MicroRNA-135a-induced formation of CD133+ subpopulation with cancer stem cell properties in cervical cancer.

Cancer stem cells (CSCs) play significant roles in tumor initiation. MicroRNA-135a (miR-135a) induced the formation of a CD133+ subpopulation from a human papillomavirus-immortalized cervical epithelial cell line. Compared with the CD133- cells, the CD133+ cells expressed higher levels of miR-135a and OCT4, exhibited significantly higher tumorsphere forming capacity and the time required for tumorsphere formation was shortened in the second generation. Serum induction suppressed the expression of CD133, OCT4 and miR-135a, but increased expression of involucrin in the miR-135a-induced CD133+ cells. The miR-135a-induced CD133+ cells were tumorigenic in a limiting dilution approach in vivo. The cells expressed significantly higher level of active ß-catenin and OCT4 than the CD133- counterpart. Wnt3a enhanced the expression of OCT4 and CD133 in cervical cancer cells but failed to enhance CD133 transcription in normal cervical cells. Wnt3a stimulation also increased tumorsphere size and self-renewal of miR-135a-induced CD133+ subpopulation. Wnt/ß-catenin inhibition suppressed tumorsphere formation while Wnt3a partially nullified the inhibitory effect. Taken together, miR-135a induced the formation of a subpopulation of cells with CSC properties both in vitro and in vivo and the Wnt/ß-catenin signaling pathway is essential to maintain its tumorigenicity.

Myometrial Cells Stimulate Self-Renewal of Endometrial Mesenchymal Stem-Like Cells Through WNT5A/ß-Catenin Signaling.

Human endometrium undergoes cycles of proliferation and differentiation throughout the reproductive years of women. The endometrial stem/progenitor cells contribute to this regenerative process. They lie in the basalis layer of the endometrium next to the myometrium. We hypothesized that human myometrial cells provide niche signals regulating the activities of endometrial mesenchymal stem-like cells (eMSCs). In vitro coculture of myometrial cells enhanced the colony-forming and self-renewal ability of eMSCs. The cocultured eMSCs retained their multipotent characteristic and exhibited a greater total cell output when compared with medium alone culture. The expression of active ß-catenin in eMSCs increased significantly after coculture with myometrial cells, suggesting activation of WNT/ß-catenin signaling. Secretory factors in spent medium from myometrial cell culture produced the same stimulatory effects on eMSCs. The involvement of WNT/ß-catenin signaling in self-renewal of eMSCs was confirmed with the use of WNT activator (Wnt3A conditioned medium) and WNT inhibitors (XAV939 and inhibitor of Wnt Production-2 [IWP-2]). The myometrial cells expressed more WNT5A than other WNT ligands. Recombinant WNT5A stimulated whereas anti-WNT5A antibody suppressed the colony formation, self-renewal, and T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcriptional activities of eMSCs. Moreover, eMSCs expressed FZD4 and LRP5. WNT5A is known to activate the canonical WNT signaling in the presence of these receptor components. WNT antagonist, DKK1, binds to LRP5/6. Consistently, DKK1 treatment nullified the stimulatory effect of myometrial cell coculture. In conclusion, our findings show that the myometrial cells are niche components of eMSCs, modulating the self-renewal activity of eMSCs by WNT5A-dependent activation of WNT/ß-catenin signaling. Stem Cells 2019;37:1455-1466.

Polymer-based precipitation preserves biological activities of extracellular vesicles from an endometrial cell line.

