犬新孢子虫miRNAs的鉴定与分析

doi:10.11843/j.issn.0366-6964.2024.07.027

Abstract

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

CLC Number:

S852.723

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.

Figures/Tables 7

Table 1

Fig. 1

Table 2

Fig. 2

Fig. 3

Fig. 4

Fig. 5

References 22

1	蒋金书. 动物原虫病学[M]. 北京: 中国农业大学出版社, 2000: 294- 295.
	JIANG J S . Veterinary protozoology[M]. Beijing: China Agricultural University Press, 2000: 294- 295.
2	王建中, 张恒, 李晓光, 等. 奶牛犬新孢子虫流产诊疗防[J]. 中国乳业, 2023, (9): 18- 27.
	WANG J Z , ZHANG H , LI X G , et al. The diagnosis and treatment of Neospora caninum-induced abortion in cows[J]. China Dairy, 2023, (9): 18- 27.
3	梁馨一, 葛冰洁, 赵鹏. 抗新孢子虫病药物研究进展[J]. 吉林畜牧兽医, 2020, 41 (11): 11-12, 14. doi: 10.3969/j.issn.1672-2078.2020.11.007
	LIANG X Y , GE B J , ZHAO P . Research progress of anti-neosporiasis drugs[J]. Jilin Animal Husbandry and Veterinary Medicine, 2020, 41 (11): 11-12, 14. doi: 10.3969/j.issn.1672-2078.2020.11.007
4	DUBEY J P , BUXTON D , WOUDA W . Pathogenesis of bovine neosporosis[J]. J Comp Pathol, 2006, 134 (4): 267- 289. doi: 10.1016/j.jcpa.2005.11.004
5	FILIPOWICZ W , BHATTACHARYYA S N , SONENBERG N . Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight?[J]. Nat Rev Genet, 2008, 9 (2): 102- 114. doi: 10.1038/nrg2290
6	CHENG L C , TAVAZOIE M , DOETSCH F . Stem cells: from epigenetics to microRNAs[J]. Neuron, 2005, 46 (3): 363- 367. doi: 10.1016/j.neuron.2005.04.027
7	SUH M R , LEE Y , KIM J Y , et al. Human embryonic stem cells express a unique set of microRNAs[J]. Dev Biol, 2004, 270 (2): 488- 498. doi: 10.1016/j.ydbio.2004.02.019
8	CIMMINO A , CALIN G A , FABBRI M , et al. miR-15 and miR-16 induce apoptosis by targeting BCL2[J]. Proc Natl Acad Sci USA, 2005, 102 (39): 13944- 13949. doi: 10.1073/pnas.0506654102
9	LIU Y S , LIN H Y , LAI S W , et al. MiR-181b modulates EGFR-dependent VCAM-1 expression and monocyte adhesion in glioblastoma[J]. Oncogene, 2017, 36 (35): 5006- 5022. doi: 10.1038/onc.2017.129
10	DE CORREIA M C , GJORGJIEVA M , DOLICKA D , et al. Deciphering miRNAs' action through miRNA editing[J]. Int J Mol Sci, 2019, 20 (24): 6249. doi: 10.3390/ijms20246249
11	HE X , PAN W Q . Host-parasite interactions mediated by cross-species microRNAs[J]. Trends Parasitol, 2022, 38 (6): 478- 488. doi: 10.1016/j.pt.2022.02.011
12	LIU G , JIA L J , SHAO Q Y , et al. MicroRNA profiling of Neospora caninum tachyzoites (NC-1) using a high-throughput approach[J]. Parasitol Res, 2021, 120 (6): 2165- 2174. doi: 10.1007/s00436-021-07155-2
13	陶德良, 赵姗姗, 陈曦, 等. 宿主抗新孢子虫感染的免疫机制研究进展[J]. 动物医学进展, 2022, 43 (12): 104- 108. doi: 10.3969/j.issn.1007-5038.2022.12.019
	TAO D L , ZHAO S S , CHEN X , et al. Progress on immune mechanisms of hosts against infection with Neospora caninum[J]. Progress in Veterinary Medicine, 2022, 43 (12): 104- 108. doi: 10.3969/j.issn.1007-5038.2022.12.019
14	LARSON E T , OJO K K , MURPHY R C , et al. Multiple determinants for selective inhibition of apicomplexan calcium-dependent protein kinase CDPK1[J]. J Med Chem, 2012, 55 (6): 2803- 2810. doi: 10.1021/jm201725v
15	吴亮, 王晓, 苏丹华, 等. 弓形虫入侵的宿主细胞骨架重塑触发线粒体重新分布[J]. 中国人兽共患病学报, 2015, 31 (11): 1001- 1004. doi: 10.3969/j.issn.1002-2694.2015.11.003
	WU L , WANG X , SU D H , et al. Cytoskeleton rearrangement and mitochondria re-distribution of host cells in Toxoplasma gondii invasion[J]. Chinese Journal of Zoonoses, 2015, 31 (11): 1001- 1004. doi: 10.3969/j.issn.1002-2694.2015.11.003
16	MOTA C M , OLIVEIRA A C M , DAVOLI-FERREIRA M , et al. Neospora caninum activates p38 MAPK as an evasion mechanism against innate immunity[J]. Front Microbiol, 2016, 7, 1456. doi: 10.3389/fmicb.2016.01456
17	LI S , GONG P T , ZHANG N , et al. 14-3-3 protein of Neospora caninum modulates host cell innate immunity through the activation of MAPK and NF-κB pathways[J]. Front Microbiol, 2019, 10, 37. doi: 10.3389/fmicb.2019.00037
18	NAQVI A R , SHANGO J , SEAL A , et al. Viral miRNAs alter host cell miRNA profiles and modulate innate immune responses[J]. Front Immunol, 2018, 9, 433. doi: 10.3389/fimmu.2018.00433
19	MENINGHER T , BARSHESHET Y , OFIR-BIRIN Y , et al. Schistosomal extracellular vesicle-enclosed miRNAs modulate host T helper cell differentiation[J]. EMBO Rep, 2020, 21 (1): e47882. doi: 10.15252/embr.201947882
20	TRAN N , RICAFRENTE A , TO J , et al. Fasciola hepatica hijacks host macrophage miRNA machinery to modulate early innate immune responses[J]. Sci Rep, 2021, 11 (1): 6712. doi: 10.1038/s41598-021-86125-1
21	RICCIARDI A , BENNURU S , TARIQ S , et al. Extracellular vesicles released from the filarial parasite Brugia malayi downregulate the host mTOR pathway[J]. PLoS Negl Trop Dis, 2021, 15 (1): e0008884. doi: 10.1371/journal.pntd.0008884
22	BUCK A H , COAKLEY G , SIMBARI F , et al. Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity[J]. Nat Commun, 2014, 5, 5488. doi: 10.1038/ncomms6488

