The primary human epidermal melanocytes were isolated from healthy juvenile foreskin tissues, a total of 60 specimens, collected after circumcision between September 2017 and May 2019 (donor age range, 13-19 years). After removing the subcutaneous tissue, the samples were cut into small pieces and incubated with 0.25% dispase at 4°C overnight to separate the epidermis and dermis (18). The present study was approved by the Ethical Committee of the Renmin Hospital of Wuhan University. Written informed consent was obtained from each participant before enrollment. Melanocytes were cultured with complete Medium 254 supplemented with Human Melanocyte Growth Supplement and 100 U/ml penicillin/streptomycin (all from Cascade Biologics) in a humidified atmosphere at 37°C and 5% CO₂. Cells used in the present experiments were from passages 2 to 6. Cells were seeded in 6-well plates at a population density of 5×10⁵. Before irradiation, the medium was removed and warm PBS was used to wash the cells. The cells were treated with single or repeated exposures to UVB using a high dose targeted phototherapy system (The Daavlin Company) with a maximum wavelength at 311 nm. The cumulative dose of UVB irradiation is indicated in the figure legends. Cells of the UVB-unexposed control were treated in same way but without UVB exposure.

Total RNAs were purified using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the protocol of the manufacturer. cDNAs were synthesized from total RNAs (1.5 µg) using a Moloney murine leukemia virus reverse transcriptase first strand kit (Invitrogen; Thermo Fisher Scientific, Inc.). RT-PCR was performed under the following cycle parameters: Predenaturation at 95°C for 2 min, followed by 35 cycles of 95°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec. PCR was performed in triplicate with SYBR-Green PCR core reagents (Applied Biosystems; Thermo Fisher Scientific, Inc.), 50 ng cDNA and 1 µM forward and reverse primers for the following human genes: p53 (forward: 5′-GTC TAC CTC CCG CCA TAA-3′; reverse: 5′-GCA AGC AAG GGT TCA AAG-3′), TRPM1 (forward: 5′-TAC ACG CTA TTT CCC CGA TGA-3′; reverse: 5′-GCG CGT GAT CTT TTG AAC TTG-3′), MMP9 (forward: 5′-GCT ACC ACC TCG AAC TTT GAC-3′; reverse: 5′-TCA GTG AAG CGG TAC ATA GGG-3′) and the housekeeping gene β-actin (ACTB; forward: 5′-AGC GAG CAT CCC CCA AAG TT-3′; reverse: 5′-GGG CAC GAA GGC TCA TCA TT-3′) was used as an endogenous internal control for the normalization of p53, TRPM1, and MMP9 mRNA expression. For qPCR analysis of miR-211, 10 ng total RNA was used in a TaqMan miRNA assay according to the manufacturer′s protocol. Analysis of miRNA was carried out using a CFX96 Detection System (Bio-Rad Laboratories, Inc.) in triplicate and the SYBR-Green Supermix. miR-211 specific primers: (Forward: 5′-TGC GCT TCC CTT TGT CAT CCT T-3′; reverse: 5′-CTC AAG TGT CGT GGA GTC GG C AA-3′) loop primer: 5′-GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACA GGC GAA G-3′). The expression of miR-211 was normalized to the U6 small nuclear RNA (forward: 5′-CTC GCT TCG GCA GCA CAT-3′; reverse: 5′-AAC GCT TCA CGA ATT TGC GT-3′). All primers used in this study were synthesized and supplied by Invitrogen; Thermo Fisher Scientific, Inc. qPCR was performed using an ABI 7500 system with the following cycle parameters: Denaturation at 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec, 60°C for 4 sec and 72°C for 30 sec. The purity of each PCR product was checked by dissociation curve analysis, as well as by running each sample on 1% agarose gels with Goldview (cat. no. 110214; SBSGene). Fold-change values were calculated using the formula of 2^−ΔΔCq (19).

Cells were harvested, washed in PBS and lysed in extraction buffer containing 1% Nonidet P-40, 0.01% SDS and a protease inhibitor cocktail (Roche Diagnostics). Protein contents were determined using a bicinchoninic acid assay kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of each protein extract (20 µg per lane) were resolved using 10% SDS-PAGE. Following transblotting onto Immobilon-P membranes (EMD Millipore) and blocking with 5% nonfat milk in saline buffer for 1 h at room temperature, the membranes were incubated with antibodies to p53 (cat. no. ab179477; Abcam; 1:1,000), TRPM1 (cat. no. ab72154; Abcam; 1:1,500), MMP9 (cat. no. ab76003; Abcam; 1:1,000) and GAPDH (cat. no. ab37168; Abcam; 1:10,000) for 1 h at room temperature. The membranes were then washed and incubated with HRP-conjugated goat anti-rabbit IgG (AS1107; Aspen Biological; 1:10,000) for 1 h at room temperature. Each membrane was then washed again and specific immunoreactive bands were visualized using an enhanced chemiluminescent reaction (ECL kit; Amersham; GE Healthcare). Signal intensities were quantified using ImageJ software version 1.47 (National Institutes of Health) and were normalized to GAPDH. All data were obtained from more than two independent experiments carried out in triplicate.

