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Effects of miR-214-5p and miR-21-5p in hypoxic endometrial epithelial-cell-derived exosomes on human umbilical cord mesenchymal stem cells

sarah Jonas

BACKGROUND

Thin endometrium seriously affects endometrial receptivity, resulting in a significant reduction in embryo implantation, and clinical pregnancy and live birth rates, and there is no gold standard for treatment. The main pathophysiological characteristics of thin endometrium are increased uterine arterial blood flow resistance, angiodysplasia, slow growth of the glandular epithelium, and low expression of vascular endothelial growth factor, resulting in endometrial epithelial cell (EEC) hypoxia and endometrial tissue aplasia. Human umbilical cord mesenchymal stem cells (HucMSCs) promote repair and regeneration of damaged endometrium by secreting microRNA (miRNA)-carrying exosomes. However, the initiation mechanism of HucMSCs to repair thin endometrium has not yet been clarified.

AIM

To determine the role of hypoxic-EEC-derived exosomes in function of HucMSCs and explore the potential mechanism.

METHODS

Exosomes were isolated from normal EECs (EEC-exs) and hypoxia-damaged EECs (EECD-exs), before characterization using Western blotting, nanoparticle-tracking analysis, and transmission electron microscopy. HucMSCs were cocultured with EEC-exs or EECD-exs and differentially expressed miRNAs were determined using sequencing. MiR-21-5p or miR-214-5p inhibitors or miR-21-3p or miR-214-5p mimics were transfected into HucMSCs and treated with a signal transducer and activator of transcription 3 (STAT3) activator or STAT3 inhibitor. HucMSC migration was assessed by Transwell and wound healing assays. Differentiation of HucMSCs into EECs was assessed by detecting markers of stromal lineage (Vimentin and CD13) and epithelial cell lineage (CK19 and CD9) using Western blotting and immunofluorescence. The binding of the miRNAs to potential targets was validated by dual-luciferase reporter assay.

RESULTS

MiR-21-5p and miR-214-5p were lowly expressed in EECD-ex-pretreated HucMSCs. MiR-214-5p and miR-21-5p inhibitors facilitated the migratory and differentiative potentials of HucMSCs. MiR-21-5p and miR-214-5p targeted STAT3 and protein inhibitor of activated STAT3, respectively, and negatively regulated phospho-STAT3. MiR-21-5p- and miR-214-5p-inhibitor-induced promotive effects on HucMSC function were reversed by STAT3 inhibition. MiR-21-5p and miR-214-5p overexpression repressed HucMSC migration and differentiation, while STAT3 activation reversed these effects.

CONCLUSION

Low expression of miR-21-5p/miR-214-5p in hypoxic-EEC-derived exosomes promotes migration and differentiation of HucMSCs into EECs via STAT3 signaling. Exosomal miR-214-5p/miR-21-5p may function as valuable targets for thin endometrium.

Key Words: Endometrial epithelial cells; Exosomes; Human umbilical cord mesenchymal stem cells; MiR-214-5p/miR-21-5p; Signal transducer and activator of transcription 3

 

Core Tip: Thin endometrium is a primary cause of repeated implantation failure and infertility. In this study, with the help of microRNA sequencing, we investigated the role of hypoxic endometrial epithelial cell (EEC)-derived exosomes in cell function of human umbilical cord mesenchymal stem cells and explored the potential mechanism. Eventually, we found that lowly-expressed miR-21-5p and miR-214-5p in hypoxic EEC-derived exosomes promoted migration and differentiation of human umbilical cord mesenchymal stem cells into EECs via signal transducer and activator of transcription 3 signaling. Exosomal miR-214-5p and miR-21-5p may function as valuable targets for thin endometrium.