MicroRNA-212 regulates endometrial receptivity on spheroid attachment through down-regulation of olfactomedin 1 and C-terminal binding protein 1 expressions

Abstract

Embryo implantation is one of the most critical events for successful human reproduction. In human, it occurs between days 20 to 24 (as known as window of implantation, WOI) of the menstrual cycle and requires a receptive endometrium that cross-talks with the competent blastocyst for embryo apposition, attachment and invasion.

The receptivity of human endometrium is mainly regulated by maternal steroid hormones and possibly embryo-derived factor(s). In the presence of progesterone, the estrogen-primed human endometrium undergoes extensive secretory transformation to produce various factors/cytokines for embryo implantation. The receptive endometrium is further supported by human Chorionic Gonadotropin (hCG), an embryo derived protein that has been shown to modulate endometrial microenvironment for embryo implantation.

MicroRNAs (MiRNAs) are ~ 18-22 nucleotide-long non-coding RNA molecules that bind to their target mRNAs at the 3’-untranslated region (UTR) and regulate gene expression resulting in translational inhibition or transcript degradation. Recent studies demonstrated that hCG up-regulated miR-212 and miR-132 expressions and miRNAs in turn down-regulated the expression of olfactomedin 1 (Olfm1) and C-terminal binding protein 1 (Ctbp1) in mouse ovaries. Whether similar regulatory mechanism exists in human endometrial cells to regulate endometrial receptivity remains largely unknown. I therefore, hypothesized that embryo-derived hCG induces endometrial receptivity through miRNA-212 and miR-132 mediated down-regulation of OLFM1 and CTBP1 expression during embryo implantation.

In-silico analysis revealed the 3’UTR of OLFM1 and CTBP1 contain the seeding sites for miR-212 and miR-132. Dual Luciferase Reporter (DLR) assay of miR-212 and miR-132 on the wild-type (WT) OLFM1 and CTBP1 3’UTR reporter constructs (WT-OLFM1 and WT-CTBP1) and the mutated (MT) constructs (MT-OLFM1 and MT-CTBP1) were performed. The effects of hCG and progesterone on the expression on miRNA (miRNA-212 and miRNA-132), mRNA and protein (OLFM1 and CTBP1) expressions were studied using endometrial epithelial cells (Ishikawa) and Fallopian tubal epithelial cells (OE-E6/E7). Spheroid attachment assays were performed by coculturing spheroids of human choriocarcinoma Jeg-3 (blastocyst surrogates) with monolayers of Ishikawa or OE-E6/E7 cells.

The DLR assay performed in HeLa cells co-transfected with the WT or MT 3’UTR reporter constructs of OLFM1 and CTBP1, and either the precursor or inhibitor of miRNA-212/miRNA-132 confirmed the regulatory role of miR-212 on OLFM1 and CTBP1 expressions. HCG (25 IU/ml) significantly increased the expression of miRNA-212 and miRNA-132 but decreased OLFM1 (mRNA and protein), and CTBP1 (protein) expressions in Ishikawa and OE-E6/E7 cells. Progesterone (0.6, 6, 60 nM) did not change the expression of miRNA-212 or miRNA-132 in Ishikawa cells, but at 60 nM, it significantly down-regulated OLFM1 and CTBP1 protein expressions. HCG (25 IU/ml), as well as miR-212 precursor over-expression in Ishikawa cells significantly increased Jeg-3 spheroid attachment rate. MiR-212 precursor transfection significantly down-regulated OLFM1 and CTBP1 protein expression in the Ishikawa cells.

Taken together, embryo-derived hCG induces endometrial receptivity through down-regulation of OLFM1 and CTBP1 expression by miR-212 and enhances spheroid attachment. Results from this study shed light on the role of embryo-derived factor in regulating endometrial receptivity and embryo implantation. Whether similar regulation occurs in vivo or with human embryos remains to be investigated.