猪GV/MⅡ期卵母细胞miRNAs表达谱及Npm2基因相关miRNAs筛选

miRNAs Expression Profile of Porcine GV/MⅡ-Stage Oocytes and Screening of miRNAs Related to Npm 2 Gene

旨在探索体外成熟前后猪卵母细胞miRNAs表达谱,并筛选参与调控Npm2基因表达的miRNAs。本试验回收屠宰场猪卵巢,收集GV期卵母细胞,经体外成熟培养后获得MⅡ期卵母细胞,分别提取约130个GV期和MⅡ期卵母细胞总RNA进行Illumina HiSeqTM 2500测序,获取miRNA测序数据,每个处理重复3次,筛选差异表达microRNAs并进行靶基因GO和KEGG聚类分析。结果表明,本研究成功构建了GV期和MⅡ期猪卵母细胞miRNAs表达谱,GV期和MⅡ期卵母细胞差异表达miRNAs有95个,具有相似表达模式的miRNA聚为一类,对95个差异表达miRNAs进行靶基因预测,共得到3 967个靶基因,经GO和KEGG富集分析表明,这些靶基因被富集于5 194个GO条目和212个信号通路,其中参与卵母细胞减数分裂成熟的信号通路有2个。在差异表达miRNAs中筛选到5个与Npm2基因相关的miRNAs。采用qRT-PCR对其中2个miRNAs进行验证,证实其表达趋势与测序结果一致。本研究筛选了GV期和MⅡ期卵母细胞差异表达miRNAs,推测差异表达的miRNAs可能通过代谢、卵母细胞减数分裂相关通路、孕激素介导的卵母细胞成熟通路等途径在卵母细胞体外成熟过程中发挥作用,并筛选出调控Npm2基因表达的miRNAs,研究结果可为进一步阐明miRNA对Npm2基因的调控及其在卵母细胞成熟过程中的作用提供依据。

The aim of this study was to explore the miRNAs expression profile of porcine oocytes before and after in vitro maturation,and then screen miRNAs involved in regulating Npm 2 gene expression.Porcine ovaries were collected at a local slaughterhouse and transported to the laboratory.GV-stage oocytes were collected and matured in vitro to obtain MⅡ-stage oocytes.Total RNA of 130 GV-or MⅡ-stage oocytes were extracted respectively and subjected to small RNA sequencing by Illumina HiSeqTM 2500.Each group was repeated 3 times.The differentially expressed miRNAs was screened,and GO and KEGG analysis were performed on the target genes.The results showed that the miRNAs expression profiles of GV-and MⅡ-stage oocytes were successfully constructed.Ninety five differentially expressed miRNAs were screened in GV-and MⅡ-stage oocytes,and miRNAs with similar expression patterns were clustered into one group.Total 3967 target genes were obtained from 95 differentially expressed miRNAs.GO and KEGG analysis showed that these target genes were enriched in 5194 GO terms and 212 KEGG pathways.Among these,there were two pathways related to oocyte maturation.Five miRNAs related to Npm 2 gene were screened from the differentially expressed miRNAs.Two miRNAs expression were examined by qRT-PCR,and their expression trend was consistent with the sequencing results.The differentially expressed miRNAs were screened from the GV-and MⅡ-stage oocytes,it was speculated that miRNAs play a role in the process of in vitro maturation of oocytes via metabolism,oocyte meiotic related pathways and progesterone mediated oocyte maturation pathway.And miRNAs regulating Npm 2 gene expression were screened.The result of this study can provide a foundation for further study the regulation mechanism of miRNAs on Npm 2 gene and their roles in oocyte maturation.