expression of miR‐200c, which mediates melanogenesis in exosomes secreted by keratinocytes in vitiligo lesions, is down‐regulated, which may be one of the pathogenesis in vitiligo. Therefore, keratinocyte‐derived exosomal miR‐200c may be a potential target for the treatment of vitiligo.

locally affect proliferation and activity of melanocytes. For example, UVB can stimulate CRH gene expression and peptide production in melanocytes which can affect epidermal proliferation of keratinocytes and melanocytes. 8 Androgens and estrogens can regulate proliferation and melanogenesis in cultured melanocytes. Neuron growth factor (NGF) stimulates melanocyte dendrite formation and prolongs melanocytes survival after UV injury. 9 L-tyrosine and L-dihydroxyphenylalanine (L-DOPA), as substrates and intermediates of melanogenesis, not only induce but also positively regulate the process of melanogenesis. 10 Some studies have demonstrated that keratinocyte-derived factors are inadequate and that the ultrastructure of keratinocytes has changed in vitiligo. The alteration of keratinocytes may be the cause of the disappearance of melanocytes in vitiligo. [11][12][13] Exosomes are nano-sized extracellular vesicles that are released from almost all cell types in a physical or pathological state. 14 These can mediate cell-to-cell communication by transferring proteins and various nucleic acids, including mRNAs, miRNAs and small regulatory RNAs (sRNAs), to neighbouring cells or distant cells. 15,16 Current studies have found that keratinocyte-derived exosomes modulated melanogenesis in melanocytes. 17,18 MicroRNAs (miRNAs) are small endogenous non-coding RNAs that play a key role in regulating gene expression at the post-transcriptional level. 19 It was identified that miR-3196 in exosomes derived from keratinocytes enhanced the gene expression of microphthalmia-associated transcription factor (MITF) in melanocytes. Mir-203, in contrast to miR-3196, increases the tyrosinase (TYR) expression, without modifying the gene expression of MITF. 17 However, some miRNAs from keratinocyte exosomes negatively regulate melanogenesis in melanocytes, such as miR-330-5p and miR-675. 20,21 In vitiligo, we investigated whether exosomes derived from keratinocytes still have the ability to modulate melanogenesis in melanocytes. Our study revealed that exosomes secreted by VLK have a weaker capacity for promoting melanogenesis of melanocytes.
Furthermore, the down-regulated expression of miR-200c carried by exosomes from keratinocytes in vitiligo lesions to target melanocytes led to the decrease in tyrosinase activity and melanogenesis-related gene expression.

| Sample collection, ethical statement, and cell culture
Normal human epidermal melanocytes (NHEM) and normal human epidermal keratinocytes (NHEK) were isolated from male adult foreskins obtained at circumcision. The epidermis was separated from the dermis and treated with 0.25% trypsin after digesting foreskins by 0.25% dispase at 4°C for 18 hours. Under a negative pressure of 250 mm Hg, suction blisters of depigmented skin in vitiligo were obtained, and keratinocytes in the vitiligo lesions (VLK) were further isolated. All participants provided a signed informed consent.

| Exosome isolation
The exosomes were isolated from the conditioned medium of keratinocytes. Briefly, the supernatant was filtered through a 0.22-μm syringe filter and centrifuged at 120 000 g for 90 minutes (4°C). Then, the pellet was washed with phosphate-buffered saline (PBS) before another 90 minutes of ultracentrifugation at 120 000 g (4°C). In the last step, the pellet was resuspended and stored at −80°C. All steps of ultracentrifugation are performed using the Optima™ XPN-100.

| Immunofluorescence (IF)
The exosomes were labelled using green fluorescent membrane dye PKH67 (PKH67GL; Sigma-Aldrich, St. Louis, MO, USA), according to the protocol. Cells were cultured in Medium 254 containing exosomal solution labelled PKH67 or negative control solution for 24 hours at 37°C in six-well plates. Then, the slides were washed for three times with PBS, fixed with 4% formaldehyde for 20 minutes and washed for three times again with PBS. Afterwards, the cell nuclei were stained using DAPI fluorescent stain (D9542, Sigma-Aldrich). The images were acquired using the ImageXpress Micro ® Confocal High-Content Imaging System (Molecular Devices).
Afterwards, the cell lysate was collected after centrifugation at 13 000 g for 10 minutes at 4°C. Next, 0.1% L-DOPA was added to dilute the sample to 1 mL, and 200 μL per well was added to the 96-well plate and incubated at 37°C for 30 minutes. The absorbance value was measured by the enzyme mark instrument at 475 nm, and the tyrosinase activity (normalized to control) in melanocytes was

| Melanin content assay
After the melanocytes were treated with exosomes (25 μg/mL) for 72 hours, these were digested with 0.25% trypsin and washed twice with PBS. Then, the cells were dissolved in 1 mol/L NaOH at 80°C for 30 minutes, and the melanin content was measured as OD at 475 nm.

