The expression profiles of miRNAs in exosomes derived from H2O2-treated and untreated HaCaT cells
Vitiligo is a skin disease characterized by the destruction of epidermal melanocytes due to oxidative stress and autoimmune response. As the main responder to oxidative stress, keratinocytes facilitate melanocyte loss under oxidative stress by inducing melanocyte death and recruiting antigen-specific CD8+ T cell to skin to destroy melanocytes. It has been proved that keratinocytes secrete abundant functional exosomes, but the role of exosomes secreted from keratinocytes under oxidative stress in vitiligo pathogenesis is unknown. We found that oxidative stress enhanced the secretion of exosomes from keratinocytes. These exosomes (OS-Exos) administration aggravated melanocyte loss and CD8+ T cell infiltration in the epidermis of tail in the vitiligo mouse model, thereby driving vitiligo progression. OS-Exos suppressed the survival of melanocytes while promoting the proliferation and activation of CD8+  T cells in vitro. As miRNAs contained in OS-Exos might be responsible for the functions of OS-Exos on melanocytes and CD8+ T cells, we employed small RNAs-seq analysis to screen miRNAs enriched in OS-Exos as compared to those in Exos (from untreated HaCaT cells). The hierarchical clustering analysis and the volcano plot showed the distinct signatures of known miRNAs as well as novel miRNAs between OS-Exos and Exos. In total, 276 differentially expressed miRNAs were discovered, with 134 and 142 miRNAs displaying significant up- or down-regulation, respectively, in OS-Exos compared with Exos (P<0.05; fold change=2. The above data suggest that miRNAs enriched in OS-Exos might contribute to the facilitatory role of OS-Exos in vitiligo progression. Overall design: Human keratinocyte cell line HaCaT was treated with 0.4mM H2O2 for 24h and exosomes in the culture media were extracted using ultracentrifugation. Exosomes derived from H2O2-treated or untreated HaCaT were indicated as OS-Exos or Exos, respectively. The control group is Exos and the experimental group is  OS-Exos. Each group has 3 replicates.