Extracellular vesicles (EVs) are membrane-bound vesicles released by cells and act as media for transfer of proteins, small RNAs and mRNAs to distant sites. They can be isolated by different methods. However, the biological activities of the purified EVs have seldom been studied. In this study, we compared the use of ultracentrifugation (UC), ultra-filtration (UF), polymer-based precipitation (PBP), and PBP with size-based purification (PBP+SP) for isolation of EVs from human endometrial cells and mouse uterine luminal fluid (ULF). Electron microscopy revealed that the diameters of the isolated EVs were similar among the tested methods. UF recovered the highest number of EVs followed by PBP, while UC and PBP+SP were significantly less efficient (P<0.05). Based on the number of EVs-to-protein ratios, PBP had the least protein contamination, significantly better than the other methods (P<0.05). All the isolated EVs expressed exosome-enriched proteins CD63, TSG101 and HSP70. Incubation of the trophoblast JEG-3 cells with an equal amount of the fluorescence-labelled EVs isolated by the studied methods showed that many of the PBP-EVs treated cells were fluorescence positive but only a few cells were labelled in the UC- and UF-EVs treated groups. Moreover, the PBP-EVs could transfer significantly more miRNA to the recipient cells than the other 3 methods (P<0.05). The PBP method could isolate EVs from mouse ULF; the diameter of the isolated EVs was 62±19 nm and expressed CD63, TSG101 and HSP70 proteins. In conclusion, PBP could best preserve the activities of the isolated EVs among the 4 methods studied and was able to isolate EVs from a small volume of sample. The simple setup and low equipment demands makes PBP the most suitable method for rapid EV assessment and isolation of EVs in clinical and basic research settings.

A meta-analysis of the impact of human leukocyte antigen-G on the outcomes of IVF/ICSI.

This analysis was performed to determine whether the presence of soluble human leukocyte antigen-G (sHLA-G) in embryo culture medium is predictive of clinical outcomes in IVF treatment. The outcomes of implantation, clinical pregnancy, multiple pregnancy and miscarriage, between groups with and without sHLA-G in embryo culture media, were analysed. Fifteen studies with a total of 6170 cases were included. Ten of them were prospective studies while five were retrospective studies. Embryo culture media with sHLA-G were associated with significantly higher implantation rate and clinical pregnancy rate when compared with those without; the odd ratios (ORs) were 2.66 [95% confidence interval (CI): 1.75-4.06, P < 0.00001], 3.79 (95% CI: 2.69-5.33, P < 0.00001), respectively. There was no significant difference in the rate of multiple pregnancy (OR: 1.87, 95% CI: 0.55-6.31) and miscarriage (OR: 0.77, 95% CI: 0.52-1.16). The results suggested that the presence of sHLA-G in the embryo culture medium favoured higher implantation rate and pregnancy rate. However, the conclusion needs to be consolidated by further clinical studies using a more precise method of determination of sHLA-G and research on the physiological and molecular mechanisms of the beneficial effect of sHLA-G on early embryo development and implantation.

MicroRNA and Embryo Implantation.

PROBLEM: In mammals, implantation involves interactions between an activated blastocyst and a receptive endometrium. There are controversies on the role of microRNAs in preimplantation embryo development. The actions of endometrial microRNAs on implantation are beginning to be understood. METHOD OF STUDY: Review of literature on microRNAs in preimplantation embryos and endometrium. RESULTS: Emerging evidence suggests a role of microRNAs in blastocyst activation and implantation. Differential expression of microRNAs is found between receptive and non-receptive endometria. Members of the let-7, miR-200, miR-30 families, and the miR-17-92 clusters are more commonly found to be associated with endometrial receptivity. Experimental studies show that the targets of the differentially expressed microRNAs affect endometrial receptivity, decidualization, and embryo implantation. Free and exosome/microvesicle containing microRNAs have been detected in human and ovine uterine luminal fluid (ULF). They may serve as mediators of embryo-endometrium dialog. Some observations suggest that the microRNAs in ULF may be used as biomarkers in infertility treatment. CONCLUSION: MicroRNAs in endometrium and blastocysts are involved in the implantation process.

Annexin A2 Acts as an Adhesion Molecule on the Endometrial Epithelium during Implantation in Mice.

