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

Abstract

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

CLC Number:

S828.3

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.

Figures/Tables 9

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

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