| Quantitative real-time PCR
The total RNA of the control and treated cells or exosomes was isolated using the RNeasy Mini kit (Qiagen, Hilden, Germany), the total RNA of exosomes was isolated using the exoRNeasy Midi Kit (Qiagen, Hilden, Germany), and the cDNA was synthesized using HiScript II Q RT SuperMix for qRT-PCR (R223-01, Vazyme).
Quantitative real-time PCR for mRNA and miRNA expression analysis was carried out using QuantStudio TM 7 Flex (Thermo Fisher Scientific), and this was normalized using β-actin (for mRNA) or RNU6 (for miRNA). The data were analysed using the delta Ct method. The primers used for the present study are listed in Table 1.
The total proteins of lysates or exosomes were separated by SDS polyacrylamide gels and transferred onto PVDF membranes (Millipore, USA). Then, these membranes were blocked with 5% BSA for one hour at room temperature and incubated with primary antibodies overnight at 4°C. Afterwards, the secondary antibodies were added. The blots were observed using the Azure Biosystems

| MiRNA sequencing
The total RNAs were extracted from exosomes isolated from the conditioned medium of keratinocytes. The miRNA library preparation and sequencing were performed by a commercial service (Ribobio, China). The 3′ and 5′ splices were successively connected.
Then, the reverse transcription was carried out to the cDNA, followed by PCR amplification. The cDNA library was sequenced using the Illumina HiSeq 2500 platform.

| Transmission electron microscopy (TEM)
Exosomes resuspended in PBS were fixed with 4%PFA (at 1:1 dilution) and deposited on formvar-carbon-coated grids. Then, these copper grids were fixed with 1% glutaraldehyde for 5 minutes and washed with ddH 2 O for eight times after washing the PBS droplets.
Afterwards, negative staining with uranium acetate was performed for 10 minutes, followed by absorption of the excess liquid with a filter paper. The samples were observed using a transmission electron microscopy at 80 kV.

| Nanoparticle tracking analysis (NTA)
Nanoparticle tracking analysis (NTA) was used to analyse the size distribution of exosomes through the ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany). The exosomal samples were diluted to a concentration of 1 × 10 8 particles/mL with filtered PBS. The moving particles were recorded in video for five times, for 30 seconds each. Then, the data were analysed using the ZetaView 8.04.02 software.

| Luciferase assay
The sequences for the SOX1 3' UTR that contained the potential wide-type or mutant binding sites of miR-200c were constructed Kit (Promega, Madison, WI, USA) was used to detect the luciferase activity of these samples.

| Statistical analysis
All data were expressed as mean ± standard deviation (SD). Oneway ANOVA was used to analyse the statistical significance of the difference in three or more than three groups, and t test was used to calculate the statistical difference between the two groups. The Diego, CA, USA). P < 0.05 was considered statistically significant.

| Exosomes secreted by VLK exhibit a weaker capacity in promoting melanogenesis of melanocytes than those secreted by NHEK
To characterize the exosomes derived from NHEK and VLK, ultracentrifugation was applied to isolate exosomes from the culture supernatants of keratinocytes. Based on TEM, vesicles that contained the typical cup-shaped structure of exosomes were observed in both healthy and patient samples ( Figure 1A). NTA showed that the average diameter of exosomes from NHEK was 120 ± 1.3 nm and that of exosomes from VLK was 136.2 ± 4.0 nm ( Figure 1B). The exosome size was slightly larger in keratinocytes from vitiligo lesions than keratinocytes from normal human epidermis (P < 0.05) ( Figure 1C). Exosomal protein markers, including CD63 and TSG101, were identified in exosomes from both NHEK and VK by Western blot ( Figure 1D).
It has been reported that exosomes released by normal keratinocytes can modulate melanocyte pigmentation. In order to compare the capacity of exosomes secreted by NHEK and VLK in promoting the melanogenesis of melanocytes, exosomes from both NHEK and VK were added into melanocytes obtained from healthy controls (HCs). Initially, through the immunofluorescence assay performed with PKH-67-labelled exosomes, the ability of exosomes to enter the melanocytes was identified ( Figure 1E). Then, it was observed that the intracellular melanin content and TYR activity of melanocytes incubated with patient exosomes were significantly lower than those of melanocytes incubated with NHEK exosomes ( Figure 1F,G). Accordingly, melanocytes treated with exosomes from VLK presented with lower mRNA and protein levels of MITF, TYR, TRP1 and TRP2, when compared to those treated with exosomes from NHEK ( Figure 1H,I). Taken together, these data suggest that exosomes secreted by VLK exhibit a weaker capacity in promoting the melanogenesis of melanocytes than those secreted by NHEK.

| MiR-200c is down-regulated in exosomes from VLK
Some studies have reported that exosomal miRNAs derived from keratinocytes modulate pigmentation in melanocytes. To explore whether exosomal miRNAs participate in the modulation of melanogenesis in melanocytes, the miRNA profile of VLK and NHEK exosomes was analysed. In the present study, 73 differentially expressed miRNAs (20 up-regulated and 53 down-regulated miRNAs) were identified (Figure 2A). Then, the qualified miRNA was further selected using the following criteria: (a) log 2 fold change of more than  Figure 2E). Therefore, miR-200c was chosen to further investigate its role in melanocytes.