To determine the function of Annexin A2 (Axna2) in mouse embryo implantation in vivo, experimental manipulation of Axna2 activities was performed in mouse endometrial tissue in vivo and in vitro. Histological examination of endometrial tissues was performed throughout the reproduction cycle and after steroid treatment. Embryo implantation was determined after blockage of the Axna2 activities by siRNA or anti-Axna2 antibody. The expression of Axna2 immunoreactivies in the endometrial luminal epithelium changed cyclically in the estrus cycle and was upregulated by estrogen. After nidatory estrogen surge, there was a concentration of Axna2 immunoreactivities at the interface between the implanting embryo and the luminal epithelium. The phenomenon was likely to be induced by the implanting embryos as no such concentration of signal was observed in the inter-implantation sites and in pseudopregnancy. Knockdown of Axna2 by siRNA reduced attachment of mouse blastocysts onto endometrial tissues in vitro. Consistently, the number of implantation sites was significantly reduced after infusion of anti-Axna2 antibody into the uterine cavity. Steroids and embryos modulate the expression of Axna2 in the endometrial epithelium. Axna2 may function as an adhesion molecule during embryo implantation in mice.

Two-dimensional liquid chromatography with tandem mass spectrometry-based proteomic characterization of endometrial luminal epithelial surface proteins responsible for embryo implantation.

OBJECTIVE: To identify endometrial epithelial cell surface proteins essential for blastocysts implantation. DESIGN: Isolation of cell-surface labeled prereceptive (pregnancy day 1) and receptive (pregnancy day 4) mouse endometrial proteins coupled to two-dimensional liquid chromatography with tandem mass spectrometry. SETTING: University research laboratory. ANIMAL(S): Sexually mature female imprinting control region (ICR) mice. INTERVENTION(S): Labeling, purification, and identification of endometrial luminal surface proteins with differentially expressing proteins determined by significant analysis of a microarray algorithm and selected differentially expressed proteins verified by immunohistochemistry and functional assay. MAIN OUTCOME MEASURE(S): Investigation in endometrial luminal surface proteome of prereceptive and receptive endometria of the expression of four of the differentially expressed proteins and functional analysis of aminopeptidase N in a three-dimensional blastocyst-endometrial coculture model. RESULT(S): We identified 104 cell surface proteins from prereceptive and receptive pregnant mouse endometria and found that 27 were statistically significantly up-regulated and 18 were statistically significantly down-regulated in the receptive endometrium. Immunohistochemical analysis of four of the differentially expressed proteins in the endometrium showed concordant results. Functional assay showed that blastocyst attachment was statistically significantly reduced upon inhibition of aminopeptidase N. CONCLUSION(S): The luminal cell surface proteome of the prereceptive and receptive endometria differs, and aminopeptidase N is potentially involved in embryo attachment.

miR-135a leads to cervical cancer cell transformation through regulation of ß-catenin via a SIAH1-dependent ubiquitin proteosomal pathway.

Human papillomaviruses (HPVs) is the principal etiological agent of cervical cancer (CC). However, exposure to the high-risk type HPV alone is insufficient for tumor formation, and additional factors are required for the HPV-infected cells to become tumorigenic. Dysregulated microRNAs (miRNAs) expression is frequently observed in cancer but their roles in the formation of CC have not been fully revealed. In this study, we compared the expression of miR-135a in laser capture microdissected cervical specimens and confirmed overexpression of the miRNA in malignant cervical squamous cell carcinoma compared with precancerous lesions. Transient force-expression of miR-135a induced growth in low-density culture, anchorage-independent growth, proliferation and invasion of a HPV-16 E6/E7-immortalized cervical epithelial cell line, NC104-E6/E7. The observed effects were due to the inhibitory action of miR-135a on its direct target seven in absentia homolog 1 (SIAH1) leading to upregulation of ß-catenin/T cell factor signaling. miR-135a force-expression enhanced the growth of HeLa- and NC104-E6/E7-derived tumor in vivo. The effect of miR-135a could be partially nullified by SIAH1 force-expression. More importantly, the expression of SIAH1 and ß-catenin correlated with that of miR-135a in precancerous and cancerous lesions of cervical biopsies. By comparing the tumorigenic activities of miR-135a in E6/E7 positive/negative cell lines and in NC104-E6/E7 with or without E6/E7 knockdown, we demonstrated that HPV E6/E7 proteins are prerequisite for miR-135a as an oncomiR. Taken together, miR-135a/SIAH1/ß-catenin signaling is important in the transformation and progression of cervical carcinoma.

