164 Micro RNAs enriched in exosome derived from keratinocytes under oxidative stress contributes to melanocyte loss in vitiligo

Abstract

Vitiligo is a skin disease characterized by the destruction of epidermal melanocytes. Oxidative stress is closely related to the development of vitiligo. As the main constituent cells in the epidermis, keratinocytes regulates the viability and the function of melanocytes, but the underlying mechanism is still not clear. We hypothesized that exosomes derived from keratinocytes under oxidative stress induce the destuction of melanocytes. We treated human keratinocytes (HaCaT) with H2O2 for 24h and extracted exosomes from culture media by ultracentrifugation. TEM images revealed that exosomes secreted by keratinocytes displayed double layer membranes and cup-like structure, and varied in size (30–200 nm), findings consistent with NTA result. The markers of exosomes, CD63 and Hsp70, can be detected by western blot. Interestingly, we found that the amount of exosomes secreted by keratinocytes was dramatically increased under H2O2 treatment, with a concentration dependent. H2O2 at 0.4mM enhanced exosomes secretion by 3-fold. Meanwhile, we found that exosomes secreted by keratinocytes were successfully intake by melanocytes after 24h co-culture. Importantly, exosomes derived from H2O2-treated keratinocytes (Exo. KC-H2O2) significantly suppressed the proliferation of melanocytes after 96h co-culture. Subsequently, micorRNAs (miRNAs)-seq data showed that the miRNAs expression profile of Exo.KC-H2O2 had distinct characteristics. The qRT-PCR results showed that there are six miRNAs enriched in Exo.KC-H2O2. Further, we proved that two of the six miRNAs inhibited proliferation through regulating MITF axis and induced caspase triggered cell apoptosis of melanocytes in both normal condition and oxidative stress. In conclusion, our data have proved that miRNAs enriched in exosomes derived from keratinocytes under oxidative stress contribute to melanocyte loss. The findings provide new insights for understanding the pathogenesis of vitiligo.