HCG regulates miR-125a-3p-CTNNBIP1-Wnt signaling pathway in human endometrial epithelial cells and in patients with repeated implantation failure

Abstract

Successful embryo implantation requires a receptive endometrium for embryo adhesion and invasion, while impaired endometrial receptivity may lead to implantation failure. Repeated implantation failure (RIF) occurs in 5-10% of women who have gone through 2-3 unsuccessful IVF treatment cycles when each cycle 4 good quality cleavage stage embryos or 2 blastocysts were transferred. The causes of RIF are diverse including multifactorial, endometrial and embryonic factors. Recent studies suggested RIF patients with impaired endometrial receptivity is associated with dysregulated microRNAs (miRNAs) and genes expressions. Previous studies from this laboratory demonstrated that human chorionic gonadotropin (hCG) stimulates miR-125a-3p expression in granulosa cells, and in silico analysis further identified miR-125a-3p regulates CTNNBIP1 and downstream Wnt signaling pathway related to endometrial receptivity. Whether embryo-derived hCG modulate endometrial miRNA expression and downstream Wnt signaling pathway in regulating endometrial receptivity remains largely unknown. Therefore, it is hypothesized that hCG regulates miR-125a-3p and affects CTNNBIP1 and Wnt signaling pathway in endometrial epithelial cells that favor embryo implantation, while dysregulation of miR-125a-3p-CTNNBIP1-Wnt signaling pathway contributed to RIF. Small RNA-sequencing of endometrial aspirate from RIF and non-RIF patients identified 87 differentially expressed miRNAs. KEGG analysis showed that Wnt signaling pathway was enriched and 105 Wnt-related genes potentially regulated by 61 out of the 87 miRNAs were identified among RIF and non-RIF patients. MiRNAs-mRNAs and protein-protein interaction network analysis revealed that many Wnt signaling pathway molecules are regulated by miR-125a-3p and CTNNBIP1. In human endometrial aspirate taken at mid-secretory phase of the cycle, the expression of miR-125a-3p was upregulated while CTNNBIP1 was downregulated. Interestingly, a lower miR-125a-3p and a higher CTNNBIP1 expression were found in the endometrium of RIF patients when compared with non-RIF patients. Moreover, the downstream Wnt signaling molecule, active β-catenin was found to be significantly downregulated in RIF patients. Furthermore, significant downregulation of endometrial estrogen receptor alpha, progesterone receptor and luteinizing hormone/chorionic gonadotropin receptor were found in RIF when compared to non-RIF patients. Using human endometrial epithelial cells, miR-125a-3p was found to be significantly higher in receptive Ishikawa than non-receptive HEC1-B cells in vitro. HCG dose-dependently upregulated miR-125a-3p in Ishikawa cells, but not for HEC1-B cells. Forced expression of miR-125a-3p by precursor significantly downregulated CTNNBIP1, while knockdown of miR-125a-3p by inhibitor significantly upregulated CTNNBIP1 expression in Ishikawa cells. Moreover, hCG stimulated active β-catenin and inhibited CTNNBIP1 expression in Ishikawa cells. Similarly, active β-catenin was significantly upregulated by miR-125a-3p precursor, but significantly downregulated by miR-125a-3p inhibitor in Ishikawa cells. Luciferase assay showed that miR-125a-3p inhibitor nullified hCG induced Wnt signaling activation in transfected Ishikawa cells. Functionally, forced expression of miR-125-3p by precursor or suppression of CTNNBIP1 by siRNA enhanced Jeg-3 spheroid (blastocyst surrogate) attachment onto Ishikawa cells. Taken together, hCG activates Wnt signaling pathway via elevated miR-125a-3p and suppressed CTNNBIP1 to promote embryo attachment, while dysregulation of miR-125a-3p and CTNNBIP is associated with impaired embryo implantation in RIF patients. Modulation of miR-125a-3p-CTNNBIP1-Wnt signaling pathway may open a new diagnostic and treatment regimen for patient with RIF. (Words count 488)