调控猪卵母细胞Npm2表达的microRNAs的筛选及信号通路研究

Npm 2 is a maternal gene existed in the mammalian oocyte and encode nucleoplasmin which is a member of molecular chaperones and play an important role in chromatin recondensation, nucleosome assemble, chromatin reprogramming. Its expression is influenced by many factors, including the microRNAs. MicroRNAs are small, non-coding regulatory RNAs and function in post-transcriptional regulation of gene expression through incomplete complementary in mammal. But the relationship between miRNAs and Npm 2 gene expression of porcine oocytes are still not reported. In this project, 1) miRNAs deep sequencing will be used and the miRNAs-mRNA regulation networks of porcine oocytes during in vitro or in vivo maturation will be constructed; 2) miRNAs related to Npm 2 gene expression and signal pathway will be screened out; 3) the regulation role of miRNAs on Npm 2 gene expression will be verified in cellular and embryonic level. The performance of this project will reveal the relationship between miRNAs and Npm 2 gene expression of porcine oocytes and regulatory pathway, discover new regulatory factor in porcine oocytes maturation and nuclear reprogramming. These results will play an important role in improving the efficiency of porcine nuclear reprogramming and promoting the study on the pig complex gene regulation network.

Npm2作为哺乳动物卵母细胞中储存的一种母源基因，其编码的核质蛋白是分子伴侣家族的成员，在染色体解聚、核小体组装、染色体重编程方面发挥重要的作用，其表达调控受多种因素影响，包括microRNAs。MiRNAs是一类具有调控功能的单链非编码RNA，在哺乳动物中通过不完全互补方式，起到转录后调控靶基因表达的作用，但miRNAs对猪卵母细胞Npm2基因表达的调控仍未见报道。本项目采用miRNAs深度测序技术，构建体内外成熟前后卵母细胞miRNAs-mRNA调控网络，筛选与Npm2基因表达相关的miRNAs及其信号通路，在细胞及胚胎水平验证miRNAs对Npm2基因表达的调控作用。通过本项目实施，揭示miRNAs与猪卵母细胞Npm2基因表达的关系及调控途径，发现新的猪卵母细胞成熟及克隆胚重编程的关键调节因子，将对提高猪克隆胚染色体重编程效率、促进对猪复杂的基因表达调控网络的理解具有重要意义。

本项目旨在构建miRNAs-mRNA调控网络，揭示miRNAs与猪卵母细胞Npm2基因表达的相互关系，发现新的猪卵母细胞成熟及体细胞核移植重构胚重编程的关键调节因子。收集猪GV期和体外成熟MⅡ期卵母细胞进行miRNAs测序，筛选到95个差异表达miRNAs，其中上调miRNAs33个，下调miRNAs62个。对差异表达miRNAs进行靶基因预测，共预测到3967个靶基因，GO和KEGG富集分析表明靶基因主要参与代谢通路、PI3K-Akt、AMPK等信号通路。从差异表达miRNAs中筛选到5个与Npm2基因表达相关的miRNAs，其中上调miRNA2个，下调miRNA3个。选择自体表达量高且极显著下调miR-423-3p和GV期完全不表达而MII期卵母细胞极显著上调的miR-7138-5p进行qPCR验证，其表达趋势与测序结果一致。双荧光素酶报告实验表明miR-423-3p与Npm2基因无结合位点，miR-7138-5p与Npm2 3’UTR有结合位点。猪卵母细胞成熟培养液分别添加不同浓度的miR-423-3p和miR-7138-5p模拟物和抑制物，结果表明添加不同浓度miR-423-3p模拟物对卵母细胞成熟率和孤雌胚胎体外发育率无显著影响，添加150nM miR-423-3p抑制物后孤雌胚胎体外发育率显著升高，推测miR-423-3p可能通过调控其他基因表达参与卵母细胞体外成熟。添加200nM miR-7138-5p模拟物极显著提高孤雌胚胎体外发育率，添加miR-7138-5p抑制物显著降低孤雌胚胎体外发育率。分别将不同浓度miR-423-3p和miR-7138-5p模拟物和抑制物注入GV期卵母细胞再进行体外成熟培养，得到相类似的结果。本项目研究结果可为今后进一步研究miRNA对Npm2基因表达调控提供基础，为优化卵母细胞体外成熟培养体系、提高体细胞核移植效率提供支撑。