| Background: Vitiligo is a pigmentary disorder caused by the decrease and dysfunction of melanocytes,and it is mainly divided into segmental,non-segmental,limited,and mixed vitiligo.The pathogenesis of vitiligo is complex and has not been fully elucidated.Among the four types of vitiligo,segmental vitiligo(SV)is considered to have relatively independent pathogenesis due to its unique unilateral distribution characteristics and stable development process after rapid progression.As vehicles for intercellular communication,exosomes regulate physiological and pathological functions by delivering their cargoes to target cells.It has expanded a whole new field for studying the mechanisms of various diseases,and exploring molecular diagnostic markers and therapeutic applications.Recently,circulating exosomal microRNA(miRNA)has been used as a potential biomarker for some disorders However,there are no reports on the identification of circulating exosomal miRNA expression profiles in SV patients and their involvement in the pathogenesis of the disease.Objective: This study aimed to screen for differentially expressed miRNAs in circulating exosomes of SV patients and to explore the mechanism by which circulating exosomal miRNAs enter the skin lesions locally and lead to the damage and dysfunction of melanocytes,thus establishing a new biomarker of SV to guide clinical practice.Methods: Firstly,sequencing and analyzation of circulating exosomal miRNAs in circulating exosomes of patients with SV and healthy controls were conducted.The significantly differentially expressed miRNAs were identified and validated by expanding the samples.And then,the obtained candidate miRNA was bioinformatically analyzed to explore their downstream molecules and related pathways.Further,assays were performed using skin tissue and cell co-culture system in vitro to verify whether the candidate circulating exosomal miRNA could enter target cells in localized skin and repress downstream genes to exert functions.Finally,after regulating the expression of the candidate miRNA,various molecular biology techniques such as dual-luciferase reporter assay were used to detect and demonstrate their regulatory mechanisms on downstream molecules and their functions.Results: This study found that circulating exosomal miR-493-3p was differentially elevated in SV patients,and its expression was positively correlated with disease activity.Moreover,miR-493-3p was highly expressed in the epidermis of the perilesions in SV patients.Dopamine metabolic pathway was enriched in miR-493-3p target genes,and dopamine concentration was also elevated at perilesional skin of SV patients.In vitro cell co-culture experiments revealed that epidermal keratinocytes could take up circulating exosomes from SV patients,and the miR-493-3p delivered by them could regulate dopamine metabolism.In the co-culture system of keratinocytes and melanocytes,miR-493-3p was demonstrated to inhibit melanocyte proliferation and melanin synthesis and to promote oxidative stress and apoptosis of melanocytes by affecting keratinocyte function.Identified by bioinformatic analysis,the co-transcription factor heterogeneous nuclear ribonucleoprotein U(hnRNPU)and dopamine hydrolase catechol-O-methyltransferase(COMT)is the downstream pathway genes of miR-493-3p that regulate dopamine metabolism.The downregulated expression of both hnRNPU and COMT was verified at perilesional skin of SV patients.Cellular experiments further demonstrated that miR-493-3p regulated dopamine metabolism in keratinocytes through the hnRNPU-COMT axis.Conclusion: This study demonstrates the specific mechanism that circulating exosomal miR-493-3p regulates the dopamine metabolism in keratinocytes by targeting the hnRNPU-COMT axis to play a role in SV pathogenesis,which provides a potential therapeutic target for SV.Circulating exosomal miR-493-3p may become a clinical marker of SV disease progression. |
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