MicroRNA Let-7a and dicer are important in the activation and implantation of delayed implanting mouse embryos.

STUDY QUESTION: Does Let-7a have a functional role in modulating dicer expression to activate dormant mouse blastocysts for implantation? SUMMARY ANSWER: Let-7a post-transcriptionally regulates dicer expression altering microRNA expression to affect the implantation competency of the activated blastocysts. WHAT IS KNOWN ALREADY: The Let-7a microRNA is up-regulated during blastocyst dormancy and its forced-expression suppresses embryo implantation in vitro and in vivo. Dicer is a Let-7 target, which processes pre-microRNA to mature microRNA. STUDY DESIGN, SIZE, DURATION: The effects on the expression of Let-7a and dicer in dormant blastocysts during the first 12 h after estradiol-induced activation, and the relationship between Let-7a and dicer in preimplantation embryos were determined. The effects on the microRNA expression and embryo implantation in vivo in dicer-knockdown mouse 5-8 cell embryos and dormant blastocysts at 1 h post estradiol activation were also studied. PARTICIPANTS/MATERIALS, SETTING, METHODS: ICR female mice at 6 weeks of age were ovariectomized on Day 4 of pregnancy to generate the delayed implantation model. Mouse 5-8 cell embryos and/or dormant blastocysts at 1 h after estradiol injection were electroporated with dicer siRNA and Let-7a precursor or Let-7a inhibitor. At 48 h post electroporation, the Let-7a expression, dicer transcripts and proteins in the embryos were determined using qPCR and immunostaining/western blotting, respectively. All experiments were repeated at least three times. MAIN RESULTS AND THE ROLE OF CHANCE: Estradiol injection down-regulated Let-7a and up-regulated dicer in the dormant blastocysts during the first 12 h post-activation. Dicer knockdown at 1 h post-activation of blastocysts suppressed EGFR expression, attenuated EGF binding and compromised implantation of the transferred embryos. Let-7a transcriptionally regulated dicer by binding to the 3'-UTR of dicer in trophoblast cells. Dicer knockdown in blastocysts suppressed mature Let-7a expression and compromised implantation. LIMITATIONS, REASONS FOR CAUTION: Gain- and loss-of-function approaches were used by analyzing transient expressions of transfected microRNA modulators or genes. The consequence of the Let-7a-dicer interaction on pregnancy remains to be determined. The study used the mouse as a model and the applicability of the observed phenomena in humans warrants further investigation. WIDER IMPLICATIONS OF THE FINDINGS: Our results indicate that the Let-7a-dicer interaction leads to differential microRNA expression in dormant blastocysts after estradiol activation. Because the expression pattern of Let-7a in human blastocysts is similar to that in mouse blastocysts, our observation that the Let-7a-dicer interaction has a role in regulating the implantation potential of the mouse blastocysts could be applicable to humans. STUDY FUNDING/COMPETING INTEREST(S): This project is supported partly by a research grant from the Research Grant Council to W.S.B.Y. The authors have no competing interests to declare.

Identification of CD147 (basigin) as a mediator of trophoblast functions.

