[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 SacⅠ and 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.%【目的】预测和验证羊驼 TGF-β1是 miR-663的靶基因之一，并对 miR-663介导的 TGF-β1在黑色素生成过程中的作用进行研究。【方法】利用 Targetscan、RNAhybrid 和 RNA22在线预测 miR-663的靶基因，并对靶基因3′UTR 区可能的 miR-663的作用位点进行分析。利用 DNAMAN 软件分析比对羊驼、人和牛等哺乳动物 TGF-β1-3′UTR 区的相似性。利用 SacⅠ和 XbaⅠ将羊驼 TGF-β1基因的3′UTR 区插入 pmirGLO 构建双荧光报告载体 pmirGLO-TGF-β1-3′UTR 并与 miR-663 mimic 共转染293T 细胞，通过测定荧光素酶活性来验证 miR-663的直接靶基因是 TGF-β1。在羊驼黑色素细胞中通过转染 miR-663 mimic 来过表达 miR-663，并利用 qRT-PCR 和 Western blotting 检测 miR-663过表达后细胞中 TGF-β1、Smad3、Smad4、Smad7和β-catenin 分别在 mRNA 和蛋白水平的变化及黑色素含量的变化。【结果】软件预测显示 miR-663有68个保守的靶基因，包含74个保守的靶位点和44个非保守靶位点。TGF-β1是 miR-663的靶基因之一，且已被证明与毛囊发育和黑色素生成相关。不同物种的 TGF-β1基因的3′UTR 区相似性较高且均存在多个保守的 miR-663靶位点。克隆了羊驼 TGF-β1基因3′UTR 区发现存在3个保守的 miR-663靶位点。成功构建包含3个 miR-663作用位点的 pmirGLO-TGF-β1-3′UTR 双荧光报告载体并与 miR-663 mimic 共转染293T 细胞。双荧光素酶报告基因检测结果显示：与对照组相比，试验组 pmirGLO-TGF-β1-3′UTR + miR-663 mimic 荧光素酶活性下调31.01%，表明 TGF-β1是 miR-663直接的靶基因。在羊驼黑色素细胞中转染 miR-663 mimic 后：miR-663表达量上调334倍，TGF-β1、β-catenin、Smad4基因的表达量分别下调89%、41%、34%；TGF-β1蛋白表达量下降了21%，β-catenin 蛋白表达量无明显差异；黑色素细胞中的黑色素含量下降42%。【结论】TGF-β1是 miR-663的直接靶基因。miR-663通过调控 TGF-β1的表达而影响 TGF-β/Smad和 Wnt 信号通路，进而影响羊驼黑色素细胞中的黑色素生成。
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