title: Reduced MiR-675 in exosome in H19 RNA-related melanogenesis via MITF as a direct target.
abstract: 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.
authors: Department of Dermatology, Dongguk University Ilsan Hospital, Gyenoggi-do, South Korea.
authors: Nan-Hyung
authors: NH
authors: Kim
authors: Department of Dermatology, Dongguk University Ilsan Hospital, Gyenoggi-do, South Korea.
authors: Soo-Hyun
authors: SH
authors: Choi
authors: Department of Dermatology, Dongguk University Ilsan Hospital, Gyenoggi-do, South Korea.
authors: Chang-Hyun
authors: CH
authors: Kim
authors: College of Pharmacy, Dongguk University, Seoul, Korea.
authors: Chang Hoon
authors: CH
authors: Lee
authors: Bioscience Institute, AmorePacific Corporation R&D Center, Gyeonggi-do, South Korea.
authors: Tae Ryong
authors: TR
authors: Lee
authors: Department of Dermatology, Dongguk University Ilsan Hospital, Gyenoggi-do, South Korea. Electronic address: leeay@duih.org.
authors: Ai-Young
authors: AY
authors: Lee
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D017209:Apoptosis; D002469:Cell Separation; D004817:Epidermis / Q000378:metabolism; D055354:Exosomes / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006801:Humans; D015603:Keratinocytes / Q000166:cytology; D008544:Melanocytes / Q000166:cytology*; D051379:Mice; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D051739:Microphthalmia-Associated Transcription Factor / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D062085:RNA, Long Noncoding / Q000235:genetics*; D060149:Tetraspanin 30 / Q000378:metabolism
chemical_list: D020413:3' Untranslated Regions; C094335:H19 long non-coding RNA; C548379:MIRN675 microRNA, human; C574758:MIRN675 microRNA, mouse; C497234:MITF protein, human; D035683:MicroRNAs; D051739:Microphthalmia-Associated Transcription Factor; C497235:Mitf protein, mouse; D062085:RNA, Long Noncoding; D060149:Tetraspanin 30