STUDY QUESTION: Does CD147 regulate trophoblast functions in vitro? SUMMARY ANSWER: CD147 exists as a receptor complex on human trophoblast and regulates the implantation, invasion and differentiation of trophoblast. WHAT IS KNOWN ALREADY: CD147 is a membrane protein implicated in a variety of physiological and pathological conditions due to its regulation of cell-cell recognition, cell differentiation and tissue remodeling. Reduced placental CD147 expression is associated with pre-eclampsia, but the mechanism of actions remains unclear. STUDY DESIGN, SIZE, DURATION: A loss of function approach or functional blocking antibody was used to study the function of CD147 in primary human cytotrophoblasts isolated from first trimester termination of pregnancy and/or in the BeWo cell line, which possesses characteristics of human cytotrophoblasts. PARTICIPANTS/MATERIALS, SETTING METHODS: CD147 expression was analyzed by immunofluorescence staining and western blotting. CD147-associated protein complex on plasma membrane were separated by blue native gel electrophoresis and identified by reversed-phase liquid chromatography coupled with quadrupole time-of-flight hybrid mass spectrometer. Cell proliferation and invasion were determined by fluorometric cell proliferation assays and transwell invasion assays, respectively. Matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) activities were measured by gelatin gel zymography and uPA assay kits, respectively. Cell migration was determined by wound-healing assays. Cell fusion was analyzed by immunocytochemistry staining of E-cadherin and 4',6-diamidino-2-phenylindole. The transcripts of matrix proteinases and trophoblast lineage markers were measured by quantitative PCR. Extracellular signal-regulated kinase (ERK) activation was analyzed by western blot using antibodies against ERKs. MAIN RESULTS AND THE ROLE OF CHANCE: CD147 exists as protein complexes on the plasma membrane of primary human cytotrophoblasts and BeWo cells. Several known CD147-interacting partners, including integrin ß1 and monocarboxylate transporter-1, were identified. Suppression of CD147 by siRNA significantly (P < 0.05) reduced trophoblast-endometrial cell interaction, cell invasion, syncytialization, differentiation and ERK activation of BeWo cells. Consistently, anti-CD147 functional blocking antibody suppressed the invasiveness of primary human cytotrophoblasts. The reduced invasiveness was probably due to the restrained (P < 0.05) enzyme activities of MMP-2, MMP-9 and uPA. LIMITATIONS, REASONS FOR CAUTION: Most of the above findings are based on BeWo cell lines. These results need to be confirmed with human first trimester primary cytotrophoblast. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study on the role of CD147 in trophoblast function. Further investigation on the function of CD147 and its associated protein complexes will enhance our understanding on human placentation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported in part by the University of Hong Kong Grant 201011159200. The authors have no competing interests to declare.

Development and characterization of an endometrial tissue culture model for study of early implantation events.

OBJECTIVE: To improve and characterize an endometrial tissue culture model. DESIGN: Experimental study of the characteristics of mouse endometrial tissue cultured on amniotic membrane matrix. SETTING: University research laboratory. ANIMAL(S): Sexually mature female ICR mice. INTERVENTION(S): Histologic examination of the cultured endometrial tissues. The attachment rates of the cultured tissues to implantation blastocysts under various conditions were determined. MAIN OUTCOME MEASURE(S): Morphometric analysis of the cultured tissues. Blastocyst attachment rate and expression of decidualization markers cylcooxygenase-2, connexin 43, and peroxisome proliferator-activated receptor δ. RESULT(S): Endometrial tissues could be grown on amniotic membrane matrix for 3 days with morphometric parameters similar to those in the in vivo pseudopregnant control. The cultured tissues responded to the surrounding steroid environment. Morphometric assessment indicated that medium containing 63.5 nmol/L P and 0.9 nmol/L E(2) provided the best support. The condition allowed attachment of approximately 60% of the cocultured blastocysts. A small percentage of the attached blastocyst started to penetrate the luminal epithelium within 28 hours. The attachment rate was significantly reduced with prior treatment of the cultured endometrium with anti-leukemia inhibitory factor antibody. The attached blastocyst induced decidualization around the attachment site. CONCLUSION(S): The model is useful for the study on implantation in the mouse.

Study of transforming growth factor alpha for the maintenance of human embryonic stem cells.

Human embryonic stem cells (hESCs) have great potential for regenerative medicine as they have self-regenerative and pluripotent properties. Feeder cells or their conditioned medium are required for the maintenance of hESC in the undifferentiated state. Feeder cells have been postulated to produce growth factors and extracellular molecules for maintaining hESC in culture. The present study has aimed at identifying these molecules. The gene expression of supportive feeder cells, namely human foreskin fibroblast (hFF-1) and non-supportive human lung fibroblast (WI-38) was analyzed by microarray and 445 genes were found to be differentially expressed. Gene ontology analysis showed that 20.9% and 15.5% of the products of these genes belonged to the extracellular region and regulation of transcription activity, respectively. After validation of selected differentially expressed genes in both human and mouse feeder cells, transforming growth factor α (TGFα) was chosen for functional study. The results demonstrated that knockdown or protein neutralization of TGFα in hFF-1 led to increased expression of early differentiation markers and lower attachment rates of hESC. More importantly, TGFα maintained pluripotent gene expression levels, attachment rates and pluripotency by the in vitro differentiation of H9 under non-supportive conditions. TGFα treatment activated the p44/42 MAPK pathway but not the PI3K/Akt pathway. In addition, TGFα treatment increased the expression of pluripotent markers, NANOG and SSEA-3 but had no effects on the proliferation of hESCs. This study of the functional role of TGFα provides insights for the development of clinical grade hESCs for therapeutic applications.

