中国农业科学 ›› 2015, Vol. 48 ›› Issue (1): 165-173.doi: 10.3864/j.issn.0578-1752.2015.01.16

• 畜牧·兽医 • 上一篇    下一篇

miR-663通过靶向TGF-β1调控羊驼黑色素细胞的黑色素生成

贾小云1,金雷皓1,苗潋涓1,丁娜1,范瑞文2,董常生2   

  1. 1山西农业大学生命科学学院,山西太谷 030801
    2山西农业大学动物科技学院,山西太谷 030801
  • 收稿日期:2014-04-01 出版日期:2015-01-01 发布日期:2015-01-01
  • 通讯作者: 董常生,E-mail:cs_dong@sxau.edu.cn
  • 作者简介:贾小云,Tel:0354-6287191;E-mail:gssjxy@hotmail.com。金雷皓,E-mail:venus_jx@126.com。贾小云与金雷皓为同等贡献作者。
  • 基金资助:
    教育部高等学校博士学科点专项科研基金(20111403120006)、中国博士后科学基金普通资助和特别资助(20100481306、2012T50246)、山西省高等学校131人才项目、山西省人才引进开发专项、山西省高等学校优秀青年学术带头人项目

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

摘要: 【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。

关键词: 羊驼黑色素细胞;双荧光素酶报告载体;黑色素;miR-663;TGF-&beta, 1

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