Exosomes derived from mesenchymal stem cells increase the viability of damaged endometrial cells via the miR-99b-5p/PCSK9 axis.

Abstract

To investigate whether exosomes derived from bone marrow mesenchymal stem cells repair damaged endometrial stromal cells through the miR-99b-5p/PCSK9 axis. Exosomes derived from BMSCs (BMSC-exos) were isolated by ultracentrifugation and characterized using transmission electron microscopy and nanoflow cytometry. A mifepristone-induced EnSC injury model was established in vitro, and the uptake of BMSC-exos assessed. EnSCs were divided into three groups: the normal group (ctrl), EnSC injury group (model) and BMSC-exo treatment group. The effects of BMSC-exos on EnSC proliferation, apoptosis, and vascular endothelial growth factor (VEGF) expression were assessed by coculturing MSC-exos with endometrial cells. Furthermore, high-throughput sequencing was used to identify differentially expressed genes (DEGs). Through bioinformatics analysis, RT‒qPCR, western blotting, the CCK8 assay, immunohistochemistry and dual luciferase experiments, the potential mechanism by which BMSC-exos derived miRNAs repair EnSC injury was studied. BMSC-exos expressed the marker proteins CD9 and CD63. Laser confocal microscopy showed that BMSC-exos could enter damaged EnSCs. In the BMSC-exos-EnSC coculture group compared with the model group, BMSC-exos significantly increased the proliferation of damaged EnSC and inhibited cell apoptosis in a dose-dependent manner. The expression levels of Caspase-3, Caspase-9, Bax and VEGF mRNA were significantly downregulated in the BMSC-exos-EnSC coculture group, while Bcl-2 expression was upregulated. We identified 28 overlapping DEGs between the model and ctrl groups, and between the BMSC-exo and model groups. Transfection with miR-99b-5p mimics significantly decreased PCSK9 gene expression and inhibited the expression of the autophagy related proteins Beclin-1 and LC3-II/I and apoptosis, thereby promoting EnSC proliferation. Transfection with a miR-99b-5p inhibitor showed the opposite effects. Beclin-1, LC3-II/I and PCSK9 expression in the thin endometrium was significantly increased. miR-99b-5p promoted cell proliferation by targeting PCSK9. BMSC-exos promoted endometrial proliferation, and miR-99b-5p inhibited cell apoptosis and promoted EnSC proliferation by targeting PCSK9, providing a new target for the treatment of thin endometrium.