犬新孢子虫miRNAs的鉴定与分析

doi:10.11843/j.issn.0366-6964.2024.07.027

摘要/Abstract

摘要：

为鉴定犬新孢子虫(Neospora caninum)特异表达的miRNAs，并研究其在宿主细胞中的表达情况，本研究收集N. caninum速殖子感染的山羊子宫内膜上皮细胞(caprine endometrial epithelial cells，EECs)24和48 h的样本，用Illumina高通量测序平台和生物信息学分析犬新孢子虫特异表达的miRNAs及其体外表达情况，并研究了一个上调的miRNA novel3_mature对犬新孢子虫在山羊EECs增殖的影响。结果发现，共鉴定出3 404个犬新孢子虫特异表达的miRNAs(155个新预测miRNAs)，其中77个(47个上调和30个下调)miRNAs显著差异表达；随机选取7个显著差异表达的miRNAs进行实时荧光定量PCR分析，结果与高通量测序结果一致；预测分析发现61个miRNAs的3 978个靶基因，功能富集分析发现这些靶基因可能主要参与MAPK信号通路、突触导向、黏着斑、cMAP信号通路、钙信号通路等；转染novel3_mature的模拟物显著促进犬新孢子虫在山羊EECs中的增殖。犬新孢子虫存在多个特异表达的miRNAs，其在虫体增殖中发挥重要作用，研究成果为深入理解犬新孢子虫胞内生存提供了新的思路。

关键词: 犬新孢子虫, 山羊子宫内膜上皮细胞, miRNAs, novel3_mature, 增殖

Abstract:

To identify specific expressed miRNAs of Neospora caninum and to investigate their expression in host cells, the samples of caprine endometrial epithelial cells (EECs) infected with tachyzoites of N. caninum at 24 and 48 h were collected. Specific expressed miRNAs of N. caninum and their in vitro expression were analyzed by using Illumina highthroughput sequencing technology and bioinformatics analysis. The effect of an up-regulated miRNA, novel3_mature, on the propagation of N. caninum in caprine EECs was also studied. The results showed that a total of 3 404 specific expressed miRNAs (155 novel predicted miRNAs) of N. caninum were identified. Of them, 77 (47 up- and 30 down-regulated) miRNAs were significantly differentially expressed. The consistent results were found with highthroughput sequencing by using quantitative real-time PCR analysis of seven significantly differentially expressed miRNAs randomly selected. Predictive analysis found 3 978 target genes of 61 miRNAs. Functional enrichment analysis showed that these target genes were possible involved in MAPK signaling pathway, axon guidance, focal adhesion, cAMP signaling pathway, calcium signaling pathway. Transfection of novel3_mature mimics significantly promoted the propagation of N. caninum in caprine EECs. Many specific expressed miRNAs were encoded by N. caninum and they played important roles in the propagation of N. caninum, providing a novel way to understand the intracellular survival of N. caninum.

Key words: Neospora caninum, caprine endometrial epithelial cells, miRNAs, novel3_mature, propagation

中图分类号:

S852.723

李京宇, 陈金铭, 张明一, 赵姗姗, 陶德良, 宋军科, 杨新, 樊莹莹, 赵光辉. 犬新孢子虫miRNAs的鉴定与分析[J]. 畜牧兽医学报, 2024, 55(7): 3085-3093.

Jingyu LI, Jinming CHEN, Mingyi ZHANG, Shanshan ZHAO, Deliang TAO, Junke SONG, Xin YANG, Yingying FAN, Guanghui ZHAO. Identification and Analysis of miRNAs of Neospora caninum[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(7): 3085-3093.

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引物名称 Primer name	序列(5′→ 3′) Sequence
cfa-miR-219-3p	AGAATTGTGGCTGGACATCTGT
novel105_mature	AGAGGGATGTTTAGCCGGGAAGT
novel114_mature	AGGACGAGAGGACTGTGCCTT
novel97_mature	ATTATGCAAGGCCGACGGG
pma-miR-29d-3p	CGTAGCACCATATGAAATCGGTGTC
sha-miR-24	TGGCTCAGTTCAGCAGGAA
aca-let-7f-1-3p	CGCGGCTATACAATCTACTGTCTTT
u6-R	GGAACGATACAGAGAAGATTAGC
u6-F	TGGAACGCTTCACGAATTTGCG

miRNA名称 miRNA name	序列(5′→ 3′) Sequence
novel1_mature	ATATACTACGAGTTGGACTACTGGA
novel100_mature	AGAGACACTCACGAGCCT
novel105_mature	AGAGGGATGTTTAGCCGGGAAGTC
novel114_mature	AGGACGAGAGGACTGTGCCTTCTT
novel119_mature	GGCCAGGATGAAACAAAAGAATCT
novel12_mature	GTGTCGGAGCCCGTAAGCC
novel136_mature	AAGCTAGTGAACGCCATGACCG
novel2_mature	ACATCGACAGCGGCTGCTACTCGC
novel21_mature	CGGGAGGATGAGAAGCATCTT
novel3_mature	GCGCATGCGCGTCAAGCCCTTCCT
novel51_mature	ACGTGGCTGCCGGGAATCTGCT
novel57_mature	TGAGATGAGGAAAGAGAAGTCCGAG
novel73_mature	ACACGTGCCGGAGACACTCGTTCC
novel81_mature	ACGGGACTGGAGCCCAAGGCGT
novel82_mature	GACACCGGTCCGGCGGATGGGCT
novel93_mature	ACGCGTGCATTTGTCAGACT
novel97_mature	GCAAGGCCGACGGGGCTGTCTCT

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