Abstract: 【Abstract】 Objective To investigate the role of microRNA-25-5p （miR-25-5p） in melanogenesis, and to explore its underlying mechanisms. Methods Target genes of miR-25-5p were predicted using the TargetScan database. The interaction between miR-25-5p and the 3' untranslated region （3' UTR） of the RAB11B gene （a member of RAS oncogene family） was validated through a dual-luciferase reporter assay. Post-inflammatory hyperpigmentation （PIH） models were established in female C57BL/6J mice （6 - 8 weeks old） and female brown guinea pigs （4 - 6 weeks old） through daily broadband ultraviolet B （UVB） irradiation on the dorsal skin of the mouse ear or shaved dorsal skin of guinea pigs, while untreated mice and untreated dorsal skin areas of guinea pigs served as control groups. During modeling, these experimental animals received intradermal injections of a miR-25-5p agomir or a miR control agomir. Changes in skin pigmentation were observed, and skin tissue samples were harvested for further analysis after modeling. Melanin content in skin tissues was evaluated using Masson-Fontana staining. Expression of RAB11B and tyrosinase （TYR） in skin tissues was determined using immunohistochemical staining and quantitative real-time PCR （qPCR）. Primary human melanocytes were isolated from discarded normal foreskin tissues of healthy males after circumcision. Both primary human melanocytes and human MNT1 melanoma cells were transfected with miR-25-5p mimics or miR control mimics. Relative expression levels of miR-25-5p and RAB11B mRNA were quantified by qPCR using the 2-ΔΔCt calculation method. In MNT1 cells, miR-25-5p and RAB11B were co-overexpressed to assess their effect on the mRNA expression of RAB11B and TYR. Statistical analysis was conducted using t test or one-way analysis of variance followed by Tukey's post hoc test for multiple comparisons. Results The bioinformatic prediction and dual-luciferase reporter assay confirmed a binding site for miR-25-5p in the 3′ UTR of the RAB11B gene. In both animal models, the treatment with the miR-25-5p agomir significantly reduced local skin pigmentation compared to the control groups; Masson-Fontana staining showed a marked decrease in the density of melanin granules in the epidermis and dermis in the miR-25-5p agomir groups compared with the miR control agomir groups （mice: 0.050 ± 0.005 vs. 0.087 ± 0.008; guinea pigs: 0.067 ± 0.015 vs. 0.110 ± 0.013; both P < 0.05）. Immunohistochemical staining revealed significantly lower expression of RAB11B in mouse skin tissues in the miR-25-5p agomir group than in those in the miR control agomir group （both P < 0.05）. qPCR revealed significantly lower mRNA expression of RAB11B and TYR in skin tissues of guinea pigs in the miR-25-5p agomir group than in those in the miR control agomir group （both P < 0.05）. Similarly, RAB11B mRNA expression significantly decreased in the miR-25-5p mimics group compared with the miR control mimics group in primary human melanocytes and MNT1 cells （both P < 0.05）. In human MNT1 melanoma cells, miR-25-5p overexpression could suppress TYR mRNA expression, whereas co-overexpression of miR-25-5p and RAB11B could reverse this suppression. Conclusion Overexpression of miR-25-5p could alleviate UVB-induced post-inflammatory hyperpigmentation and inhibit melanogenesis, likely by targeted suppression of RAB11B expression.

Key words: Skin pigmentation, Models, animal, Melanogenesis, miR-25-5p, RAB11B, Tyrosinase