The Influence Of MicroRNA-663to Melanin Synthesis In Melanocytes Of Alpaca

Coat color is the most important economic characters of wooler. In mammals, the coat color is formed by the melanin, which is produced by melanocytes. Melanin type and relative quantity makes the coat color from deep to shallow. The production of melanin is closely related with the growth of hair follicle cycle. TGF-β1control hair follicles to the regression phase shift through inhibiting cell proliferation and promoting cell apoptosis. MicroRNAs are including21-24nucleotides length which negatively involved in post-transcriptional regulation of gene expression. In our previous study, miR-663had diffrent expression in the skin tissue of diffren coat color alpacas. The objective of this study is to identify the target gene of miR-663and investigate the role of miR-663in melanin synthesis in alpaca melanocytes. The study includ:(1) The target gene of miR-663was predicted and selected by bioinformatics.(2) The TGF-β1was cloned and made analysis of expression and location.(3) The target gene of miR-663was validated by pmirGLO dual luciferase reporter assays.(4) The overexpression and repression of miR-663were achieved by transfecting melanocytes with synthetic miR-663mimic or miR-663inhibitor, respectively. The mRNA and protein levels and melanin assay of TGF-β1、Smad3、Smad4、Smad7and melanin synthesis genes were analyzed by qRT-PCR or Western blotting, respectively.The results are followings:(1) A potential target gene of miR-663predicted by informatics is TGF-β1.(2) Sequence analysis indicated alpaca TGF-β1had typical structures of TGF-β family, showing high similarity with related proteins from other mammals.(3) miR-663was identified to be expressed higher in white skin than brown skin in alpaca, and TGF-β1had different expression in different tissues. TGF-β1was located in melanocytes of alpaca membranes and matrix.(4) Alpaca TGF-β1was the direct target of miR-663, which was validated by Luciferase reporter assays.(5) When miR-663was inhibited by transfecting into melanocytes, the expression levels of TGF-β1and the genes related to melanin synthesis increased both of mRNA level and protein level, and the content of melanin increased in melanocytes.(6) When miR-663was overexpressed by transfecting into melanocytes, the expression levels of TGF-β1and the genes related to melanin synthesis reduced both of mRNA level and protein level, and the content of melanin decreased. In conclusion, TGF-β1is the direct target gene of miR-663. miR-663may function as a potent regulator of pigmentation by regulating TGF-β1and other several important melanogenesis-related genes, directly or indirectly. The manipulating of miR-663levels may be a promising approach in coat color modification.