miR-191通过调控C/EBPβ转录影响猪前体脂肪细胞分化  

miR-191 May Regulate Pig Preadipocyte Differentiation by Targeting The Transcription Factor C/EBPβ

作　　者：刘帅[1] 宁小敏[1] 李美航[1] 仇杨[1] 李艳杰[1] 董培越[1] 杨公社[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,杨凌712100

出　　处：《生物化学与生物物理进展》2013年第2期165-176,共12页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(31072014);西北农林科技大学"创新团队建设计划"资助项目~~

摘　　要：对实验室前期Solexa结果进行深入挖掘,结合生物信息学分析从不同发育阶段猪皮下脂肪组织差异表达的miRNAs中筛选出高丰度差异极显著的候选miR-191.采用腺病毒过表达miR-191,实时定量PCR、Western blot及双荧光素酶报告基因检测等技术方法,初步研究miR-191对猪前体脂肪细胞分化的影响.结果发现,miR-191随着猪前体脂肪细胞的分化表达量逐渐增加.与对照组相比,过表达miR-191的猪前体脂肪细胞中miR-191转录本显著增加,并引起CCAAT增强子结合蛋白β(C/EBPβ)、PPARγ和aP2的mRNA水平降低,抑制了猪前体脂肪细胞分化.同时,Western blot结果显示,与对照组相比过表达miR-191的猪前体脂肪细胞在48 h C/EBPβ蛋白水平下降了55%.更重要的是,通过TargetScan等算法正向筛选以及MicroInspector反向筛选联合获得miR-191候选靶基因,经双荧光素酶报告基因检测结果证实,miR-191可直接作用于C/EBPβ3′UTR,从而降低萤火虫荧光素酶活性.综上所述,miR-191可能通过抑制脂肪细胞分化早期标志基因C/EBPβ的表达,从而抑制了猪前体脂肪细胞的分化.Based on our previous solexa sequencing data,combining with bioinformatics analysis of microRNA expression changes in porcine adipose tissue from different growth phases in a model of obesity,we found the expression level of miR-191 was significantly different in developing swine adipose tissue.In this research,we successfully overexpressed miR-191＇s transcripts by recombinant adenovirus in primary cultured porcine preadipocytes,and then investigated the impact on differentiation of pig preadipocyte by Real-time quantitative PCR,Western blot.The results showed that miR-191＇s expression level gradually increased during differentiation.The miR-191 transcripts increased dramatically when miR-191 overexpressed compared to the control group,which caused the decrease of C/EBPβ,PPARγ and aP2＇s mRNA level and the sequentially repression of preadipocytes differentiation.Meanwhile,Western blotting results indicated that the expression of C/EBPβ protein in primary cultured porcine preadipocytes that overexpressed miR-191 was 55% lower than that in control cells after 48 h post-transfection.Importantly,we predicted that C/EBPβ was miR-191＇s target gene through bioinformatics software TargetScan and MicroInspector.This is conformed by dual luciferase reporter vector system assay which showed that miR-191 can target C/EBPβ 3′ untranslated region（3′UTR） directly and then decrease its expression.In conclusion,this study indicates that miR-191 can attenuate pig preadipocytes defferetiation possibly by repressing the gene expression of C/EBPβ which is one of the early marker gene of adipocytes differentiation.

关 键 词：miR-191 C EBPβ 猪前体脂肪细胞 表达谱 靶基因预测 腺病毒 双荧光素酶报告基因检测系统 

分 类 号：Q78[生物学—分子生物学] S82[农业科学—畜牧学]