The animal experiments were approved by the Animal Ethics Committee of Yangzhou University, China (approval No. 202103283) and were strictly implemented according to the Guide for the Care and Use of Laboratory Animals.

Skin samples from the backs of Rex rabbits were collected and immersed in phosphate buffered saline (PBS) containing 3% cyanine/streptomycin. These tissues were cut into strips measuring 0.25 cm×0.5 cm and digested overnight at 4°C in 0.25% dispase II (Gibco, Grand Island, NY, USA). The epidermis and dermis were then separated, retaining only the dermal tissue. To isolate and culture DPCs, the dermis was minced and digested with 1 mg/mL collagenase (Gibco, USA) at 37°C for 1 h. The digest was then filtered through a 70 μm mesh and centrifuged to collect the cells. DPCs were cultured with mesenchymal stem cell medium (MSCM) (Sciencell, San Diego, CA, USA) at 37°C in 5% (v/v) CO₂. MCs were isolated as described previously with slight modification [25,26]. Collagenase digestion was conducted after additional dissection of hair follicles from the dermis. After 70 μm and 40 μm filtration, the cells were then collected by centrifugation. MCs were cultured with M254 medium (Gibco, USA) containing HMGS-2 (Gibco, USA) at 37°C in 5% (v/v) CO₂.

The following antibodies were used: anti-vimentin (VIM) antibody at 1:800 for western blotting (WB) and 1:200 for immunofluorescent (IF) (BM0135; BOSTER, Wuhan, China), anti-α-smooth muscle actin (SMA) antibody at 1:800 for WB and 1:200 for IF (BM0002; BOSTER, China), anti-SOX2 antibody at 1:250 for IF (66411-1-Ig; Proteintech, Wuhan, China), anti-Versican antibody at 1:250 for IF (DF10007; Affinity, Jiangsu, China), anti-tyrosinase-related protein 2 (TYRP2) antibody at 1:200 for IF (AF5303; Affinity, China), anti-TYR antibody at 1:1,000 for WB and 1:200 for IF (Ab6211; Abcam, Cambridge, MA, USA), anti-microphthalmia-associated transcription factor (MITF) antibody at 1:1,000 for WB and 1:200 for IF (SC515925; Santa Cruz, CA, USA), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody at 1:10,000 for WB (60004-1-Ig; Proteintech, China), anti-CD9 antigen (CD9) antibody at 1:5,000 for WB (60232-1-Ig; Proteintech, China), anti-tumor susceptibility gene 101 (PTSG101) antibody at 1:5,000 for WB (28283-1-AP; Proteintech, China), anti-SOX10 antibody at 1:1,000 for WB (66786-1-Ig; Proteintech, China), HRP-conjugated Affinipure Goat Anti-Mouse IgG(H+L) at 1:10,000 for WB, HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) at 1:10,000 for WB and 1:200 for IF (SA 00001-2; Proteintech, China), Cy3-conjugated Affinipure Goat Anti-Mouse IgG(H+L) at 1:2,000 for IF (SA00009-1; Proteintech, China) and Cy3-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) at 1:2,000 for IF (SA00009-2; Proteintech, China).

Samples were lysed with a solution containing PMSF and centrifuged at 13,000 g at 4°C for 5 min to collect the supernatants. The protein concentration was determined using the BCA kit (Beyotime, Shanghai, China). After denaturation, the protein was transferred to the PVDF membrane by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using SurePAGE Bis-Tris 10×8, 4% to 20% 10 wells (GenScript, Jiangsu, China). Primary antibodies were diluted according to the manufacturer’s instructions and incubated at 4°C overnight. The second antibody was incubated at room temperature for 1 to 2 h. After ECL hypersensitive luminescence processing, the images were collected.

When cells reached 50% confluency, they were fixed with 4% paraformaldehyde for 30 min. Triton X-100 (Solarbio, Beijing, China) was used for permeabilization at 37°C for 20 to 30 min. The cells were blocked with 1% bovine serum albumin (BSA) for 1 h. Cells were incubated with the primary antibody at 4°C for overnight and with a Cy3-labeled secondary antibody at room temperature for 1 to 2 h. After 4’,6-diamidino-2-phenylindole (DAPI) staining, images were captured under an inverted fluorescence microscope (IX71; Olympus, Tokyo, Japan).

When the confluency reached to approximately 80% to 90%, MCs were fixed with 4% paraformaldehyde at room temperature for 20 min. Subsequently, the cells were incubated with 0.1% L-3,4-dihydroxyphenylalanine (L-DOPA) (Sigma, China) dissolved in 1% Triton X-100 (Solarbio, China) at 37°C for 3 h. The images were captured under a microscope (IX71; Olympus, Japan).

