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

doi:10.11843/j.issn.0366-6964.2024.11.021

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (11): 5035-5049.doi: 10.11843/j.issn.0366-6964.2024.11.021

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

李河林¹^,³(), 蒋玉芬¹^,³(), 程娜¹^,³, 韩宇辰¹^,³, 霍晓颖²^,³, 苏宏鼎³, 常悦¹^,³, 方玉珠²^,³, 王配¹^,³, 贾宝瑜²^,³, 魏红江²^,³^,*(), 成文敏¹^,³^,*()

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

收稿日期:2024-03-29 出版日期:2024-11-23 发布日期:2024-11-30
通讯作者: 魏红江,成文敏 E-mail:sonnyspike@163.com;2962181956@qq.com;hongjiangwei@126.com;cheng_8097@163.com
作者简介:李河林(1998-)，男，云南昆明人，硕士，主要从事动物胚胎生物技术研究，E-mail: sonnyspike@163.com
蒋玉芬(1993-)，女，云南曲靖人，硕士，主要从事动物胚胎生物技术研究, E-mail：2962181956@qq.com
第一联系人：
李河林和蒋玉芬为同等贡献作者
基金资助:
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)

The Study of Regulatory Effect of Differentially Expressed microRNAs on the Npm2 Expression in Pig Oocytes

Helin LI¹^,³(), Yufen JIANG¹^,³(), Na CHENG¹^,³, Yuchen HAN¹^,³, Xiaoying HUO²^,³, Hongding SU³, Yue CHANG¹^,³, Yuzhu FANG²^,³, Pei WANG¹^,³, Baoyu JIA²^,³, Hongjiang WEI²^,³^,*(), Wenmin CHENG¹^,³^,*()

1. College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China
2. College of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, China
3. Yunnan Key Laboratory of Porcine Gene Editing and Xenotransplantation, Kunming 650201, China

Received:2024-03-29 Online:2024-11-23 Published:2024-11-30
Contact: Hongjiang WEI, Wenmin CHENG E-mail:sonnyspike@163.com;2962181956@qq.com;hongjiangwei@126.com;cheng_8097@163.com

摘要/Abstract

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表达影响卵母细胞体外成熟及早期胚胎体外发育，可为猪卵母细胞microRNAs与靶基因复杂调控网络研究提供基础。

关键词: 猪, 卵母细胞, microRNA, Npm2

Abstract:

This study aimed to explore the regulatory effect of differentially expressed microRNAs on Npm2 (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 Npm2. 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 Npm2 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 MⅡ oocytes (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 Npm2, while the fluorescence intensity was significantly down-regulated (P < 0.05). There was no binding site between miR-296-5p, miR-423-3p and Npm2, 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-7138-5p mimics (P < 0.05). The expression of Npm2 mRNA in MⅡ oocytes was significantly higher than that in GV oocytes, and after the addition of the optimal concentration of miR-150, miR-296-3p, miR-296-5p, miR-7138-5p mimics and/or inhibitors, the expression of Npm2 in MⅡ oocytes had significant differences compared with the GV oocytes (P < 0.05). There was no significant difference in the expression of Npm2 mRNA after miR-32 and miR-423-3p mimics/inhibitors in MⅡ oocytes compared with control group (P>0.05). These findings indicated that the screened miRNAs may affect oocyte maturation and early embryo development in vitro by regulating Npm2 expression.

Key words: pig, oocyte, microRNA, Npm2

中图分类号:

S828.3

李河林, 蒋玉芬, 程娜, 韩宇辰, 霍晓颖, 苏宏鼎, 常悦, 方玉珠, 王配, 贾宝瑜, 魏红江, 成文敏. 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[J]. 畜牧兽医学报, 2024, 55(11): 5035-5049.

Helin LI, Yufen JIANG, Na CHENG, Yuchen HAN, Xiaoying HUO, Hongding SU, Yue CHANG, Yuzhu FANG, Pei WANG, Baoyu JIA, Hongjiang WEI, Wenmin CHENG. The Study of Regulatory Effect of Differentially Expressed microRNAs on the Npm2 Expression in Pig Oocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(11): 5035-5049.

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引物名称 Primer name	引物序列(5′→3′) Primer sequence	注释 Exegesis
miR-32-RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCAACT	miR-32反转录引物
miR-32-qPF	AGCCAGCGTATTGCACATTAC	miR-32上游引物
miR-32-qPR	AGTGCAGGGTCCGAGGTATT	miR-32下游引物
miR-150-RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCACTGG	miR-150反转录引物
miR-150-qPF	GCGTCTCCCAACCCTTGTA	miR-150上游引物
miR-150-qPR	AGTGCAGGGTCCGAGGTATT	miR-150下游引物
miR-296-5p-RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAGGA	miR-296-5p反转录引物
miR-296-5p-qPF	AGAGGGCCCCCCCCAA	miR-296-5p上游引物
miR-296-5p-qPR	AGTGCAGGGTCCGAGGTATT	miR-296-5p下游引物
miR-296-3p-RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGAAAG	miR-296-3p反转录引物
miR-296-3p-qPF	AACAATAGGGTTGGGCGGA	miR-296-3p上游引物
miR-296-3p-qPR	GTCGTATCCAGTGCAGGGT	miR-296-3p下游引物
U6-qPF	CTCGCTTCGGCAGCACA	U6上游引物
U6-qPR&RT	AACGCTTCACGAATTTGCGT	U6下游引物/反转录引物
NPM2-qPF	GCTTAGCACGATTTGCCTGG	Npm2上游引物
NPM2-qPR	CCACTGAGGAACACAGGTCC	Npm2下游引物
GAPDH-qPF	AGGGCATCCTGGGCTACACT	GAPDH上游引物
GAPDH-qPR	TCCACCACCCTGTTGCTGTAG	GAPDH下游引物

试剂 Reagent	使用量/μL Usage amount	终浓度 Final concentration
2×miRNA qPCR master mix	10	1×
Forward Primer(10 μmol·L^-1)	0.5	0.25 μmol·L^-1
Reverse Primer(10 μmol·L^-1)	0.5	0.25 μmol·L^-1
DNA模板Template DNA	1~2
ROX Reference Dye(L)/(H)	1
无酶水RNase-free water	up to 20

试剂 Reagent	使用量/μL Usage amount	终浓度 Final concentration
TB Green Premix Ex Taq II(Tli RNaseH Plus)(2×)	12.5	1×
PCR Forward Primer(10 μmol·L^-1)	1	0.4 μmol·L^-1
PCR Reverse Primer(10 μmol·L^-1)	1	0.4 μmol·L^-1
DNA模板Template DNA(< 100 ng)*2	2
无酶水RNase-free water	8.5
总量Total	25

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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 Npm2 Expression in Pig Oocytes

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