Reduced MiR-675 in Exosome in H19 RNA-Related Melanogenesis via MITF as a Direct Target

• Published in: Journal of investigative dermatology Vol. 134; no. 4; pp. 1075 - 1082
• Main Authors: Kim, Nan-Hyung, Choi, Soo-Hyun, Kim, Chang-Hyun, Lee, Chang Hoon, Lee, Tae Ryong, Lee, Ai-Young
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.04.2014; Elsevier Limited
• Subjects: 3' Untranslated Regions; Animals; Apoptosis; Cell Separation; Epidermis - metabolism; Exosomes - metabolism; Female; Fibroblasts - cytology; Fibroblasts - metabolism; Flow Cytometry; Gene Expression Regulation; Humans; Keratinocytes - cytology; Melanocytes - cytology; Mice; Mice, Inbred C57BL; Microphthalmia-Associated Transcription Factor - metabolism; MicroRNAs - genetics; MicroRNAs - metabolism; Oligonucleotide Array Sequence Analysis; RNA, Long Noncoding - genetics; Tetraspanin 30 - metabolism
• ISSN: 0022-202X; 1523-1747
• DOI: 10.1038/jid.2013.478

H19 non-coding RNA downregulation stimulates melanogenesis in melasma patients. However, its mechanism is unclear. In this study, the potential role of a H19 microRNA, miR-675, in melanogenesis was examined. Real-time PCR using cultured normal human skin keratinocytes, melanocytes, and fibroblasts with or without H19 knockdown showed accompanying changes between expression levels of H19 and those of miR-675 in keratinocytes. MiR-675 was also detected in concentrated culture supernatants and showed expression levels parallel with those of cell lysates. In addition to RNase resistance, FACS analysis showed anti-CD63-positive exosomes in culture supernatants, suggesting miR-675 could be released extracellularly and delivered to neighboring cells without degradation. In western blot analysis, the miR-675 mimic reduced the expression of microphthalmia-associated transcription factor (MITF) and phosphorylation of cAMP-responsive element-binding protein, extracellular signal–regulated kinase and apoptosis signal–regulating kinase, whereas these expressions were increased by the miR-675 inhibitor. Although H19 was not a miR-675 target, luciferase reporter assay showed a direct binding of miR-675 to 3′-untranslated region of MITF. In addition, localized in vivo miR-675 overexpression in mouse using a cationic polymer transfection reagent showed reduced mRNA expression levels of MITF, tyrosinase, tyrosine-related protein-1 (Trp-1), and Trp-2. Collectively, the results suggest that miR-675 derived from keratinocytes could be involved in H19-stimulated melanogenesis using MITF as a target of miR-675.