miR-663通过靶向TGF-β1调控羊驼黑色素细胞的黑色素生成  

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

作　　者：贾小云[1] 金雷皓 苗潋涓 丁娜[1] 范瑞文[2] 董常生[2] 

机构地区：[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

出　　处：《中国农业科学》2015年第1期165-173,共9页Scientia Agricultura Sinica

基　　金：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

摘　　要：【目的】预测和验证羊驼TGF-β1是mi R-663的靶基因之一,并对mi R-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mi R-663的靶基因,并对靶基因3′UTR区可能的mi R-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pmir GLO构建双荧光报告载体pmir GLO-TGF-β1-3′UTR并与mi R-663 mimic共转染293T细胞,通过测定荧光素酶活性来验证mi R-663的直接靶基因是TGF-β1。在羊驼黑色素细胞中通过转染mi R-663 mimic来过表达mi R-663,并利用q RT-PCR和Western blotting检测mi R-663过表达后细胞中TGF-β1、Smad3、Smad4、Smad7和β-catenin分别在m RNA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mi R-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mi R-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mi R-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mi R-663靶位点。成功构建包含3个mi R-663作用位点的pmir GLO-TGF-β1-3′UTR双荧光报告载体并与mi R-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pmir GLO-TGF-β1-3′UTR+mi R-663 mimic荧光素酶活性下调31.01%,表明TGF-β1是mi R-663直接的靶基因。在羊驼黑色素细胞中转染mi R-663 mimic后:mi R-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mi R-663的直接靶基因。mi R-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�[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-6

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-Β1 

分 类 号：S829.9[农业科学—畜牧学]