Proteomic analysis reveals platelet factor 4 and beta-thromboglobulin as prognostic markers in severe acute respiratory syndrome.

Previously, we reported that proteomic fingerprints were present in sera of patients with severe acute respiratory syndrome (SARS), and could separate patients into subgroups with different prognoses. In the present study, we examined the prognostic values of the SARS-associated proteomic features by biostatistical analysis, and deciphered the identities of those with prognostic values. Data of 20 SARS-associated serum proteomic features and ten serological variables from 38 SARS adult patients before treatment were subjected to multivariate logistic regression. Proteomic features of m/z 6634, m/z 7769, m/z 8635, and m/z 8865 were identified as independent prognostic markers. After purification by cation-exchange chromatography and gel electrophoresis, proteomic features of m/z 7769 and m/z 8865 were found to be platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG) by tandem mass spectrometry, respectively. The associations of decreased serum PF4 and increased serum beta-TG levels with poor prognosis were confirmed by Western blot. Previous studies suggest that PF4 and beta-TG are involved in the pathogenesis of acute respiratory distress syndrome (ARDS) in a negative and positive way, respectively. Our results suggest that PF4 and beta-TG may also play similar roles in the development of ARDS in SARS patients.

Involvement of microRNA lethal-7a in the regulation of embryo implantation in mice.

MicroRNAs interact with multiple mRNAs resulting in their degradation and/or translational repression. This report used the delayed implantation model to determine the role of miRNAs in blastocysts. Dormant blastocysts in delayed implanting mice were activated by estradiol. Differential expression of 45 out of 238 miRNAs examined was found between the dormant and the activated blastocysts. Five of the nine members of the microRNA lethal-7 (let-7) family were down-regulated after activation. Human blastocysts also had a low expression of let-7 family. Forced-expression of a family member, let-7a in mouse blastocysts decreased the number of implantation sites (let-7a: 1.1±0.4; control: 3.8±0.4) in vivo, and reduced the percentages of blastocyst that attached (let-7a: 42.0±8.3%; control: 79.0±5.1%) and spreaded (let-7a: 33.5±2.9%; control: 67.3±3.8%) on fibronectin in vitro. Integrin-ß3, a known implantation-related molecule, was demonstrated to be a target of let-7a by 3'-untranslated region reporter assay in cervical cancer cells HeLa, and Western blotting in mouse blastocysts. The inhibitory effect of forced-expression of let-7a on blastocyst attachment and outgrowth was partially nullified in vitro and in vivo by forced-expression of integrin-ß3. This study provides the first direct evidence that let-7a is involved in regulating the implantation process partly via modulation of the expression of integrin-ß3.

Sperm-borne microRNA-34c is required for the first cleavage division in mouse.

In mammals, the sperm deliver mRNA of unknown function into the oocytes during fertilization. The role of sperm microRNAs (miRNAs) in preimplantation development is unknown. miRNA profiling identified six miRNAs expressed in the sperm and the zygotes but not in the oocytes or preimplantation embryos. Sperm contained both the precursor and the mature form of one of these miRNAs, miR-34c. The absence of an increased level of miR-34c in zygotes derived from α-amanitin-treated oocytes and in parthenogenetic oocytes supported a sperm origin of zygotic miR-34c. Injection of miR-34c inhibitor into zygotes inhibited DNA synthesis and significantly suppressed first cleavage division. A 3' UTR luciferase assay and Western blotting demonstrated that miR-34c regulates B-cell leukemia/lymphoma 2 (Bcl-2) expression in the zygotes. Coinjection of anti-Bcl-2 antibody in zygotes partially reversed but injection of Bcl-2 protein mimicked the effect of miR-34c inhibition. Oocyte activation is essential for the miR-34c action in zygotes, as demonstrated by a decrease in 3'UTR luciferase reporter activity and Bcl-2 expression after injection of precursor miR-34c into parthenogenetic oocytes. Our findings provide evidence that sperm-borne miR-34c is important for the first cell division via modulation of Bcl-2 expression.

