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Identification of miR-192 target genes in porcine endometrial epithelial cells based on miRNA pull-down

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Abstract

Introduction

MicroRNAs (miRNAs)—a class of small endogenous non-coding RNAs—are widely involved in post-transcriptional gene regulation of numerous physiological processes. High-throughput sequencing revealed that the miR-192 expression level appeared to be significantly higher in the blood exosomes of sows at early gestation than that in non-pregnant sows. Furthermore, miR-192 was hypothesized to have a regulatory role in embryo implantation; however, the target genes involved in exerting the regulatory function of miR-192 required further elucidation.

Methods

In the present study, potential target genes of miR-192 in porcine endometrial epithelial cells (PEECs) were identified through biotin-labeled miRNA pull-down; functional and pathway enrichment analysis was performed via gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment. Bioinformatic analyses were concurrently used to predict the potential target genes associated with sow embryo implantation. In addition, double luciferase reporter vectors, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), and Western blot were performed to verify the targeting and regulatory roles of the abovementioned target genes.

Results

A total of 1688 differentially expressed mRNAs were identified via miRNA pull-down. Through RT-qPCR, the accuracy of the sequencing data was verified. In the bioinformatics analysis, potential target genes of miR-192 appeared to form a dense inter-regulatory network and regulated multiple signaling pathways, such as metabolic pathways and the PI3K-Akt, MAPKs, and mTOR signaling pathways, that are relevant to the mammalian embryo implantation process. In addition, CSK (C-terminal Src kinase) and YY1 (Yin-Yang-1) were predicted to be potential candidates, and we validated that miR-192 directly targets and suppresses the expression of the CSK and YY1 genes.

Conclusion

We screened 1688 potential target genes of miR-192 were screened, and CSK and YY1 were identified as miR-192 target genes. The outcomes of the present study provide novel insights into the regulatory mechanism of porcine embryo implantation and the identification of miRNA target genes.

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Funding

This research was funded by the National Natural Science Foundation of China (No. 31960645).

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Authors and Affiliations

  1. College of Animal Science and Technology, Shihezi University, 221 North Fourth Road, Shihezi, 832000, China

    Qingchun Li, Ruonan Gao, Su Xie, Xiaomei Sun, Hongbing Gong, Fan He, Yishan Sun, Shihao Lu, Xin Chen, Mengfan Qi, Mingguo Li & Tao Huang

  2. Gembloux Agro-Bio Tech (ULiège-GxABT), University of Liège, Gembloux, Belgium

    Yansen Chen

  3. Key Laboratory of Animal Breeding and Reproduction of Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China

    Su Xie

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  1. Qingchun Li
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Contributions

TH guided this work and reviewed the article. QL finished the article. RG, FH, YS, SL, XC, MQ and ML, help to finish the experiment. YC, SX, and XS collected relevant information. QL modified the figures. The final draft was read and approved by all of the writers.

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Correspondence to Tao Huang.

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Li, Q., Gao, R., Chen, Y. et al. Identification of miR-192 target genes in porcine endometrial epithelial cells based on miRNA pull-down. Mol Biol Rep 50, 4273–4284 (2023). https://doi.org/10.1007/s11033-023-08349-w

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