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

[1]	CHEN Huixian, CHEN Yajie, WANG Xianmei, WANG Lifang, LIU Qun, LIU Jing. Identification of the Cross-reacting Antigen MIC17A of Toxoplasma gondii and Neospora caninum and the Study of Its Cross-immune Protective Efficacy in Mice [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(7): 2300-2306.
[2]	YIN Yanling, HUANG Shuang, YAO Qian, WU Jiangping, GUO Haochen, YANG Xin, SONG Junke, ZHAO Guanghui. The Mechanisms of Circular RNA ciRS-7 Affecting the Propagation of Cryptosporidium parvum in HCT-8 Cells via Targeting miR-219a-5p [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 3989-3999.
[3]	ZHANG Ke, YAN Jimeng, LIU Yaowen, LI Honghui, CHEN Qiang, LI Zhuo, SU Yanhua, PU Shaoxia, WANG Haizhen, WEI Hongjiang, JIA Baoyu, CHENG Wenmin. miRNAs Expression Profile of Porcine GV/MⅡ-Stage Oocytes and Screening of miRNAs Related to Npm2 Gene [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(4): 996-1009.
[4]	ZHANG Lihuan, MA Yueyue, LIU Wenyan, LAN Wutao, ZHU Zhiwei. microRNA-96-5p Targets MITF Gene in Alpaca Melanocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(6): 1229-1237.
[5]	WANG Shufang, PAN Yangyang, REN Duanyang, ZHAO Yanyan, LI Baojun, QIAO Liying, LIU Wenzhong. Regulation of SCD1 Gene Expression by miR-200c and miR-429 in Ovine Subcutaneous Adipocytes [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(7): 1347-1357.
[6]	SHEN Peng-lei, QIN Xi-ling, YIN Qian, GUO Zhen-wei, DENG Kai, DU Shan-shan, YIN Na, RUAN Zi-yun, SHI De-shun, LU Feng-hua. Characterization of the Expression of miRNAs in Cumulus Cells during GV/MⅡ Stages of Buffalo Oocyte [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(5): 836-845.
[7]	HAO Jun-fang，HUANG Kai，WANG Yue-ying，DU Li-li，ZHONG Kai. Expression of Inflammation-related miRNAs in the LPS-induced Inflammation in Murine Mammary Epithelial Cells and Construction of miR-223 Eukaryotic Expression Vector [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(1): 183-189.
[8]	CHEN Chen，HU Xiong-gui，ZHU Ji，REN Hui-bo，DENG Yuan，PENG Ying-lin. Progress on the Research of miRNAs Associated with Fat Development in Pigs [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(12): 2117-2126.
[9]	QIAO Junwen;SU Xiaoou;WANG Yiqin;ZHOU Xiangmei;YANG Jianmin;YIN Xiaomin;MA Liying;ZHAO Deming. The Quantitative Research on the mRNA Transcription Level of the Prion Receptor-37kDa/67kDa LRP/LR in Ovine Tissues [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2009, 40(7): 1043-1047.

Identification and Analysis of miRNAs of Neospora caninum

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 7

References 22

Related Articles 9

Recommended Articles

Metrics

Comments