Abstract

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.