A solution of 1 mol/L NaOH solution containing 10% dimethyl sulfoxide (DMSO) was added to the samples and incubated at 80°C for 1 h. The samples were then added to 96-well plates (100 μL/wells), and three parallel triplicates were used for each sample. The absorbance was measured at 405 nm by a microplate reader (Infinite M200 PRO; TECAN, Männedorf, Switzerland). The relative melanin content was determined using the formula: % = (test_A405 – blank_A405)/(control_A405 – blank_A405)×100%.

DPC culture medium was collected and centrifuged to obtain the supernatant. An ultrafiltration tube was used to concentrate the cell supernatant. Thereafter, half the volume of total exosome isolation reagent (from cell culture media) (ThermoFisher, Waltham, MA, USA) was added, mixed thoroughly, and incubated at 2°C to 8°C overnight. Centrifugation was performed at 10,000 g at 4°C for 1 h. The pellet was collected, resuspended in PBS, and identified as exosomes from DPCs.

The membrane dye CM-Dil (Beyotime, China) was added and incubated in the dark for 20 min. PBS was added and centrifuged at 3,000 g at 4°C for 10 min. The supernatant was then collected, and exosomes were extracted again according to the exosome extraction procedure. Then, 20 μL exosomes were dropped into the electron microscope copper mesh grid and retained for a while (>1 min). After negative staining with uranyl acetate solution for 10 min, the samples were dried at room temperature. They were then observed and photographed under a biological transmission electron microscope (HT-7700; Hitachi, Tokyo, Japan).

After that, based on nanoparticle tracking analysis (NTA), the particle size of the extracted exosomes was analyzed. The sample pool of the ZetaView nanoparticle tracking analyzer was cleaned with deionized water and calibrated with polystyrene microspheres (100 nm). After the sample pool had been cleaned with PBS, exosomes were detected.

Transwell chambers (Corning, New York, USA) were utilized to establish a co-culture system. DPCs were seeded into the upper chambers, while MCs were seeded into the lower culture plates for 24 h. Following the transfection of DPCs with Cy3-miR-222-3p mimics (NC), the transwell chamber was positioned over the culture plate containing fresh medium. The red fluorescence of Cy3 in MCs was observed after 48 h culture. Subsequently, the transwell chamber was removed, and MCs were collected for IF staining using TYR antibody.

Total RNA was extracted from the tissues, cells, and exosomes using an RNAsimple total RNA extraction kit (TIANGEN, China). RNA was reverse transcribed into cDNA using HiScript III qRT SuperMix (Vazyme, China). Quantitative real-time polymerase chain reaction (RT-qPCR) was then performed according to the instructions of ChamQ SYBR qPCR Master Mix (Vazyme, China), with GAPDH as the internal reference. The RNA was reverse-transcribed into miRNA cDNA based on miRcute Plus miRNA First-Strand cDNA kit (TIANGEN, China). miRNA expression was detected using the miRcute Plus miRNA qPCR kit (SYBR Green) (TIANGEN, China), with small nuclear RNA U6 (U6) as the internal reference. The information about primers used is shown in Table 1.

MCs were collected 24 h after the transfection and inoculated into 96-well plates (100 μL/well) to culture for 0 h, 24 h, 48 h, 72 h. Thereafter, a cell counting kit-8 (CCK-8) kit (Vazyme, China) was used, and a CCK-8 solution (10 μL/well) was added to the cells. Each sample was assayed in triplicate. The absorbance at 450 nm was measured by a microplate reader (Infinite M200 PRO; TECAN, Switzerland) after incubation for 4 h to record the cell proliferation.

The samples were treated with Annexin V-FITC apoptosis detection kit (Vazyme, China). Annexin V-FITC and PI staining solution were added and incubated at the room temperature in the dark. Apoptosis rates were analyzed using flow cytometry (FACSAria SORP; Becton Dickinson, Franklin Lakes, NJ, USA), with each sample assayed in triplicate. The apoptosis rate was calculated by the sum of the percentage of Q2 and Q3 quadrants.

SOX10 3′-UTR target fragment (WT) and mutant fragment (MUT), with TGTAGC mutated to ACATCG, were inserted into the pmirGLO vector at SacI and XhoI restriction sites. The miR-222-3p mimics/inhibitor (NC), WT/MUT, and pRL-TK vector were co-transfected in RAB-9 (ATCC CRL-1414, Rockville, MD, USA). The luciferase activity was detected using a dual luciferase reporter assay kit (Vazyme, China).

