小鼠miR-151-3p靶基因筛选和鉴定  

Prediction and Identification of Mouse miR-151-3p Target Genes

作　　者：魏欢[1] 李仲文[1] 沈清武[1] 

机构地区：[1]西北农林科技大学动物科技学院,动物脂肪沉积与肌肉发育实验室,陕西杨凌712100

出　　处：《西北农业学报》2014年第5期23-31,共9页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：西北农林科技大学人才引进基金(Z111021006)

摘　　要：采用生物信息软件系统分析miR-151-3p的保守性,并筛选出与小鼠肌肉发育和肿瘤发生相关靶基因:蛋白激酶3(serine/threonine kinase 3,Akt 3),Twist碱性螺旋-环-螺旋转录因子(twist basic helix-loophelix 1,Twist 1)和T型电压依赖性钙通道α1g亚基(calcium channel,voltage-dependent,T type,alpha 1g subunit,Cacna1g)。采用实时定量PCR及双荧光素酶报告基因检测技术对以上靶基因进行初步验证,结果表明,超表达miR-151-3p显著抑制Akt 3mRNA表达,而Twist 1和Cacna1g mRNA表达变化不显著;双荧光素酶报告系统分析发现,miR-151-3p显著抑制野生型Akt 3报告载体相对荧光素酶活性,突变Akt 3 3′UTR与miR-151-3p结合的位点后,相对荧光素酶活性得到恢复,而Twist 1和Cacna1g基因双荧光素酶报告载体无此现象,结果证实Akt 3是miR-151-3p的靶基因。Bioinformatics software was used to analyze the conservation of miR-151-3p and predict its three putative targets including serine/threonine kinase 3（Akt 3）,twist basic helix-loop-helix 1（Twist 1）and calcium channel,voltage-dependent,T type,alpha 1g subunit（Cacna1 g）,which were likely involved in mouse myogenesis and tumorigenesis.Preliminary test for identifying the target of miR-151-3p was conducted with real-time PCR and dual-luciferase report system.The results showed that the mRNA expression level of Akt 3was significantly inhibited by overexpression of miR-151-3p,but the expression levels of Twist 1 and Cacna1 g were not changed significantly.The dual-luciferase activity system analysis indicated that the relative luciferase activity of Akt 3wilde type 3′UTR report vector was significantly decreased,and it could be recovered by mutating the binding sites of miR-151-3p on Akt 3 3′UTR;but similar results could not be detected in the report vectors of Cacna 1 gor Twist 1.In conclusion,Akt 3is the target gene of miR-151-3p in mouse.

关 键 词：miR-151-3p 靶基因 双荧光素酶报告基因检测系统 

分 类 号：Q75[生物学—分子生物学]