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https://doi.org/10.5713/ab.22.0422    [Accepted] Published online May 4, 2023.
Serum exosomal miR-192 serves as a potential detective biomarker for early pregnancy screening in sows
Ruonan Gao1  , Qingchun Li1  , Meiyu Qiu1,2  , Su Xie1,3  , Xiaomei Sun1  , Tao Huang1,4,* 
1College of Animal Science and Technology, Shihezi University, 221 North Fourth Road, Shihezi 832000, China
2Institute of Biotechnology, Xinjiang Academy of Animal Science, Urumqi, 830000, China
3Key Laboratory of Animal Breeding and Reproduction of Minstry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China
4Xinjiang Pig Breeding Engineering Technology Research Center, Xinjiang Tecon Husbandry S&T Co. Ltd, Changji, 831100, China
Correspondence:  Tao Huang,
Received: 4 November 2022   • Revised: 5 January 2023   • Accepted: 19 April 2023
Abstract
Objective
The study was conducted to screen differentially expressed miRNAs in sows at early pregnancy by high-throughput sequencing and explore its mechanism of action on embryo implantation.
Methods
The blood serum of pregnant and non-pregnant Landrace×Yorkshire sows were collected 14 days after artificial insemination, and exosomal miRNAs were purified for high throughput miRNA sequencing. The expression patterns of 10 differentially expressed (DE) miRNAs were validated by qRT-PCR. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) quantified the abundance of serum exosomal miR-192 in pregnant and control sows, and the diagnostic power was assessed by receiver operating characteristic (ROC) analysis. The target genes of DE miRNAs were predicted with bioinformatics software, and the functional and pathway enrichment analysis was performed on Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) terms. Furthermore, a luciferase reporter system was used to identify the target relation between miR-192 and integrin alpha 4 (ITGA4), a gene influencing embryo implantation in pigs. Finally, the expression levels of miRNAs and the target gene ITGA4 were analyzed by qRT-PCR, and western blot, with the proliferation of BeWo cells detected by CCK-8.
Results
A total of 221 known miRNAs were detected in the libraries of the pregnant and non-pregnant sows, of which 55 were up-regulated and 67 were down-regulated in the pregnant individuals compared with the non-pregnant controls. From these, the expression patterns of 10 DE miRNAs were validated. The qRT-PCR analysis further confirmed a significantly higher expression of miR-192 in the serum exosomes extracted from pregnant sows, when compared to controls. ROC analysis revealed that miR-192 provided excellent diagnostic accuracy for pregnancy (area under the ROC curve [AUC] = 0.843; P>0.001). The dual-luciferase reporter assay indicated that miR-192 directly targeted ITGA4. The protein expression of ITGA4 was reduced in cells that overexpressed miR-192. Overexpression of miR-192 resulted in the decreased proliferation of BeWo cells and regulated the expression of cell cycle-related genes.
Conclusions
Serum exosomal miR-192 could serve as a potential biomarker for early pregnancy in pigs. miR-192 targeted ITGA4 gene directly, and miR-192 can regulate cellular proliferation.
Keywords: Biomarkers; Early pregnancy; Exosomal miRNAs; ITGA4; miR-192
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