Exosomes Regulate Epithelial Mesenchymal Transition of Endometrial Cells in Mice with Recurrent Spontaneous Abortion

Background Recurrent spontaneous abortion (RSA) is a common reproductive disorder closely linked with the weakened epithelial-mesenchymal transition (EMT) in the endometrium during embryo implantation. Endometrium-derived exosomes and miRNAs they transport are linked with RSA via the involvement in cellular communication and EMT, although the specific mechanisms have not yet fully revealed. Objective Focusing on the function of exosomes in EMT, this study aims to validate the regulatory role of endometrium-derived exosomes and miRNA-221 they transport in EMT of endometrial cells in RSA mice. Methods From October 2021 to May 2022, a total of 24 eight-week-old female mice in the specific pathogen-free (SPF) level were randomly assigned into the control group (n=12) and RSA group (n=12) for modeling. After RSA modeling, male and female mice were housed together. On the 4th day of pregnancy, female mice were sacrificed for isolating endometrial epithelial cells. They were cultured for extracting exosomes, which were identified by transmission electron microscopy and Western blotting. Cell proliferation, invasion, and migration abilities were evaluated using CCK-8 assay, Transwell assay, and fluorescence-activated cell sorting (FACS) before and after exosome interventions. Expression levels of EMT-related genes [E-cadherin, N-cadherin, Vimentin, matrix metalloproteinase 7 (MMP-7), matrix metalloproteinase 9 (MMP-9) ] and exosome marker proteins [CD63, CD81, tumor susceptibility gene 101 (TSG101) ] were measured. Expression levels of EMT marker proteins (CD24-APC, CD44- FITC) in exosome-treated cells were analyzed. Transfection of miRNA-221 mimics and inhibitors was performed to analyze the effects of exosome-derived miRNA-221 on EMT of endometrial epithelial cells in RSA mice. Relevant analyses were conducted using Western blot and dual-luciferase reporter assay. Results Abnormal EMT was observed in the endometrial epithelial cells of mice in RSA group, and the mouse endometrial epithelial cells were able to secrete exosomes. The proliferation rate, migration ability, invasion ability, and CD44+/CD24+ ratio of cells in the RSA group treated with exosomes were significantly higher than those measured in cell supernatant of RSA group without exosome intervention (P<0.05). Compared with those in the cell supernatant of RSA group, induction of exosomes significantly downregulated EMT marker protein E-cadherin, but upregulated stromal marker proteins N-cadherin and Vimentin, as well as matrix marker proteins MMP-7 and MMP-9 (P<0.05). The expression level of miRNA-221 in the RSA group was significantly lower than that of the control group (P<0.05). Compared with those transfected with miRNA negative control, transfection of miRNA-221 mimic significantly activated the phosphatidylinositol 3-kinase (PI3K) -AKT pathway, nuclear factor-kappa B (NF-κB) pathway, and p38 pathway (P<0.05), but downregulated mRNA level of PTEN (P<0.05). Transfection of miRNA-221 inhibitor significantly upregulated mRNA level of PTEN (P<0.05) . Conclusion Endometrium-derived exosomal miRNA-221 negatively regulates the PTEN gene, activates the PI3K-AKT pathway, promotes EMT in endometrial epithelial cells, and influences the pregnant outcome of RSA mice.