Scientia Agricultura Sinica ›› 2015, Vol. 48 ›› Issue (1): 165-173.doi: 10.3864/j.issn.0578-1752.2015.01.16

• ANIMAL SCIENCE·VETERINARY SCIENCERE • Previous Articles     Next Articles

Melanin Synthesis of Alpaca Melanocytes Regulated by miR-663 Through Targeting TGF-β1

JIA Xiao-yun1, JIN Lei-hao1, MIAO Lian-juan1, DING Na1, FAN Rui-wen2, DONG Chang-sheng2   

  1. 1College of Life Science, Shanxi Agricultural University, Taigu 030801, Shanxi
    2College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, Shanxi
  • Received:2014-04-01 Online:2015-01-01 Published:2015-01-01

Abstract: 【Objective】 The objective of the present study is to identify the target genes of miR-663 and investigate the role of miR-663 in melanin synthesis in alpaca melanocytes.【Method】 The potential targets and binding sites of TGF-β1 were predicted and analyzed by Targetscan, RNAhybrid and RNA22. The similarity of 3′UTR of TGF-β1 sequences from various species was analyzed by DNAMAN. The dual-luciferase construct of pmirGLO-TGF-β1-3′UTR was created by inserting partial TGF-β1 3′UTR into the pmirGLO vector by Sacand XbaⅠ restriction sites. The regulation of TGF-β1 by miR-663 was validated by co-transfecting pmirGLO-TGF-β1-3′UTR construct with miR-663 mimic into 293T cells. The over-expression of miR-663 was achieved by transfecting melanocytes with miR-663 mimic. The mRNA and protein levels of TGF-β1, Smad3, Smad4, Smad7 and β-catenin in melanocytes transfected with miR-663 mimic were analyzed by qRT-PCR or Western blotting, respectively. The effects of miR-663 on melanin synthesis were evaluated by measuring the melanin content of the cells.【Result】There are 68 potential targets for miR-663 predicted by bioinformatics, including 74 conserved binding sites and 44 less conserved binding sites. DNAMAN analysis showed that all 3′UTR sequences of TGF-β1 from analyzed species are highly conserved and enriched potential target sites. One of the potential targets of miR-663 is TGF-β1, which is involved in the development of hair follicle as well as melanin pigmentation. The alpaca 3′UTR sequence of TGF-β1 contains three miR-663 potential binding sites. To confirm the regulation of TGF-β1 by miR-663 through its 3′UTR, a dual-luciferase reporter vector pmirGLO-TGF-β1-3′UTR was successfully constructed and co-transfected into 293T cells with miR-663 mimic. The luciferase assay experiments showed that the luciferase activity was 31.01% lower in cells co-transfected with pmirGLO-TGF-β1-3′UTR and miR-663 mimic than that in control cells, suggesting TGF-β1 is a direct target of miR-663. When miR-663 mimic was transfected into melanocytes, the mRNA level of miR-663 increased to 345% but those of TGF-β1, β-catenin and Smad4 were reduced by 89%, 41%, and 34%, respectively. The protein level of TGF-β1 was reduced by 21%. The contents of melanin were significantly reduced by 42%.【Conclusion】TGF-β1 is a direct target gene of miR-663. Overexpression of miR-663 led to the decreased expression of TGF-β1 both at protein and mRNA levels. The miR-663 may influence/affect synthesis through regulation of TGF-β1 directly as well as TGF-β/Smad and Wnt signal pathways indirectly in skin melanocytes of Alpaca.

Key words: Alpaca melanocytes, dual-luciferase reporter vector, melanin, miR-663, TGF-β1

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