The influence of different regulatory patterns of miRNA-target genes on the pigmentation of the Chinese soft-shelled turtle (Pelodiscus sinensis)

MicroRNA(miRNAs) are key post-transcriptional regulators of gene expression and play a role in body color formation in aquatic animals. However, their functional roles in body color formation in Chinese soft-shelled turtle (Pelodiscus sinensis) remain largely uncharacterized. Here, we combined in vitro and in vivo approaches to dissect the regulatory mechanisms of miR-18a-3p_3 and miR-221 on pigmentation-related genes. Dual-luciferase reporter assays confirmed direct binding between miR-18a-3p_3 and the 3'UTR of Sox10, as well as between miR-221 and Mitf. Mutations in the predicted binding sites abolished these interactions, validating the specificity of targeting. Subsequently, we successfully isolated and cultured melanocytes from P. sinensis embryos using a specialized medium, enabling functional validation of miRNA effects on pigmentation gene expression. Transfection of miR-18a-3p_3 antagomir and miR-221 agomir into primary melanocytes modulated the expression of Sox10 and Mitf, respectively, and altered the transcription of downstream melanogenic genes including Tyr, Tyrp1, and Gpnmb. In vivo administration of miRNA modulators (antagomir/agomir) confirmed these regulatory patterns in turtle tissues, though tissue-specific expression differences were less pronounced than in cultured cells, suggesting context-dependent regulatory complexity. Collectively, we speculated that miR-18a-3p_3 might be negatively modulating Sox10, while miR-221 positively regulated Mitf, thereby coordinating melanin synthesis in P. sinensis. These findings provide novel insights into the molecular basis of body color variation in turtles and expand our understanding of miRNA-mediated pigmentation regulation in aquatic vertebrates.