水牛卵母细胞GV/MⅡ期对应卵丘细胞miRNA差异表达分析

摘要

本研究旨在建立水牛卵母细胞体外成熟培养前后GV/MⅡ(Germinal vesicle,GV)/(MetaphaseⅡ,MⅡ)期对应的卵丘细胞miRNA数据库,筛选两个时期差异表达的miRNAs,为进一步阐明miRNA通过调控卵丘细胞中基因表达进而在卵母细胞成熟过程中发挥的重要作用提供科学依据.分别收集水牛GV/MⅡ期的卵丘细胞,提取总RNA,利用Solexa测序技术获取小RNA测序数据,并进行生物信息学分析.结果表明:本研究成功构建了GV/MⅡ期卵丘细胞miRNA的表达谱,发现相对于GV期卵丘细胞,miRNA在MⅡ期卵丘细胞中表达差异显著上调的有24个,表达差异显著下调的有45个.采用qRT-PCR技术对随机筛选的8个miRNAs进行验证,证实其表达水平和RNA-seq分析结果相一致.结合靶基因预测和KEGG富集分析,获得了276条显著富集的信号通路;同时对富集前20的信号通路进行归纳总结,推测差异表达的miRNAs主要是通过细胞增殖、凋亡,物质代谢,细胞连接等途径在卵丘细胞中发挥作用.%The aim of this study was to establish the miRNA database of cumulus cells in GV/M Ⅱ stages of buffalo oocytes during maturation in vitro,and then analyze the differential-expression miRNAs of cumulus cells between GV and M Ⅱ stages of oocytes to provide scientific basis for further understanding the important role of cumulus cells in buffalo oocytes maturation.Total RNAs of cumulus cells in GV/M Ⅱ stages were extracted,respectively,and subjected to small RNA sequencing by Solexa sequencing,the miRNA data were then analyzed through bioinformatics.The results indicated that the miRNA expression profiles in GV/M Ⅱ cumulus cells were successfully constructed.There were 24 up-regulated and 45 down-regulated miRNAs in cumulus cells in M Ⅱ stage compared with those in GV stage.The expression profiles of 8 randomly selected differential-expression miRNAs examined by qRT-PCR was agreement with those by RNA sequencing.Total of 276 enriched signaling pathways related to the miRNA of the cumulus cells were analyzed by miRNA target gene prediction and KEGG enriched pathways.The top 20 enriched signaling pathways were summarized and we found that the differential-expression miRNAs played roles in cumulus mainly through cell proliferation and apoptosis,material metabolism,cell junction as well as other unknown pathways.