Mouse double minute homologue 2 (MDM2) downregulation by miR-661 impairs human endometrial epithelial cell adhesive capacity

Abstract

Human blastocysts that fail to implant following IVF secrete elevated levels of miR-661, which is taken up by primary human endometrial epithelial cells (HEECs) and impairs their adhesive capability. MicroRNA miR-661 downregulates mouse double minute homologue 2 (MDM2) and MDM4 in other epithelial cell types to activate p53; however, this has not been examined in the endometrium. In this study MDM2 protein was detected in the luminal epithelium of the endometrium, the site of blastocyst attachment, during the mid secretory receptive phase of the menstrual cycle. The effects of miR-661 on gene expression in and adhesion of endometrial cells was also examined. MiR-661 overexpression consistently downregulated MDM2 but not MDM4 or p53 gene expression in the Ishikawa endometrial epithelial cell line and primary HEEC. Adhesion assays were performed on the real-time monitoring xCELLigence system and by co-culture using Ishikawa cells and HEECs with HTR8/SVneo trophoblast spheroids. Targeted siRNA-mediated knockdown of MDM2 in endometrial epithelial cells reduced Ishikawa cell adhesion (P<0.001) and also reduced HTR8/SVneo trophoblast spheroid adhesion to Ishikawa cells (P<0.05) and HEECs (P<0.05). MDM2 overexpression using recombinant protein treatment resulted in enhanced HTR8/SVneo trophoblast spheroid adhesion to Ishikawa cells (P<0.01) and HEECs (P<0.05). This study highlights a potential new mechanism by which human blastocyst-secreted miR-661 reduces endometrial epithelial cell adhesion; via downregulation of MDM2. These findings suggest that MDM2 contributes to endometrial-blastocyst adhesion, implantation and infertility in women.