Exosome Derived From Keratinocyte With Mir-330-5p Suppresses Melanocytes Pigmentation Via Targeting TYR

Pigmentation is controlled by complex mechanisms.Evidences suggest that miRNAs can regulate pigmentation.However,its mechanism has not been fully elucidated.Exosomes are one of the most potent intercellular communicators,which are able to communicate with adjacent or distant cells.In this study,we revealed a novel mechanism that regulating pigmentation involving exosomes,miRNAs and the crosstalk between keratinocytes and melanocytes.The inhibitory effects of miR-330-5p on pigmentation were analyzed by using a variety of molecular biology technology.Futhermore,we take a reaserch of the expression and localization of some exosome specific markers in keratinocytes and melanocytes.Finally,melanocytes were co-cultured with exosomes that overexpression of miR-330-5p.The changes of melanin content in melanocytes and the expression of target gene TYR and its associated genes TYRP1,TYRP2 and MITF were detected after co-culture.The results are as follows:(1)QRT-PCR and western blotting techniques were used to analyze the relative expression of TYR,TYRP1 and TYRP2 in melanocytes after overexpression of miR-330-5p.The results show that miR-330-5p can suppress the production of melanin and expression of TYR and its downstream genes in melanocytes.(2)The exosome of keratinocytes was identified by immunofluorescence,qRT-PCR and western blotting.The results of immunofluorescence showed the TSG101 was express in keratinocytes.The results of western blotting of exosome proteins extracted from keratinocytes showed that both TSG101 and CD9 were positive expression.Finally,qRT-PCR analysis showed that overexpression of miR-330-5p in keratinocytes resulted in high expression of miR-330-5p in exosomes.(3)The interaction between exosomes derived from keratinocytes and melanocytes was analyzed by fluorescence in situ hybridization,immunofluorescence,qRT-PCR and western blotting.The results demonstrated that exosomes derived from keratinocytes with overexpression of miR-330-5p caused a significant increase of miR-330-5p in melanocytes.And exosomes derived from keratinocytes overexpressed with miR-330-5p induced a significant decrease in the production of melanin and expression of TYR in melanocytes.These findings suggest that keratinocytes crosstalk with melanocytes in the epidermal melanin unit via exosome carried miRNAs.These studies reveal an important role of exosomes in melanocytes pigmentation which opens a new pathway of melanogenesis.