Study On The Effect Of Exosomal MiRNA-222-3p From The Hair Follicle Dermal Papilla Cells On Melanin Production In Melanocytes

Melanin synthesis occurs mainly in melanocytes(MCs),which determine the pigmentation of mammalian skin and hair.Dermal papilla cells(DPCs)serve as signaling centers during hair follicle development.The microenvironment they create can affect the function of MCs and regulate hair pigment deposition.Exosomes(Exos)are crucial participants in intercellular microenvironment formation.They transport regulatory proteins,mRNA,and miRNA,which serve as communication carriers facilitating "dialogue" between cells.Furthermore,the potential involvement of DPC-Exos in hair pigment deposition and their impact on communication with MCs require further investigation.Rex rabbits,whose economic value lies in their quality fur and rich color,are valuable animal models for pigment deposition studies.In this study,DPCs and their exosomes isolated from black and white rex rabbits were used to investigate the effect of DPC-Exos on MC melanin production.Hair follicle-derived MCs were used as recipient cells in this study.In addition,exosomal miRNA-222-3p from DPCs was selected as the research subject to analyze its effect on MC melanin production and regulatory relationship with the SOX10 target gene.This revealed an inhibitory mechanism on MC melanin production attributable to exosomal miRNA-222-3p from DPCs.1.Isolation and Identification of Rex Rabbits DPCsDPCs were isolated from both black and white skin samples from Rex rabbits using a two-step digestion method and rat tail collagen-coated plates to enhance cell adhesion efficiency.The isolated primary cells displayed mostly spindle-shaped and irregular morphology,and after 3 days of growth,radial cell growth was observed,followed by cell aggregation after 5 days.Immunofluorescence staining and Western blotting confirmed the specific expression of VIM and α-SMA proteins in the isolated cells,indicating their identity as DPCs.2.Isolation and Identification of hair follicle-derived melanocytes from Rex rabbitsIn this study,skin samples were collected from Rex rabbits to perform TYRP1 immunohistochemical and TYR immunofluorescence experiments.Our results confirmed the presence of MCs in both the hair papilla and outer root sheath of Rex rabbit dermis.We performed a three-step enzyme digestion process on the dermis,including overnight digestion at 4℃ with a neutral enzyme,digestion at 37℃ with collagenase,and trypsin digestion.To culture the isolated cells,we used double cell filters of 70μm and 40μm and MC selective medium M254.The isolated cells exhibited a bipolar or tripolar shape and strong refraction.We identified the primary cells through various methods,including indirect immunofluorescence staining of MITF,TYR,and TYRP2,L-DOPA staining,and the use of iconic proteins SOX10,MITF,and TYR.These tests confirmed the successful isolation of hair follicle-derived MCs,providing material support for further verification of the relationship between DPCs and MCs.3.Extraction and Identification of DPCs-Exos and its effect on melanogenesis.Exosomes extracted from black and white Rex rabbit DPCs were isolated through precipitation method and characterized using NTA.The particle size of DPCs-Exos ranged from 30-150nm.Electron microscopy analysis revealed that the extracted DPCs-Exos exhibited a cup-shaped or round shape.Western Blot analysis was performed to confirm the presence of CD9 and TSG101 in the extracted DPCs-Exos.The internalization of DPC-Exos by MCs was confirmed by adding CM-Dil tagged DPC-Exos.The melanin production in MCs was significantly higher when treated with black Rex rabbit DPCs-Exos compared to white Rex rabbit DPCs-Exos(P<0.01).4.Effect of Exosomal miRNA-222-3p from Dermal Papilla Cells on Melanin Production in Melanocytes.miR-222-3p expression was evaluated using RT-qPCR in Rex rabbit skin of different coat colors.A significantly higher expression of miR-222-3p was observed in white Rex rabbit skin(P<0.01).DPCs-Exos were collected from both black and white Rex rabbits and total RNA was extracted.The miR-222-3p expression was significantly higher in the white Rex rabbit DPCs-Exos compared to the black Rex rabbit DPCs-Exos(P<0.01).Furthermore,Cy3-labeled miR-222-3p mimics were transfected into DPCs to establish a co-culture system of DPCs and MCs using the Transwell chamber.Red fluorescence was observed to accumulate in MCs,and quantitative analysis revealed a significant upregulation of miR-222-3p expression in MCs(P<0.01).MCs were transfected with miR-222-3p mimics and inhibitor.Cell proliferation,apoptosis,and melanin production were measured using CCK8,Annexin V-FITC,and NaOH with negative control(NC)as a reference.Inhibition of miR-222-3p promoted MCs proliferation and inhibited apoptosis(P<0.01),while significantly increasing melanin content(P<0.01).Bioinformatic analysis revealed that SOX10 was the target gene of miR-222-3p.Using a double luciferase system and fixed-point mutation test,it was demonstrated that miR222-3p mimics significantly inhibited the activity of SOX10-3’UTR-WT(P<0.01).The inhibitory effect of miR-222-3p mimics on the expression of SOX10 mRNA and proteinconfirmed their targeting relationship(P<0.01).DPCs-derived exosomes carrying miR-222-3p negatively regulate MCs proliferation and melanin production by specifically targeting the transcription factor SOX10.The selection of miR-222-3p as the miRNA candidate for functional analysis was based on previous research by the group and related sequencing support.To evaluate the impact of miR-222-3p on melanocytes,we measured its expression in Rex rabbit skin of various colors.We found that its expression differed significantly in white Rex rabbit skin,as compared to black,brown,protein yellow,and protein blue.A comparison was made between the expression of miR-222-3p in exosomes derived from black and white Rex rabbit DPCs.We found that its expression was significantly higher in exosomes derived from white Rex rabbit DPCs,as compared to those derived from black Rex rabbit DPCs.We transfected melanocytes with miR-222-3p mimics and inhibitors and assessed their proliferation and apoptosis rates(P<0.01).Inhibition of miR-222-3p resulted in increased proliferation and decreased apoptosis of melanocytes,and a significant increase in melanin content was observed(P<0.01).SOX10 was predicted as the target gene for miR-222-3p,which we verified through a luciferase experiment measuring the targeting relationship between ocu-miR-222-3p and SOX10.Our findings suggest that exosomes derived from DPCs carrying miR-222-3p can down-regulate melanocyte proliferation by targeting SOX 10,a transcription factor crucial for melanin regulation.