Wnt activation downregulates olfactomedin-1 in Fallopian tubal epithelial cells: a microenvironment predisposed to tubal ectopic pregnancy.

Ectopic pregnancy (EP) occurs when the embryo fails to transit to the uterus and attach to the luminal epithelium of the Fallopian tube (FT). Tubal EP is a common gynecological emergency and more than 95% of EP occurs in the ampullary region of the FT. In humans, Wnt activation and downregulation of olfactomedin-1 (Olfm-1) occur in the receptive endometrium and coincided with embryo implantation in vivo. Whether similar molecular changes happen in the FT leading to EP remains unclear. We hypothesized that activation of Wnt signaling downregulates Olfm-1 expression predisposes to EP. We investigated the spatiotemporal expression of Olfm-1 in FT from non-pregnant women and women with EP, and used a novel trophoblastic spheroid (embryo surrogate)-FT epithelial cell co-culture model (JAr and OE-E6/E7 cells) to study the role of Olfm-1 on spheroid attachment. Olfm-1 mRNA expression in the ampullary region of non-pregnant FT was higher (P<0.05) in the follicular phase than in the luteal phase. Ampullary tubal Olfm-1 expression was lower in FT from women with EP compared to normal controls at the luteal phase (histological scoring (H-SCORE)=1.3±0.2 vs 2.4±0.5; P<0.05). Treatment of OE-E6/E7 with recombinant Olfm-1 (0.2-5 µg/ml) suppressed spheroid attachment to OE-E6/E7 cells, while activation of Wnt-signaling pathway by Wnt3a or LiCl reduced endogenous Olfm-1 expression and increased spheroid attachment. Conversely, suppression of Olfm-1 expression by RNAi increased spheroid attachment to OE-E6/E7 cells. Taken together, Wnt activation suppresses Olfm-1 expression, and this may predispose a favorable microenvironment of the retained embryo in the FT, leading to EP in humans.

miR-135A regulates preimplantation embryo development through down-regulation of E3 Ubiquitin Ligase Seven In Absentia Homolog 1A (SIAH1A) expression.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNA molecules capable of regulating transcription and translation. Previously, a cluster of miRNAs that are specifically expressed in mouse zygotes but not in oocytes or other preimplantation stages embryos are identified by multiplex real-time polymerase chain reaction-based miRNA profiling. The functional role of one of these zygote-specific miRNAs, miR-135a, in preimplantation embryo development was investigated. METHODOLOGY/PRINCIPAL FINDINGS: Microinjection of miR-135a inhibitor suppressed first cell cleavage in more than 30% of the zygotes. Bioinformatics analysis identified E3 Ubiquitin Ligase Seven In Absentia Homolog 1A (Siah1a) as a predicted target of miR-135a. Western blotting and 3'UTR luciferase functional assays demonstrated that miR-135a down-regulated the expression of Siah1 in HeLa cells and in mouse zygotes. Siah1a was expressed in preimplantation embryos and its expression pattern negatively correlated with that of miR-135a. Co-injection of Siah1a-specific antibody with miR-135a inhibitor partially nullified the effect of miR-135a inhibition. Proteasome inhibition by MG-132 revealed that miR-135a regulated proteasomal degradation and potentially controlled the expression of chemokinesin DNA binding protein (Kid). CONCLUSIONS/SIGNIFICANCE: The present study demonstrated for the first time that zygotic specific miRNA modulates the first cell cleavage through regulating expression of Siah1a.

Glycodelin-A protein interacts with Siglec-6 protein to suppress trophoblast invasiveness by down-regulating extracellular signal-regulated kinase (ERK)/c-Jun signaling pathway.

