In-Depth Analysis of miRNA Binding Sites Reveals the Complex Response of Uterine Epithelium to miR-26a-5p and miR-125b-5p During Early Pregnancy

Posttranscriptional regulation of gene expression by miRNAs likely makes significant contributions to mRNA abundance at the embryo-maternal interface. In this study, we investigated how miR-26a-5p and miR-125b-5p contribute to molecular changes occurring in the uterine luminal epithelium, which serves as the first site of signal exchange between the mother and the developing embryo. To measure de novo protein synthesis after miRNA delivery to primary uterine luminal epithelial cells, we used pulsed stable isotope labeling by amino acids (pSILACs). We found that both miRNAs alter the proteome of luminal epithelial cells, impacting numerous cellular functions, immune responses, as well as intracellular and second messenger signaling pathways. Additionally, we identified several features of miRNA-mRNA interactions that may influence the targeting efficiency of miR-26a-5p and miR-125b-5p. Overall, our study suggests a complex interaction of miR-26a-5p and miR-125b-5p with their respective targets. However, both appear to cooperatively function in modulating the cellular environment of the luminal epithelium, facilitating the morphological and molecular changes that occur during the intensive communication between the embryo and uterus at pregnancy. [Display omitted] •miR-26a-5p and miR-125b-5p influence protein synthesis in the uterine luminal epithelial cells.•Target pairing for miR-26a-5p and miR-125b-5p depends on multiple factors.•3′-supplementary sites may enhance miR-125b-5p targeting efficiency.•bindseek enables miRNA-mRNA pairing identification in nonmodel species. This study explores how miR-26a-5p and miR-125b-5p contribute to molecular changes in the uterine luminal epithelium, the primary site of communication between the mother and embryo. By measuring de novo protein synthesis following the delivery of these miRNAs to primary uterine luminal epithelial cells, we discovered that both miRNAs significantly alter the proteome, potentially influencing cellular, immune, and signaling pathways. Our findings indicate that these miRNAs cooperatively modulate the luminal epithelium, promoting essential changes for successful embryo implantation.