Analysis of microRNA expression profiles during the differentiation of chicken embryonic stem cells into male germ cells

Abstract

The differentiation of embryonic stem cells (ESCs) into germ cells in vitro could have very promising applications for infertility treatment and could provide an excellent model for uncovering the molecular mechanisms of germline generation. This study aimed to investigate the differentially expressed miRNAs (DEMs) during the differentiation of chicken ESCs (cESCs) into male germ cells and to establish a profile of the DEMs. Cells before and after induction were subjected to miRNA sequencing (miRNA-seq). A total of 113 DEMs were obtained, including 61 upregulated and 52 downregulated DEMs. GO and KEGG enrichment analyses showed that the target genes were enriched mainly in the MAPK signaling pathway, HTLV infection signaling pathway, cell adhesion molecule (CAM)-related pathways, viral myocarditis, Wnt signaling pathway, ABC transporters, TGF-β signaling pathways, Notch signaling pathways and insulin signaling pathway. The target genes of the miRNAs were related to cell binding, cell parts and biological regulatory processes. Six DEMs, let-7k-5p, miR-132c-5p, miR-193a-5p, miR-202-5p, miR-383-5p and miR-6553-3p, were assessed by qRT-PCR, and the results were consistent with the results of miRNA-seq. Based on qRT-PCR and western blot verification, miR-383-5p and its putative target gene STRN3 were selected to construct an STRN3 3'-UTR dual-luciferase gene reporter vector and its mutant vector. The double luciferase reporter activity of the cotransfected STRN3-WT + miR-383-5p mimics group was significantly lower (by approximately 46%) than that of the other five groups (p < 0.01). There was no significant difference in luciferase activity among the other 5 groups. This study establishes a DEM profile during the process of cESC differentiation into male germ cells; illustrates the mechanisms by which miRNAs regulate target genes; provides a theoretical basis for further research on the mechanisms of the formation and regulation of male germ cells; and provides an important strategy for gene editing, animal genetic resource protection and transgenic animal production.

Keywords:

• Chicken embryonic stem cells
• male germ cells
• microRNA
• dual-luciferase reporter vector
• STRN3

Author contributions

Y.Y. and Z.Z. conceived and designed the experiments. H.J. provided financial support. X.L. and Y.L. performed the experiments. X.L. and Q.Z. analyzed the data. C.L. and J.L. supplied fresh fertilized eggs to separate the ESCs.

Acknowledgements

We would like to thank the Animal Husbandry and Veterinary Institute for providing the laboratory materials.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by grants from the Basic Research Funds of Jilin Academy of Agricultural Sciences (KYJF2021ZR007).