Investigation on the hCG-miR-125a-3p-Wnt axis through CTNNBIP1 in regulating endometrial receptivity and spheroid attachment

Abstract

Successful embryo implantation (EI) is a prerequisite to establish pregnancy. During embryo implantation, the blastocyst secretes human chorionic gonadotropin (hCG) that has been known to rescue corpus luteum, but its roles on EI remains elusive. Studies from this and other laboratory suggested that hCG regulates the expression of miR-125a-3p through the binding of LH/CG receptor (LHCGR) in the granulosa cells. MiR-125a-3p microRNA (miRNA) is a small single-stranded non-coding RNA molecule that is involved in oocyte maturation, and our recent finding suggested that miR-125a-3p binds to the 3’UTR of catenin beta-interacting protein 1 (CTNNBIP1) mRNA for RNA silencing and post-transcriptional regulation of gene expression. Importantly, CTNNBIP1 is an inhibitor of the Wnt signaling pathway that play important roles on EI. However, the exact role of CTNNBIP1 on EI remains elusive. Yet, the Wnt signaling pathway is involved in many aspects of reproduction and fertility, including endometrial receptivity, EI, and placental development. This study aims to investigate the role of hCG on the expression of miR-125a-3p and the downstream target CTNNBIP1 on the regulation of Wnt-signaling molecules to spheroid attachment (mimicking EI process) using receptive (Ishikawa and RL95-2) and non-receptive (HEC1-B and AN3CA) human endometrial epithelial cells. MiRNA precursor and/or inhibitor were used to confirm the effect on spheroid attachment. CTNNBIP1 KO Ishikawa cells were established and the spheroid attachment rates are evaluated after 24 h hCG treatment. It was found that the LHCGR transcript was detectable in receptive but not non-receptive human endometrial epithelial cell lines. HCG was found to induce miR-125a-3p and active--catenin to total -catenin expression in the receptive Ishikawa cells, and no change on CTNNBIP1 expression was found in all 4 cell lines. Interestingly, hCG significantly induce spheroid attachment in non-receptive cell lines (HEC1-B and AN3CA), whereas no further increase in the spheroid attachment in receptive cell lines. Transfection of miR-125a-3p precursor and inhibitor, significantly decrease and increase CTNNBIP1 mRNA, respectively, but no change in CTNNBIP1 protein expression was found. Neither the miR-125a-3p precursor nor inhibitor or CTNNBIP1 siRNA significantly affected total β-catenin expression. The transfection of miR-125a-3p precursor and inhibitor significantly increased and decreased the spheroid attachment rate onto Ishikawa cells. The CTNNBIP1 knockout cell lines were established using the Crispr/Cas9 system, and the Sanger sequences showed a770bp deletion of the CTNNBIP1 gene when compared to the wild-type Ishikawa cells. The attachment rate was significantly increased in the CTNNBIP1 knockout cells. However, the knockout cells re-express CTNNBIP1 protein upon continuous passage. In conclusion, hCG induces mir-125a-3p expression in the Ishikawa cells which may inhibit the suppressive effect of CTNNBIP1 on Wnt-signaling, the activated Wnt-signaling pathway increases active β-catenin expression and favor spheroid attachment. Although we have established the CTNNBIP1 KO cells, the stability of the cell line changes upon culture. A more in-depth investigation is needed to delineate the hCG-miR-125a-3p-Wnt axis on EI.