The data of RT-qPCR was processed by the 2^−ΔΔCt method [27,28]. Statistical analyses were performed using the t-test and one-way analysis of variance. * Indicates significantly different (p<0.05), ** indicates extremely significant difference (p<0.01). The results have been presented as mean± standard deviation. GraphPad Prism 8 was used for the plotting.

After isolation for 24 h, primary DPCs displayed aggregative towards outwards and mostly showed long spindle-shaped or irregular shapes. After 3 days, the cells showed radial growth, but after 5 days, the cells exhibited agglutination (Figure 1a). The α-SMA and VIM proteins were identified by WB and IF staining in DPCs (Figure 2b and 2c). Additionally, the isolated MCs showed the bipolar or tripolar morphology. The MCs-specific markers MITF, TYR, and TYRP2 were then identified by IF staining (Figure 2a). Black particles were observed in MCs following L-DOPA staining (Figure 2b). MITF and TYR proteins were detected by WB analysis (Figure 2c). These results indicated that the isolated cells were DPCs and MCs, respectively.

Exosomes were isolated from DPC supernatants. NTA showed that the DPCs-exos diameter was around 120 nm, with both in the range of 30 nm to 150 nm (Figure 3a). The structures of exosomes appeared as saucer or concave hemispheres by TEM (Figure 3b). Exosome-specific protein CD9 and TSG101 were also detected in exosomes by WB (Figure 3c). So, the isolated materials were confirmed to be exosomes. To confirm the adsorption of DPCs-derived exosomes by MCs, CM-Dil labeled exosomes were added to MCs. CM-Dil-labeled exosomes were primarily observed to cluster within MCs (Figure 3d). This suggested the effective internalization of CM-Dil-labeled DPCs-exos by MCs.

The mRNA level of miR-222-3p in the skin and DPCs-exos of white and black Rex rabbits was detected by RT-qPCR. The expression of miR-222-3p was found to be higher in the skin and DPCs-exos of white Rex rabbits (p<0.01) (Figures 4a and 4b). We reasoned that miR-222-3p plays a key role in differences observed in MCs melanin synthesis caused by DPCs-exos. Furthermore, Cy3-labeled miR-222-3p mimics were transfected into DPCs, and DPCs, as well as MCs, were co-cultured in a Transwell chamber. We observed a clear red fluorescence in MCs (Figure 4c). In addition, the expression of miR-222-3p was significantly increased in comparison with NC (p<0.01) (Figure 4d). It was demonstrated that DPCs could secrete the exosomal miR-222-3p into MCs.

Next, we analyzed the effect of miR-222-3p on MCs proliferation, apoptosis, and melanin synthesis. Using CCK-8 assay and annexin V-fluorescein isothiocyanate/propidium iodide (V-FITC/PI) flow cytometry, it was found that miR-222-3p mimics significantly inhibited MCs proliferation and promote apoptosis (p<0.05), whereas miR-222-3p inhibitor exhibited an opposite effect (p<0.01) (Figure 5a–5c). It was suggested that miR-222-3p was positively correlated with MCs apoptosis. Further, it was found that after the transfection with miR-222-3p mimics, melanin content was significantly decreased (p< 0.01), whereas melanin content was significantly increased with transfection with miR-222-3p inhibitor (p<0.01) (Figure 5d). Thus, after miR-222-3p enters into MCs, its functional pathway needs further exploration.

The mature ocu-miR-222-3p sequence AGCUACAUCUG GCUACUGGGUCUC was obtained from miRBase and NCBI databases. The binding sites between ocu-miR-222-3p and SOX10 were then predicted using RNAhybrid (Figure 6a). The regulatory relationship between miR-222-3p and SOX10 was further analyzed by using a dual luciferase system and site-specific mutation techniques. When co-transfected with SOX10-3′UTR-WT and miR-222-3p mimics, the luciferase relative activity decreased markedly (p<0.01), but no significant effect on activity was observed when SOX10-3′UTR-MUT and miR-222-3p mimics were co-transfected (p>0.05). In contrast, the luciferase activity was significantly increased after co-transfection with SOX10-3′UTR-WT and miR-222-3p inhibitor (p<0.01) (Figure 6b). Furthermore, miR-222-3p mimics were transfected in MCs, resulting in a significant decrease in MCs at the RNA and protein levels (Figure 6c and 6d). In summary, after entering into the MCs, DPCs-derived exosomal miR-222-3p were able to inhibit melanin synthesis in MCs by targeting SOX10 (Figure 7).