| MiR-200c facilitates melanogenesis of melanocytes
To These results revealed that miR-200c facilitate melanogenesis of melanocytes.

| Exosomes secreted by keratinocytes regulate the activation of β-catenin in melanocytes by mediating SOX1 via miR-200c
To illustrate the mechanism of exosomes/miR-200c in regulating melanogenesis, we sought the target genes of miR-200c. SOX1, which was identified by miRDB, Targetscan and microRNA.org  Figure 5F). SOX1 has been demonstrated to down-regulate β-catenin and reverse the malignant phenotype in nasopharyngeal carcinoma. 23 Here, we uncovered that exosomes/miR-200c could thereby modulate the expression of β-catenin and the nuclear level of β-catenin via SOX1 (Figure 5F,G). Thus, these present results indicate that exosomes secreted by keratinocytes regulate the activation of β-catenin in melanocytes by mediating SOX1 via miR-200c ( Figure 6).

| D ISCUSS I ON
The process of delivering melanin from melanocytes to keratinocytes is very critical for skin protection against UVB and pigmentation. 24,25 UVB can up-regulate the expression and activity of the α-MSH receptor, the expression of POMC peptide production including that of α-MSH, β-endorphin and ACTH, to regulate skin pigmentation, to protect skin from UV induced injury, and to modulate skin immune response. 26 The decrease in melanocytes and the dysfunction of melanogenesis are two distinct characteristics of vitiligo. However, in vitiligo, both the impairment of melanocytes and the morphology and function of keratinocytes exist.
The interaction between keratinocytes and melanocytes plays an important role in maintaining the homeostasis of the epidermis. 27 Compared with normally pigmented epidermis, the apoptosis of keratinocytes is more likely to occur in depigmented suction-blistered epidermis, which can decrease the levels of keratinocytederived factors, such as SCF and basic fibroblast growth factor.
The reduction in these survival factors causes the apoptosis of melanocytes. 11  Exosomes are small intracellular vesicles with a diameter of 40-150 nm. 28 In the present study, the size of the exosomes of VLK was slightly larger than that of NHEK, although TEM and NTA revealed that both of them were within the size range indicative of exosomes.
Singh et al reported larger keratinocytes in vitiligo lesional skin, when compared to the non-lesional stratum corneum. 12 Changes in cell size are commonly observed in many disease conditions and during ageing. 29 Senescence in human cells is correlated to the increase in cell size, 30,31 which influences the structural integrity of cells, increases intracellular distances and reduces surface-to-volume (SV) ratio, leading to metabolic inefficiency and cell death. 29 In our study, the larger size of exosomes from VLK still needs to be confirmed through large sample studies, and further studies are needed to determine whether this is associated with the dysfunction in pro- Compared with NHEK exosomes, the effect of exosomes from VLK in promoting melanin production was significantly reduced. In addition, it was observed that VLK exosomes still had a slight effect in promoting pigmentation compared with the control group. The present study showed that the effect of VLK exosomes in promoting melanogenesis was weakened. However, we did not find any evidence that VLK exosomes inhibit melanogenesis in melanocytes.
Moreover, further studies are needed to analyse the processing of these melanogenesis-related proteins because of double bands showed in Western blot, which may represent glycosylation.
In recent years, miR-200c has been found to have two main functions in a variety of cancers, including melanoma. One function is to suppress the drug resistance of tumour cells, [32][33][34] while the other function is to inhibit the invasion and migration of malignant cells. 35 [45][46][47][48] In particular, SOX9 has been demonstrated to play a key role in melanogenesis in adults, and SOX10 has been found to seriously influence the development of melanocytes. Additionally, the link between SOX1 and β-catenin has been shown in various carcinomas. It has been reported that SOX1 interacts with β-catenin and induces proteasome-independent down-regulation of β-catenin. 23 In our study, the protein expression levels of total and intranuclear β-catenin were up-regulated with the down-regulation of SOX1 expression, which was consistent with previous study. Our results indicate that exosomal miR-200c derived from keratinocytes regulate the activation of β-catenin in melanocytes by inhibiting SOX1.
The relationship between β-catenin and MITF has been shown in several studies. MITF is a downstream target of Wnt signalling in the development of melanocytes. 49 Schepsky et al implied that β-catenin not only regulates the expression of MITF at the transcription level, but also directly interplays with MITF. Overexpression of β-catenin improves MITF-dependent gene expression. 50 In this study, we found that β-catenin up-regulated the gene expression of MITF and in turn, promoted the expression of TYR, TRP1 and TRP2.
In general, the present study is the first to provide evidence that exosomes secreted by VLK have a weaker capacity in regulating melanogenesis of melanocyte, when compared to those se-

CO N FLI C T O F I NTE R E S T
The authors declare that there are no competing interests associated with the manuscript.