Primary melanocytes (5×10⁵) were seeded in 6-well culture plates containing coverslips. After attachment, the cells were treated with multiple UVB exposures for a cumulative dose of 70 mJ/cm² (8.75 mJ/cm² per exposure in 5 h intervals for a total of 8 times). Subsequently, the cells were immediately fixed in 4% paraformaldehyde in PBS for 30 min at room temperature, permeabilized with 0.3% Triton X- 100 in PBS for 15 min and then blocked for 1 h at 37°C using a blocking buffer containing 10% normal goat serum (cat. no. C0265; Beyotime Institute of Biotechnology). The anti-MMP9 (cat. no. ab76003; Abcam; 1:200) antibody was diluted in blocking buffer and placed on the cells at 4°C overnight. After the incubation, cells on coverslips were washed three times in PBS and then incubated with goat anti-rabbit IgG (AS1109; Aspen Biological; 1:50) for 1 h at 37°C. Nuclei were stained using 4′6′-diamidino-2-phenylin-dole (DAPI) solution for 10 min at room temperature. Imaging was performed using an FV1200 (Olympus Corporation) confocal microscope.

Cell migration was assessed using Transwell cell culture chambers as previously described (20). Polyvinylpyrrolidone-free polycarbonate filters with an 8.0-µm pore size were pre-coated with 10 mg/ml collagen IV (C6745; Sigma-Aldrich; Merck KGaA) and placed on the lower surface of Transwell chambers (Costar, 3422; Corning, Inc.). The filters were dried overnight at room temperature, washed extensively in PBS and then dried immediately before use. Melanocytes (1×10⁵) were seeded into the upper chambers, while the lower chambers were filled with fresh complete medium supplemented with or without 0.2 nM GM6001 (HY-15768; MedChemExpress). Cell culture inserts were treated with repeated exposure to UVB as detailed above. A total of 48 h later, the filters were fixed in methanol for 15 min at room temperature and stained with crystal violet for 20 min at room temperature. Cells that had migrated into the lower surface of the membrane were counted in five microscopic fields at ×200 magnification using an automatic microscope (Olympus Corporation) at least three independent experiments.

Primary melanocytes were seeded in 6-well culture plates at a population density of 5×10⁵. miR-211-mimic and miR-negative control (miR-NC; cat. no. miR01101) were purchased from Guangzhou RiboBio Co., Ltd. After 24 h of culture, the transfection of 50 nM miR-211-mimic (cat. no. miR10022694; sequence, 5′-GCA GGG ACA GCA AAG GGG UGC-3′) was performed with RiboFECT™ CP transfection reagent (Guangzhou RiboBio Co., Ltd.) according to the manufacturer's protocol. miR-NC, which contains a sequence that lacks homology with other miRNAs, was transfected under identical conditions into melanocytes as the NC. After 48 h of transfection, the cells were harvested and assayed using qPCR and western blotting.

In order to produce a p53 expression construct, the protein coding sequence region of the human p53 gene was synthesized and inserted into BamH I/Age I restriction sites of the lentiviral expression vector GV358 that contains the EGFP gene (Shanghai GeneChem Co., Ltd.). 293T cells (Shanghai GeneChem Co., Ltd.) were transfected with a mixture of plasmids, including viral packaging plasmids and the p53 expression plasmid (GV359-p53) or the control plasmid (GV358) via Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) based on the manufacturer's protocol. The viral supernatant was collected at 48 h after transfection and was used to infect melanocytes. Evaluation of p53 expression was conducted using fluorescence microscopy at 48 h after viral infection by harvesting the cells and characterizing their levels of mRNA and protein expression by qPCR and western blotting, respectively.

