DSpace Collection:http://hdl.handle.net/10722/386192024-04-10T21:20:33Z2024-04-10T21:20:33ZInvestigation on the hCG-miR-125a-3p-Wnt axis through CTNNBIP1 in regulating endometrial receptivity and spheroid attachmentHe, Xiaoxue何晓雪http://hdl.handle.net/10722/3415882024-03-18T09:56:12Z2023-01-01T00:00:00ZTitle: Investigation on the hCG-miR-125a-3p-Wnt axis through CTNNBIP1 in regulating endometrial receptivity and spheroid attachment
Authors: He, Xiaoxue; 何晓雪
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.2023-01-01T00:00:00ZHuman stem cell derived embryo surrogates for human implantation studyRuan, Hanzhang阮含章http://hdl.handle.net/10722/3415442024-03-18T09:55:48Z2023-01-01T00:00:00ZTitle: Human stem cell derived embryo surrogates for human implantation study
Authors: Ruan, Hanzhang; 阮含章
Abstract: Implantation failure is one of the major causes leading to failed pregnancy and low success rates of assisted reproductive technology treatments. However, there is no reliable tool for assessing endometrial receptivity. Interactions between human embryo and endometrium during implantation are poorly understood. Trophoblastic spheroids (BAP-EB) derived from human stem cells resembling human blastocyst polar trophectoderm (pTE) was established in our laboratory. It was hypothesized that BAP-EB can be used for predicting clinical pregnancy outcomes and studying molecular interactions at the embryo-maternal interface.
The predictive value of BAP-EB attachment rate onto endometrial epithelial cells (EEC) on the cumulative live birth rate of an in vitro fertilization (IVF) cycle was evaluated by a prospective observation study. The results showed that BAP-EB attachment rates on EEC isolated from women aged 35 years who had a cumulative live birth within 6 months of ovarian stimulation were significantly higher than those without a live birth. No such difference was observed for women with younger ages. The data indicated that the attachment of BAP-EB onto EEC modestly predicted cumulative livebirth of women aged 35 or over.
The surface molecules involved in the BAP-EB-EEC attachment process were identified. The conditioned medium of attachment competent BAP-EB induced the expressions of catenin delta 1 (CTNND1) and E-cadherin (CDH1) on EEC. The current study was the first to report CTNND1 which maintained the cell surface integrity and stabilized CDH1 expression on EEC played a role in the early implantation processes. CDH, but not CTNND1, was located at the apical surface of EEC. Knockdown of CTNND1 and CDH1 in EEC reduced cell adhesiveness and BAP-EB attachment. Human blastocyst secretory factors like human chorionic gonadotropin (HCG) induced the translocation of CDH1 from the adherent junction to the apical surface of EEC. Most critically, the expressions of CTNND1 and CDH1 in EEC were correlated with the live birth and repeated implantation failure.
Mass spectrometry analysis identified CDH1 as the embryonic binding partner of endometrial CDH1. Intense apical CDH1 signal was located on receptive EEC and pTE-like BAP-EB. Homophilic binding of trophectodermal CDH1 with endometrial CDH1 was confirmed by atomic force microscope and antibody blocking assays. It was found that CDH1 homophilic binding was a major mediator of the attachment of BAP-EB onto EEC. The use of expanded potential stem cell derived from human embryos further confirmed the role of CDH1 during BAP-EB attachment. In addition, trophectodermal Neuropilin 1 was identified as another molecule potentially involved in the attachment process during embryo implantation.
Three-dimensional implantation models were reconstructed to better mimic the in vivo conditions of embryo implantation. While ex vivo explant culture failed to maintain the structure and viability of endometrium, an UP-CELL 3D implantation model with columnar epithelium-like Ishikawa cells stacking on multi-layered stromal cells was established. Attachment and early invasion of BAP-EB onto the Ishikawa cells were observed. The setup might potentially be used for studying the mechanistic pathways leading to implantation failure.
In summary, BAP-EB represented an embryo surrogate for predicting endometrial receptivity and studying embryo-endometrium communications during embryo implantation.2023-01-01T00:00:00ZThe regulatory role of endometrial gland on decidualization processLin, Leqian林乐千http://hdl.handle.net/10722/3359682023-12-29T04:05:16Z2023-01-01T00:00:00ZTitle: The regulatory role of endometrial gland on decidualization process
Authors: Lin, Leqian; 林乐千
Abstract: The maternal-fetus interface consists of endometrial glands, stromal cells, immune cells, trophoblasts and endothelial cells, is essential for a successful pregnancy. Defective endometrial glandular development and/or functions is associated with infertility and pregnancy complications, though the pathogenesis is unknown. This project hypothesizes that endometrial glands modulate stromal cells and immune cell functions at the fetal-maternal interface.
In this study, a human endometrial glandular organoid (EEO) system was established using primary endometrial gland tissue. The derived EEOs demonstrated the glandular epithelial origin by expressing the specific glandular markers. The resemblance between in vitro generated EEOs and the in vivo ones was supported by single-cell RNA-seq analysis. Moreover, we have cultured EEOs in hormonal conditions mimicking the proliferative phase (Estrogen, E2), secretory phase (E2+progesterone, P4) and early pregnancy (E2+P4+human chorionic gonadotropin, hCG). Our results showed that EEOs were hormone responsive and hormonal treatments can modulate their secretome.
