热应激奶牛血清miRNA表达谱及miR-181a靶基因分析

Profiling of differential expression of microRNAs in serum of heat-stresseded cows and bioinformatic analyses of miR-181a target genes

通过分析热应激奶牛和正常奶牛血清差异表达miRNA,及对重要的候选miRNA进行靶基因预测及相关生物信息学分析,探索miRNA在荷斯坦奶牛发生热应激过程中的作用及其调控机制。利用miRNA高通量测序技术对奶牛血清中miRNA表达谱进行检测和分析以筛选差异表达miRNA;用RT-qPCR方法验证4个在热应激奶牛血清与正常奶牛血清中显著差异表达的候选miRNAs;采用Targetscan生物信息学计算方法预测目标miR-181a(对应激以及免疫反应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。结果表明:miRNA高通量测序分析发现共有52个差异表达极显著miRNA(P<0.01)。候选的4个miRNAs(miR-19a、miR-19b、miR-181a、miR-1246)的RT-qPCR检测结果与测序分析结果具有很好的一致性。GO和KEGG分析结果显示,miR-181a的靶基因与应激反应以及免疫功能密切相关,并且靶基因在B细胞和T细胞受体信号通路中显著富集。结论:在热应激奶牛与正常奶牛血清中存在差异表达的miRNAs,某些重要的miRNA(如miR-181a)可能以独特的通路(如B细胞和T细胞受体信号通路)来调节奶牛热应激的发生过程。

In order to explore the role of microRNA （miRNA） in the heat-stresseded response process and its regulatory mechanism when Holstein cows occurred to heating stress, the profiling of differential expressed miRNA in serum of heat-stressed and normal Holstein cows was analyzed by Solexa deep sequencing approach and real-time PCR（RT-qPCR）. The target genes and signal path- way of miR-181a were predicted by bioinformatics software and database. Deep sequencing of bovine miRNA was employed to detect the differentially expressed miRNA between heat-stressed and normal Holstein cows serum. Then the expression of four selected miR- NAs（ miR-19a,miR-19b,miR-181a and miR-1246）in serum of heat-stressed and normal Holstein cows was also validated by RT- qPCR method. Target scan algorithm was used to predict target gene of miR-181a, and the result of gene set was analyzed by bioin- formatics of GO annotations, GO term enrichment and KEGG enrichment methods. The results revealed that there were 52 miRNAs with significant differential expression between serum of heat-stressed and normal Holstein cows（ P〈0.01 ）. The expression of rniR- 19a,miR-19b,miR-181a and miR-1246 which were also detected by RT-qPCR was in high concordance with deep sequencing results. GO function analysis showed that target genes of miR-181a were related to stress response and immune function. The results of KEGG indicated that target genes of miR-181a were mostly enriched in B cell receptor signaling pathway and T cell receptor signa- ling pathway. Conclusion ：There are specific miRNA expression profiles in serum of heat-stressed and normal Holstein cows. Some differentially expressed miRNA in serum of heat-stressed such as miR-181a may play important roles by distinct pathway in serum of heat-stressed and normal Holstein cows.