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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。
This study aimed to establish miRNA database of cumulus cell of GV / MII (MetaphaseⅡ, MⅡ) in buffalo oocytes before and after maturation, and to screen differentially expressed miRNAs in two stages. To further elucidate the effect of miRNA Provide a scientific basis for regulating the important role played by the gene expression in the cumulus cells during oocyte maturation. The cumulus cells of Buffalo GV / MII were collected and the total RNA was extracted respectively. Small RNA sequencing data were obtained by Solexa sequencing and bioinformatics analysis was performed. The results showed that the miRNA expression profile of GV / M phase Ⅱ cumulus cells was successfully constructed in this study. It was found that miRNAs significantly up-regulated in MⅡ stage cumulus cells compared with GV stage cumulus cells, and their expression differences were significantly down-regulated There are 45. QRT-PCR was used to validate 8 randomly selected miRNAs, confirming that their expression levels are consistent with the results of RNA-seq analysis. Combined with the prediction of target genes and KEGG enrichment analysis, 276 significantly enriched signaling pathways were obtained. At the same time, the signal pathways enriched in the top 20 were summarized. It is speculated that the differentially expressed miRNAs mainly through cell proliferation, apoptosis, , Cell connections and other pathways in the cumulus cells play a role.