Decidualization is essential for the establishment/maintenance of pregnancy. It involves the differentiation of endometrial stromal cells (ESCs) and the recruitment of leukocytes. To investigate the role of endometrial glands on stromal cell decidualization, a co-culture model was established using EEO and primary/telomerase-transformed human ESCs (T-HESCs). The results showed that EEO secretome suppressed the decidualization of ESCs in a time and EEO amount dependent manner. The transcriptome of the primary ESCs before and after EEO co-culture were also compared. Based on the differentially expressed genes, protein association network analysis and literature search, 9 genes in ESCs were identified as potential genes associated with the suppressive effect of EEO on decidualization. We have then employed in vitro co-culture model and in vivo endometrial hyperplasia model to validate the results and identified CEBPA (CCAAT enhancer-binding protein alpha) and EDN1 (Endothelin 1) were closely related to the suppressive effect of EEO.
Decidual macrophages (dMs) function to regulate maternal immune tolerance and remodel the placenta. However, the factors regulating the differentiation/functions of dMs remains unclear. Therefore, another objective of this project is to determine the regulatory role of EEO on macrophage functions. Human monocytes were isolated from female blood and were differentiated into macrophages using macrophage colony-stimulating factor (MCSF). The inclusion of the E2+P4-treated EEO secretome during the differentiation process increased indoleamine 2, 3-dioxygenase 1 (IDO-1) expression in the resulting macrophages. Interestingly, IDO-1 is an immunoregulatory enzyme marker of dMs. Cytokine array further demonstrated altered expressions of migration inhibitory factor (MIF), serpin family E member 1 (Serpin E1), granulocyte colony-stimulating factor (GCSF), interleukin-6 (IL-6) and macrophage inflammatory protein 1α (MIP-1α) in macrophage after EEO secretome treatment. All these cytokines are known to be related to immune tolerance and placenta development during pregnancy. Moreover, the phagocytosis of the MCSF-differentiated macrophages was induced by the secretome of hormones treated EEOs.
In conclusion, the results of this study not only demonstrated that EEO could be a reliable model to study the endometrial gland functions in humans, but also provide confirmatory evidence on the maternal regulation of stromal cell decidualization and dM functions via the endometrial glandular secretion.
(494 words)2023-01-01T00:00:00ZThe roles of DGAT2 in promoting omental metastasis of ovarian cancerZhan, Shijie詹詩杰http://hdl.handle.net/10722/3359562023-12-29T04:05:09Z2023-01-01T00:00:00ZTitle: The roles of DGAT2 in promoting omental metastasis of ovarian cancer
Authors: Zhan, Shijie; 詹詩杰
Abstract: Epithelial ovarian cancer (EOC) is one of the fatal gynecological malignancies around the globe, accounting for over 90% of ovarian cancer cases. Most patients with EOC are defectively diagnosed at advanced stages accompanied with severe omental metastasis and peritoneal dissemination, leading to a typically poor prognosis and aggressive tumor recurrence from minimal residual disease (MRD). Mounting evidence from different groups and our team has disclosed that lipid-enriched ascitic/omental microenvironments provide surplus free fatty acids for sustaining ovarian tumor growth, rendering omentum as a preferential site for ovarian cancer carcinomatosis. Therefore, understanding the molecular mechanisms associated with the stimulatory effects of the omental metastatic niche on ovarian cancer cell growth and aggressiveness is of great importance to speed up the development of novel therapeutic strategies against transcoelomic metastasis of ovarian cancer.
In this study, transcriptomic analysis, QPCR analysis and IHC analysis were assessed on specimens of omental metastatic lesions of EOC and the corresponding pair of primary tumors. DGAT2 was consistently found to be highly upregulated in the omental metastatic tumors, while DGAT1 was relatively less obvious in expression status in both primary and metastatic counterparts, implying that DGAT2 was clinically relevant to take the leading role in directing EOC carcinomatosis. Kaplan-Meier survival analysis furthermore unveiled a more favorable prognosis in EOC patients with low DGAT2 expression when compared with the high expression group, inferring a reciprocal correlation between DGAT2 level and long-term survival among ovarian cancer patients.
Exploiting omental conditioned medium (OCM) to mimics the ascitic/omental microenvironment, pharmaceutical inhibition or genetic silencing of DGAT2 significantly reduced OCM-initiated lipid droplets formation, ATP production, cell proliferation and cell migration/invasion, while accelerating the capacity of ROS stress in human ovarian cancer cell lines and primary culture of ascites-derived ovarian cancer cells. Rescue experiments with overexpression of DGAT2 in OCM-cocultured ovarian cancer cells showed increased cell proliferation and cell migration/invasion, enhanced accumulation of lipid droplets in the cytoplasmic compartment and augmented tumor colonization in ex vivo murine omental-tumor model, supporting that the upregulated DGAT2 enhanced lipid metabolic activities, proliferation and aggression of ovarian cancer cells in omental tumor microenvironment (TME). Mechanistic studies demonstrated that the inhibition of DGAT2 reduced expression of phospho-AKT and phosphor-mTOR, indicating that DGAT2 functioned as AKT upstream in promoting cancer cells growth and metastasis through activating AKT/mTOR signaling cascade.
Further investigations revealed that pharmaceutical inhibition or genetic deletion of DGAT2 sensitized RSL3-induced ferroptosis in in vitro OCM-cocultured ovarian cancer cells and in vivo tumor dissemination. Meanwhile, inhibition of DGAT2 led to downregulation of two ferroptosis executioners, ACSL4 and LPCAT3. These findings not only revealed that DGAT2 augmented tumor aggressiveness of ovarian cancer cells, but also suggested the utility of combining DGAT2 inhibitors with ferroptosis inducers as a potential target-based therapy in eradicating peritoneal metastases of EOC. Thus, findings herein shed light on the orchestrated action of DGAT2 inhibitors and combined ferroptosis initiating therapies (FITs) in enhancing therapeutic outcomes of metastatic ovarian cancer and benefiting EOC patients with peritoneal metastases.2023-01-01T00:00:00Z