热应激对羊成肌细胞部分miRNA的影响及其生物信息学分析与靶基因预测

Effect of heat stress on partial miRNA in myoblasts and bioinformatics analysis and target gene prediction of miRNA

[目的]本文旨在探究热应激对羊成肌细胞部分miRNA的影响,并对miR-23a、miR-24、miR-27a-3p和miR-30a-5p进行生物信息学分析与靶基因预测,揭示热应激下miRNA影响骨骼肌发育的调控机制。[方法]通过RT-qPCR技术研究热应激对羊成肌细胞miRNA表达量的影响;利用miRBase数据库获取不同物种中miR-23a、miR-24、miR-27a-3p和miR-30a-5p的前体序列和成熟序列,并利用MEGA X软件构建系统进化树进行相似性分析;通过TargetScan、miRDB、RNA22、miRWalk对上述miRNA进行靶基因预测,使用DAVID对预测出的共同靶基因进行GO功能与KEGG通路富集分析,最后进行miRNA、共同靶基因与KEGG通路的关联分析。[结果]热应激显著下调羊成肌细胞在增殖阶段与分化阶段miRNA的表达。miR-23a、miR-24、miR-27a和miR-30a在不同物种间的保守性较高,除了牛的miR-24以外,成熟序列基本相同,种子序列完全一致。预测出的共同靶基因主要富集在细胞增殖、分化、周期、凋亡、迁移等GO条目,以及mTOR、TGFβ、MAPK、Ras、ErbB、FoxO和PI3K-Akt等KEGG通路。关联分析表明miR-24和miR-27a-3p可能靶向NLK和TAOK1以参与MAPK和FoxO信号通路来调控骨骼肌的发育。[结论]miR-23a、miR-24、miR-27a和miR-30a在不同物种中保守性较高,热应激下调羊成肌细胞部分miRNA的表达,可能影响骨骼肌的发育。

[Objectives]This paper aimed to explore the effect of heat stress on partial miRNA in myoblasts,and analyze by bioinformatics and predict the target genes of miR-23a,miR-24,miR-27a-3p and miR-30a-5p,so as to reveal the mechanism of miRNA regulating the development of skeletal muscle under heat stress.[Methods]The expression of miRNA in myoblasts under heat stress was studied by RT-qPCR.The miRBase was used to obtain the precursor sequences and mature sequences of miR-23a,miR-24,miR-27a-3p and miR-30a-5p in different species,then MEGA X software was used to construct the phylogenetic trees for similarity analysis.The target genes of the above miRNA were predicted by TargetScan,miRDB,RNA22 and miRWalk,and the predicted common targets were analyzed by DAVID for GO function and KEGG pathway enrichment.Ultimately,the correlation among miRNA,common target genes and the KEGG pathway was analyzed.[Results]Heat stress significantly decreased the expression of miRNA in myoblasts at the proliferation and differentiation stages.The miR-23a,miR-24,miR-27a and miR-30a showed high conservation among different species,and except for miR-24 of Bos taurus,the mature sequences of four miRNA were basically the same,and the seed sequences were completely the same.The predicted common target genes were mainly enriched in the GO items,such as cell proliferation,cell differentiation,cell cycle,cell apoptosis and cell migration,as well as mTOR,TGFβ,MAPK,Ras,ErbB,FoxO,and PI3K-Akt and other KEGG pathways.Additionally,the correlation analysis showed that miR-24 and miR-27a-3p may target NLK and TAOK1 to participate in the MAPK and FoxO signaling pathways to regulate skeletal muscle development.[Conclusions]The miR-23a,miR-24,miR-27a and miR-30a were highly conserved in different species and the expression of partial miRNA was down-regulated in myoblasts under heat stress,thereby it might affect the development of skeletal muscle.