One of the essential periods of embryonic development and morphological transformation in pigs is the peri-implantation period, occurring on gestation days (D) 9, D12, and D15. circular RNAs (circRNAs), a non-coding RNA (ncRNA) family member, are suspected to play key roles in numerous biological processes. However, the role of circRNAs during early pregnancy in pigs has not yet been elucidated. To address this, we integrated RNA-seq, bioinformatics analyses, and molecular biology experiments to define the circRNA expression landscape in embryos at gestational D9, D12, and D15. A total of 6,931 circRNAs were identified, with 183, 634, and 82 differentially expressed circRNAs (DEcircRNAs) found in the differential expression analyses of the three sample pairs (D9 vs. D12, D9 vs. D15, and D12 vs. D15). Functional enrichment analysis revealed significant associations with multiple biological processes and pathways relevant to embryonic development. The intersection of these differential expression analyses yielded seven hub circRNAs. By integrating porcine miRNAs from miRBase with mRNA sequencing data, a competing endogenous RNA (ceRNA) network comprising two circRNAs, seven mRNAs, and 166 miRNAs was constructed, suggesting a potential role for circRNAs as molecular sponges that may sequester miRNAs and thereby influence mRNA expression and related biological processes. To validate the back-spliced circular structure of the circRNAs, polymerase chain reaction (PCR) amplification and Sanger sequencing were performed. Additionally, three circRNAs were randomly selected for real-time quantitative PCR (RT-qPCR) validation, and their relative expression levels were consistent with sequencing results. This study not only characterized the circRNA expression profile of pig embryos on D9, D12, and D15 of pregnancy but also established a solid foundation for further exploring the molecular mechanisms of key circRNAs in pig embryo development, thereby providing insights into the processes of implantation and embryo survival.
Gu et al. (Thu,) studied this question.