摘要
环状RNA(circular RNA,circRNA)作为竞争性内源RNA(competitive endogenous RNA,ceRNA)在细胞分化调控中发挥着重要作用。本研究旨在对猪环状RNA IGF1R(circular RNA insulin-like growth factor 1 receptor,circIGF1R)进行鉴定及分析,探明其表达规律,构建猪circIGF1R相关的ceRNA调控网络,并探究其异位表达对小鼠间充质干细胞(C3H10T1/2)成脂分化的调控作用。通过正反向引物PCR、Sanger测序、RNase R酶消化检测和qRT-PCR验证circIGF1R是胰岛素样生长因子1受体(insulin-like growth factor 1 receptor,IGF1R)第二外显子形成的circRNA,它在猪各组织中均有表达,且其表达量在脂肪组织中随日龄增加呈上升趋势;使用miRDB、TargetScan和miRWalk在线软件预测circIGF1R靶基因,运用RNAhybrid软件进行结合位点预测,使用DAVID生物信息功能分析软件对候选靶基因进行GO和KEGG富集分析,运用Cytoscape软件构建ceRNA网络,基于基因表达相关性和预测的靶标关系,绘制了GO和KEGG富集分析及构建了ceRNA网络;双荧光素酶报告基因分析证明circIGF1R及FABP 4可与ssc(Sus scrofa chromosome)-miR-133a-5p结合;成功构建circIGF1R过表达载体,在间充质干细胞C3H10T1/2中异位表达,过表达circIGF1R后关键成脂调控因子CEBPα、CEBPβ、FABP 4和PPARγ极显著升高(P<0.01),脂滴数量显著增加。本研究结果证明,circIGF1R在猪脂肪组织中存在,并且可能通过ceRNA机制正调控C3H10T1/2细胞成脂分化,为进一步研究circIGF1R调控猪前体肌内脂肪细胞成脂分化奠定理论基础。
Circular RNA(circRNA),as a competitive endogenous RNA(ceRNA),plays an important role in the regulation of cell differentiation.The purpose of this study was to identify and analyze porcine circular RNA insulin-like growth factor 1 receptor(circIGF1R),explore its expression patterns,construct a ceRNA regulatory network related to circIGF1R,and explore the regulation of its ectopic expression on adipogenic differentiation of mouse mesenchymal stem cells(C3H10T1/2)effect.Forward and reverse PCR,Sanger sequencing,RNase R enzyme digestion tests,and qRT-PCR were used to verify that circIGF1R is a circRNA formed by the second exon of insulin-like growth factor 1 receptor(IGF1R).It was expressed in all tissues of pigs,and its expression level increased with age in adipose tissues.miRDB,TargetScan and miRWalk online software were used to predict circIGF1R target genes.RNAhybrid software was used for binding site prediction.DAVID bioinformatics functional analysis software was used to perform GO and KEGG enrichment analysis on candidate target genes.Cytoscape software was used to construct the ceRNA network diagram.Based on the gene expression correlation and predicted target relationship,the GO and KEGG enrichment analysis was drawn and the ceRNA network was constructed;the dual luciferase reporter gene test was used,and we found that circIGF1R and FABP 4 can bind to ssc(Sus scrofa chromosome)-miR-133a-5p.The circIGF1R overexpression vector was successfully constructed and expressed in C3H10T1/2 cells.It was found that after overexpression of circIGF1R,the expression of key adipogenic regulatory factors CEBPα,CEBPβ,FABP 4 and PPARγincreased significantly(P<0.01),and the number of lipid droplets increased significantly.The results of this study show that circIGF1R exists in pig adipose tissues,and may positively regulate the adipogenic differentiation of C3H10T1/2 cells through the ceRNA mechanism,which lays a theoretical foundation for further research on circIGF1R regulating the adipogenic differentiation of pig precursor intramuscular adipocytes.
作者
张娜
李萌
李娇
孟珊
蔡春波
杨阳
高鹏飞
郭晓红
曹果清
李步高
ZHANG Na;LI Meng;LI Jiao;MENG Shan;CAI Chun-Bo;YANG Yang;GAO Peng-Fei;GUO Xiao-Hong;CAO Guo-Qing;LI Bu-Gao(College of Animal Sciences,Shanxi Agricultural University,Taigu 030801,Shanxi,China)
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2022年第3期333-342,共10页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金项目(No.31872336)
三晋学者人才项目(2016,2017)资助。
