西门塔尔牛肌内和皮下脂肪miRNA表达谱及miR-27b靶基因分析

摘要

[目的]通过分析西门塔尔牛肌内脂肪和皮下脂肪差异表达miRNA，及对重要的候选miRNA-27b进行靶基因预测及相关生物信息学分析，探索miRNA在动物肌内脂肪生长、发育过程中的作用及其调控机制。[方法]利用miRNA微阵列芯片技术对肌内脂肪和皮下脂肪miRNA表达谱进行检测和分析以筛选差异表达miRNA；qRT-PCR方法验证4个在肌内脂肪显著高表达的候选miRNAs；采用Targetscan生物信息学计算方法预测目标miR-27b（对肌内脂肪沉积可能起重要调节作用）的靶基因，并对靶基因进行GO注释描述、富集分析及KEGG富集。[结果]miRNA芯片分析发现肌内脂肪和皮下脂肪共有88个差异表达极显著miRNA（P＜0.01）。候选的4个在肌内脂肪高表达miRNAs（miR-140、miR-145、miR-143、miR-27b）的qRT-PCR检测结果与芯片分析结果具有很好的一致性。KEGG富集显示，目标miR-27b的预测靶基因在MAPK、Wnt、Hedgehog、TGF-beta、GnRH等信号通路中显著富集，其中在MAPK信号通路中富集度最高。[结论]牛肌内脂肪和皮下脂肪组织中确实存在一些差异表达的miRNAs，某些重要的在差异高表达miRNA（如miR-27b）可能以独特的通路（如MAPK信号）来调节牛肌内脂肪的形成过程。%[Objective] In order to explore the role of microRNA in the growth and development process and its regulatory mechanism in intramuscular fat, the profiling of differential expressed microRNA in intramuscular fat and subcutaneous fat of Simmental were analyzed by microarray and real-time PCR (qRT-PCR). The target genes and signal pathway of miRNA-27b were predicted by bioinformatics software and database.[Method]Microarray of bovine microRNA was employed to detect the differentially expressed microRNA between intramuscular adipose tissue and subcutaneous adipose tissue. Then the expression of four selected microRNAs (miR-140, miR-145, miR-143 and miR-27b) in intramuscular adipose and subcutaneous adipose was also validated by qRT-PCR method. Target scan algorithm was used to predict target gene of miR-27b, and the result of gene set was analyzed by bioinformatics of GO annotations, GO term enrichment and KEGG enrichment methods.[Result]It was revealed that there were 88 microRNAs with significant differential expression between bovine intramuscular fat and subcutaneous fat (P<0.01). The expression of miR-140, miR-145, miR-143 and miR-27b which were also detected by qRT-PCR was in high concordance with microarray results. The results of bioinformatics indicated that target genes of miR-27b were mostly enrichment in MAPK, Wnt, Hedgehog, TGF-beta, GnRH signaling pathway, especially in MAPK pathway. [Conclusion]There are specific microRNA expression profiles in bovine intramuscular adipose tissue and subcutaneous adipose tissue. Some differentially high expressed microRNA in intramuscular adipose tissue such as miR-27b may play important roles by distinct pathway in bovine intramuscular adipogenesis.