Effect of Short Tandem Target Mimic miR-5110 on Melanogenesis in Melanocytes of Alpaca (Vicugna pacos)

MicroRNAs (miRNAs) play important roles in the regulation of melanogenesis and coat color in mammals. Short tandem target mimics (STTMs) have been used to block the functions of small RNA in animals and plants. To investigate the role of miR-5110 in melanogenesis, STTM was used to block the expression of miR-5110 (STTM-miR-5110). Luciferase reporter assay data indicated the miR-5110 regulates SOX10 expression by targeting its 3′-UTR. Overexpression of STTM-miR-5110 in alpaca melanocytes downregulated the expression of miR-5110 (decreased by about 38%, p < 0.05) and upregulated SOX10 mRNA (2.2-fold, p < 0.001) and protein (1.3-fold, p < 0.05) levels. Overexpression of STTM-miR-5110 in alpaca melanocytes increased the mRNA expression of melanogenic genes, including microphthalmia transcription factor (2.0-fold, p < 0.01), tyrosinase (1.6-fold, p < 0.01), tyrosinase-related protein 1 (approximately 3.9-fold, p < 0.001) and tyrosinase-related protein 2 (1.9-fold, p < 0.01). Overexpression of STTM-miR-5110 in alpaca melanocytes increased the protein expression of melanogenic genes, including microphthalmia transcription factor (1.9-fold, p < 0.05), tyrosinase (1.3-fold, p < 0.05), tyrosinase-related protein 1 (1.8-fold, p < 0.001) and tyrosinase-related protein 2 (1.6-fold, p < 0.05). The overexpression of pGL0-STTM-miR-5110 in alpaca melanocytes increased melanin production by approximately 26% (p < 0.05), pheomelanin production by approximately 38% (p < 0.05) and eumelanin production by approximately 56% (p < 0.001). In addition, overexpression of STTM-miR-5110 in alpaca melanocytes increased the TYR activity by 37% (p < 0.01). We also identified melanin granules in alpaca melanocytes transfected with STTM-miR-5110 under Fontana-Masson staining. These results suggest that STTM-miR-5110 upregulates melanogenesis by effectively blocking miR-5110 expression.