During placentation, the cytotrophoblast differentiates into the villous cytotrophoblast and the extravillous cytotrophoblast. The latter invades the decidualized endometrium. Glycodelin-A (GdA) is abundantly synthesized by the decidua but not the trophoblast. Previous data indicate that GdA suppresses the invasion of trophoblast cell lines by down-regulating proteinase expression and activities. This study addresses the signaling pathway involved in the above phenomenon. GdA was found to suppress phosphorylation of ERKs and expression of their downstream effector c-Jun, a component of the transcription factor activator protein-1 (AP-1). The involvement of ERKs and c-Jun in suppressing trophoblast invasion and biosynthesis of proteinases was confirmed by using siRNA knockdown and pharmacological inhibitors. Desialylation reduced binding affinity of GdA toward and invasion suppressive activities on the trophoblast. Co-immunoprecipitation showed that Siglec-6 on the trophoblast was the binding protein of GdA. The binding of GdA to Siglec-6 was sialic acid-dependent. Treatment with anti-Siglec-6 antibody abolished the invasion suppressive activities of GdA. These results show that GdA interacts with Siglec-6 to suppress trophoblast invasiveness by down-regulating the ERK/c-Jun signaling pathway.

Glycodelin-A as a paracrine regulator in early pregnancy.

Glycodelin-A (GdA) is a glycoprotein secreted from the endometrial glands and decidual glandular epithelium. Given its abundance and ubiquitous distribution in the first trimester uterus, GdA may be involved in early placental development via its modulatory effect on immune and trophoblast cells. GdA inhibits activation and proliferation, and induces apoptosis of T cells. By selectively inducing Th1 cell death, GdA may shift the Th1/Th2 ratio at the feto-maternal interface. This is also achieved indirectly through enhanced expression of Fas in the Th1 cells, thus making them vulnerable to cell death through Fas ligand expressed on trophoblast, endometrial, and activated T helper cells. GdA also promotes secretion of the Th2 cytokines IL-6 and IL-13 from NK cells, and induces immunological tolerance of dendritic cells and apoptosis of monocytes. Specific glycosylation is a prerequisite for the biological activities of GdA. Reduction in α2-6 sialylation of GdA, as in gestational diabetes, is associated with impairment of its T cell apoptosis-inducing activities. This review integrates recent studies on GdA and its role as a paracrine regulator in early pregnancy.

MicroRNA-34a suppresses invasion through downregulation of Notch1 and Jagged1 in cervical carcinoma and choriocarcinoma cells.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of other genes by transcriptional inhibition or translational repression. miR-34a is a known tumor suppressor gene and inhibits abnormal cell growth. However, its role in other tumorigenic processes is not fully known. This study aimed to investigate the action of miR-34a on cell invasion. We found that miR-34a is expressed at various levels in cervical cancer (HeLa, SiHa, C4I, C33a and CaSki) and trophoblast (BeWo and JAR) cell lines. Transient forced expression of miR-34a did not affect the proliferation of these cell lines. Computational miRNA target prediction suggested that Notch1 and Jagged1 were targets of miR-34a. By using functional assays, miR-34a was demonstrated to bind to the 3' untranslated regions of Notch1 and Jagged1. Forced expression of miR-34a altered the expression of Notch1 and Jagged1 protein as well as Notch signaling as shown by the response of Hairy Enhancer of Split-1 protein to these treatments using western blot analysis. Forced expression of miR-34a suppressed the invasiveness of HeLa and JAR cells. By using gamma-secretase inhibitor (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester) that interfered Notch signaling and RNA interference that knockdown Notch1 expression, we confirmed that downregulation of Notch1 reduced the invasiveness of the cells. Transfection of intracellular domain of Notch nullifies the effect of miR-34a on the invasiveness of the cells. Besides, we identified that miR-34a affected cell invasion by regulating expression of urokinase plasminogen activator through Notch. Our results provide evidence that miR-34a inhibits invasiveness through regulation of the Notch pathway and its downstream matrix degrading enzyme.