筛选的差异表达microRNAs对猪卵母细胞Npm2基因的表达调控及作用研究  

The Study of Regulatory Effect of Differentially Expressed microRNAs on the Npm 2 Expression in Pig Oocytes

作　　者：李河林 蒋玉芬 程娜 韩宇辰 霍晓颖 苏宏鼎 常悦 方玉珠 王配[1,3] 贾宝瑜 魏红江 成文敏 LI Helin;JIANG Yufen;CHENG Na;HAN Yuchen;HUO Xiaoying;SU Hongding;CHANG Yue;FANG Yuzhu;WANG Pei;JIA Baoyu;WEI Hongjiang;CHENG Wenmin(College of Animal Science and Technology,Yunnan Agricultural University,Kunming 650201,China;College of Veterinary Medicine,Yunnan Agricultural University,Kunming 650201,China;Yunnan Key Laboratory of Porcine Gene Editing and Xenotransplantation,Kunming 650201,China)

机构地区：[1]云南农业大学动物科学技术学院,昆明650201 [2]云南农业大学动物医学院,昆明650201 [3]云南省小型猪基因编辑与异种器官移植重点实验室,昆明650201

出　　处：《畜牧兽医学报》2024年第11期5035-5049,共15页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：云南省“兴滇英才支持计划”青年人才项目(YNWR-QNBJ-2020-156);云南省基础研究专项重点项目(202201AS070078);国家自然科学基金地区项目(31660656)。

摘　　要：旨在研究卵母细胞中筛选的差异表达microRNAs对Npm2(nucleoplasmin 2)基因的调控作用。本研究采集屠宰场卵巢,收集GV期卵母细胞进行体外成熟培养,收集MⅡ期卵母细胞。使用qPCR检测筛选的差异表达miRNAs在GV、MⅡ期卵母细胞的表达水平;双荧光素酶报告试验验证预测的miRNA是否与靶基因Npm2存在结合位点;在体外成熟培养液中添加miRNAs模拟物/抑制物,验证miRNA对卵母细胞体外成熟和孤雌胚胎体外发育能力的影响。添加最适浓度的miRNAs模拟物/抑制物后,使用花生凝集素染色试验检测皮质颗粒分布和qPCR检测Npm2 mRNA表达水平。结果表明,MⅡ期卵母细胞中miR-150、miR-7138-5p表达显著高于GV期卵母细胞(P<0.05),miR-296-3p、miR-423-3p表达水平极显著低于GV期卵母细胞(P<0.01),表达趋势与测序结果一致。miR-32表达水平无显著差异(P>0.05)。双荧光素酶报告试验结果表明,miR-32、miR-7138-5p、miR-296-3p与Npm2存在结合位点,荧光强度极显著下调(P<0.01);miR-150与Npm2存在结合位点,荧光强度显著下调(P<0.05);miR-296-5p、miR-423-3p与Npm2不存在结合位点,荧光强度无显著变化(P>0.05)。通过在体外成熟培养液中添加不同浓度miRNAs模拟物/抑制物以确定最适浓度,添加miR-32抑制物、miR-296-5p模拟物、miR-296-5p抑制物、miR-423-3p模拟物、miR-423-3p抑制物、miR-7138抑制物、miR-150模拟物、miR-150抑制物后皮质颗粒荧光强度极显著下调(P<0.01);添加miR-296-3p抑制物、miR-7138-5p模拟物后皮质颗粒荧光强度显著下调(P<0.05)。MⅡ期卵母细胞Npm2 mRNA表达量极显著高于GV期卵母细胞,添加最适浓度miR-150、miR-296-3p、miR-296-5p、miR-7138-5p模拟物和/或抑制物Npm2 mRNA表达量与对照组有显著差异(P<0.05),添加miR-32、miR-423-3p模拟物/抑制物后MⅡ期卵母细胞Npm2 mRNA表达量与对照组无显著差异(P>0.05)。筛选的差异miRNA(如miR-296-3p、miR-7138-5p)可能通过调控Npm2表达�This study aimed to explore the regulatory effect of differentially expressed microRNAs on Npm 2(nucleoplasmin 2)in pig oocytes.The GV-stage oocytes were collected from slaughterhouse ovaries,cultured in vitro.The expression levels of screened miRNA in GV and MⅡstage oocytes were detected using qPCR.The dual-luciferase reporter experiment was performed to verify the binding site of predicted miRNA with target gene Npm 2.Further,miRNAs mimics/inhibitors were added to in vitro maturation medium to evaluate the effect of miRNA on the developmental ability of oocytes and parthenogenetic activated embryos.After the optimal concentration of miRNAs mimics/inhibitors were added,peanut agglutinin staining test was used to detect cortical granule distribution and Npm 2 mRNA expression level was detected using qPCR.The results showed that,compared with GV stage oocytes,the expression levels of miR-150 and miR-7138-5p were significantly higher(P<0.05)and the expression levels of miR-296-3p and miR-423-3p were significantly lower in MⅡoocytes(P<0.01),which were consistent with the previous sequencing results.There was no significant difference in the expression levels of miR-32(P>0.05).The double luciferase assay showed that miR-32,miR-7138-5p,miR-296-3p and miR-150 had binding sites with Npm 2,while the fluorescence intensity was significantly down-regulated(P<0.05).miR-150 had binding site with Npm 2 and the fluorescence intensity was significantly down-regulated(P<0.05).There was no binding site between miR-296-5p,miR-423-3p and Npm 2,as the fluorescence intensity was comparable(P>0.05).The optimal concentration of miRNAs mimics/inhibitors was determined.After addition of miR-32 inhibitor,miR-296-5p mimics,miR-296-5p inhibitor,miR-423-3p mimics,miR-423-3p inhibitor,miR-7138 inhibitor,miR-150 mimics and miR-150 inhibitor,cortical particle fluorescence intensity was significantly decreased(P<0.01).The cortical particle fluorescence intensity was significantly decreased after the addition of miR-296-3p inhibitor and miR-7

关 键 词：猪 卵母细胞 MICRORNA Npm2 

分 类 号：S828.3[农业科学—畜牧学]