Data are expressed as the mean ± standard deviation from three independent experiments. SPSS version 19.0 (IBM Corps.) and GraphPad Prism 8 (GraphPad Software, Inc.) software were used for analysis of all data. Statistical differences were determined by Student's t-test or one-way analysis of variance with Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

First, the expression profiles of the p53-TRPM1/miR-211-MMP9 axis in melanocytes treated with single or repeated exposures to UVB was examined. For the single UVB exposures, cultured human melanocytes were exposed to a single dose of UVB (0, 8.75, 17.5, 35 and 70 mJ/cm²). After 8 h, the mRNA and protein expression levels of p53, TRPM1 and MMP9 were measured using RT-qPCR and western blotting, respectively. The results (Fig. 1) showed that the expression levels of p53 and MMP9 mRNAs and proteins were upregulated in a UVB dose-dependent manner (8.75-70 mJ/cm²). Interestingly, TRPM1 mRNA and protein levels were increased at lower doses (8.75 and 17.5 mJ/cm²) but were reduced at higher doses (35 and 70 mJ/cm²) of UVB. To simulate clinical settings with multiple exposures to UVB, the cells were treated with repeated small UVB exposures for cumulative doses of 35 or 70 mJ/cm² (8.75 mJ/cm² per exposure in 5 h intervals for a total of 4 or 8 times), as shown in Fig. 2. The upregulation of p53 and MMP9 mRNA and protein levels was seen in melanocytes treated with 4 or 8 exposures to UVB, whereas mRNA and protein levels of TRPM1 were significantly decreased in cells repeatedly exposed to UVB compared with the UVB-unexposed control cells. These results demonstrated that the p53-TRPM1/miR-211-MMP9 axis is significantly activated in melanocytes by single and by repeated exposures to UVB.

To investigate whether miR-211 is involved in the regulation of MMP9 and if this plays a central role in the regulation of melanocyte migration, the capacity of UVB-exposed melanocytes to migrate on collagen IV substrate was determined. The results (Fig. 3) showed that repeated UVB exposure significantly stimulated melanocyte migration and that there was a highly significant inverse association between miR-211 expression and MMP9 protein levels in the cells following repeated exposure to UVB. The mRNA expression of miR-211 was decreased and MMP9 was upregulated. To further characterize the role of MMP9 in melanocyte migration, GM6001, a broad-spectrum MMP inhibitor, was used to block MMP9-mediated cell migration. As expected, the number of migrating cells in Transwell inserts with collagen IV-coated membranes was much lower in the presence of 0.2 nM GM6001 compared with in the untreated control and even following exposure to UVB (Fig. 3C and D). Indeed, these findings strongly suggested that the post-transcriptional regulation of the MMP9 gene by miR-211 directly affects melanocyte migration. Furthermore, to determine whether miR-211 has an effect on MMP9 expression and/or melanocyte migration, a chemically synthesized miRNA mimic was transiently transfected into melanocytes to determine the effects of miR-211. After transfection with the miR-211 mimic, the level of miR-211 was significantly increased in cells transfected with the miR-211 mimic compared with the miR-NC (Fig. 4A). Next, qPCR and western blotting were performed to assess the expression of MMP9 and found that the mRNA and protein levels of MMP9 were significantly suppressed after transfection with the miR-211 mimic (Fig. 4A and B). The Transwell assays showed that melanocyte migration was significantly reduced in the miR-211 mimic transfected cells compared with the miR-NC transfected cells (Fig. 4C). Likewise, the inhibition of the miR-211 mimic on melanocyte migration was neutralized by treatment with 0.2 nM GM6001 (Fig. 4C). Therefore, the present findings suggested that the post-transcriptional control of the MMP9 gene by miR-211 might play a critical role in the UVB-induced melanocyte migration.

In this study, the expression of endogenous p53 protein was induced by UVB irradiation (Figs. 1 and 2). To address the question of whether exogenous p53 overexpression would have a similar role in melanocyte migration, the effect of a lentiviral vector-mediated overexpression of p53 on the migration of cultured human melanocytes was examined. As shown in Fig. 5, melanocytes were transfected with p53-GFP lentiviral vectors and the transfection efficiency was monitored by GFP detection. qPCR analysis demonstrated that the relative levels of p53 and MMP9 were significantly increased in cells transfected with the p53-GFP lentiviral vector compared with those levels in the GFP vector control group, which was concomitant with decreases in TRPM1 and miR-211 levels (Fig. 5A). The effectiveness of the p53-GFP lentiviral vector was also confirmed by examining levels of the p53, TRPM1 and MMP9 proteins by western blotting analysis. The protein expression levels of p53 and MMP9 were significantly increased in cells transfected with the p53-GFP lentiviral vector compared with the control groups. Similar to the repression of miR-211 after transfection of the p53-lentiviral vector, the protein level of TRPM1 was also reduced compared with the control group. The results of immunofluorescence staining also revealed that the expression of MMP9 protein in transfected cells was increased (Fig. 5B). The Transwell assays showed that melanocyte migration was significantly increased in cells that had a lentiviral vector-mediated overexpression of p53 compared with the control group. Moreover, the over-expression of p53 induced a higher migration of melanocytes, which could be neutralized by GM6001 treatment (Fig. 5C). These data suggest that miR-211 exerts an anti-migration effect via the suppression of MMP9, whereas the overexpression of p53 in melanocytes directly reverses the inhibitory effect of miR-211 on cell migration.