JAR Cells Research Articles

Aspirin increases estrogen levels in the placenta to prevent preeclampsia by regulating placental metabolism and transport function

Preeclampsia is a unique multisystem progressive disease during pregnancy, which seriously endangers the health of pregnant women and fetuses. In clinical practice, aspirin is recommended for the prevention of preeclampsia, but the mechanism by which aspirin prevents preeclampsia has not yet been revealed. This report comprehensively evaluates the effects of aspirin on the expression and activity of placental metabolic enzymes and transporters. We found that after aspirin administration, only the expression of organic anion transporter 4 (OAT4) in the placenta showed a significant increase at both mRNA and protein levels, consistent with the results in JAR cells. Meanwhile, studies on the metabolic enzyme activity in the placenta showed a high upregulation of CYP19A1 activity. Subsequently, significant increases in endogenous substrates of OAT4 and CYP19A1 (dehydroepiandrosterone sulfate (DHEAS) and androstenedione) as well as estrone were detected in placental tissue. In summary, aspirin enhances the transport of DHEAS through OAT4 and promotes the metabolism of androstenedione through CYP19A1, thereby increasing estrogen levels in the placenta. This may be the mechanism by which aspirin prevents preeclampsia and maintains pregnancy by regulating the metabolism and transport function of the placenta.

Inhibition of human and rat placental 3β-hydroxysteroid dehydrogenases by bisphenol A analogues depends on their hydrophobicity: In silico docking analysis

Bisphenol A (BPA) and its analogues are widely used industrial chemicals. Placental 3β-hydroxysteroid dehydrogenases (3β-HSDs) catalyse the conversion of pregnenolone to progesterone. However, the potency of BPA analogues in inhibiting 3β-HSDs activity remains unclear. We investigated the inhibitory effect of 10 BPA analogues on 3β-HSDs activity using an in vitro assay and performed the structure-activity relationship and in silico docking analysis. BPH was the most potent inhibitor of human 3β-HSD1, with an IC50 value of 0.95 μM. BPFL, BPG, DABPA, BPAP, BPZ, DMBPA, and BPB also inhibited human 3β-HSD1 activity, albeit with lower potency. BPG was the most potent inhibitor of rat 3β-HSD4, with an IC50 value of 1.14 μM. BPAP, BPFL, BPG, BPH, BPZ, DABPA, and DMBPA are mixed inhibitors of human 3β-HSD1 and they significantly inhibited human JAr cells to secrete progesterone. The LogP values were inversely correlated with the inhibitory effects. Docking analysis showed that most BPA analogues bind to steroid-binding site of both 3β-HSDs. A pharmacophore containing hydrogen bond donor and hydrophobic region was generated for predicting the inhibitory strength of BPA analogues. In conclusion, this study demonstrates that some BPA analogues are potent inhibitors of 3β-HSDs and lipophilicity determines the inhibitory potency.

Overexpression of long noncoding RNA DUXAP8 inhibits ER-phagy through activating AKT/mTOR signaling and contributes to preeclampsia

Preeclampsia (PE) is a life-threatening pregnancy-specific complication with controversial mechanisms and no effective treatment except delivery is available. Currently, increasing researchers suggested that PE shares pathophysiologic features with protein misfolding/aggregation disorders, such as Alzheimer disease (AD). Evidences have proposed defective autophagy as a potential source of protein aggregation in PE. Endoplasmic reticulum-selective autophagy (ER-phagy) plays a critical role in clearing misfolded proteins and maintaining ER homeostasis. However, its roles in the molecular pathology of PE remain unclear. We found that lncRNA DUXAP8 was upregulated in preeclamptic placentae and significantly correlated with clinical indicators. DUXAP8 specifically binds to PCBP2 and inhibits its ubiquitination-mediated degradation, and decreased levels of PCBP2 reversed the activation effect of DUXAP8 overexpression on AKT/mTOR signaling pathway. Function experiments showed that DUXAP8 overexpression inhibited trophoblastic proliferation, migration, and invasion of HTR-8/SVneo and JAR cells. Moreover, pathological accumulation of swollen and lytic ER (endoplasmic reticulum) was observed in DUXAP8-overexpressed HTR8/SVneo cells and PE placental villus trophoblast cells, which suggesting that ER clearance ability is impaired. Further studies found that DUXAP8 overexpression impaired ER-phagy and caused protein aggregation medicated by reduced FAM134B and LC3II expression (key proteins involved in ER-phagy) via activating AKT/mTOR signaling pathway. The increased level of FAM134B significantly reversed the inhibitory effect of DUXAP8 overexpression on the proliferation, migration, and invasion of trophoblasts. In vivo, DUXAP8 overexpression through tail vein injection of adenovirus induced PE-like phenotypes in pregnant rats accompanied with activated AKT/mTOR signaling, decreased expression of FAM134B and LC3-II proteins and increased protein aggregation in placental tissues. Our study reveals the important role of lncRNA DUXAP8 in regulating trophoblast biological behaviors through FAM134B-mediated ER-phagy, providing a new theoretical basis for understanding the pathogenesis of PE.

Hypermethylated RASAL1’s promotive role in chemoresistance and tumorigenesis of choriocarcinoma was regulated by TET2 but not DNMTs

BackgroundPatients with choriocarcinoma (CC) accompanying chemoresistance conventionally present a poor prognosis. Whether ras protein activator like-1 (RASAL1) functions as a tumor promoter or suppressor depends on tumor types. However, the role of RASAL1 in process of chemoresistance of CC and underlying molecular mechanism remain elusive.MethodsThe expression pattern of RASAL1 in CC cells and tissues was measured using Western blotting, immunohistochemistry and qRT-PCR. Cell viability and proliferative ability were assessed by MTT assay, Tunnel assay and flow cytometric analysis. Additionally, the stemness was evaluated by the colony formation and tumor sphere formation. Methotrexate (MTX) was applied to exam the chemosensitivity of CC cells.ResultsThe expression of RASAL1 was reduced both at the protein and mRNA levels in CC tissues and cells compared to hydatidiform mole (HM) and invasive mole (IM). Loss of RASAL1 was attributed to its promoter hypermethylation and could be restored by 5-Aza. Knock-down of RASAL1 promoted the viability, proliferative potential, stemness and EMT phenotype of JEG-3 cells. However, induced overexpression of RASAL1 by 5-Aza significantly prohibited cell proliferation and stemness potential of the JAR cell. Additionally, the xenograft model indicated that knockdown of RASAL1 led to a remarkable increase of tumor volume and weight in comparison with its counterpart. Moreover, the stimulatory activity brought by decrease of RASAL1 could be deprived by β-catenin inhibitor XAV 939, yet the suppressive activity resulted from its promoter demethylation could be rescued by β-catenin activator BML-284, indicating that function of RASAL1 depends on β-catenin. Besides, the co-immunoprecipitation assay confirmed the physical binding between RASAL1 and β-catenin. Further investigations showed hypermethylated RASAL1 was regulated by TET2 but not DNMTs.ConclusionTaken together, the present data elucidated that reduced RASAL1 through its promoter hypermethylation regulated by TET2 promoted the tumorigenicity and chemoresistance of CC via modulating β-catenin both in vitro and in vivo.

Transcriptomic comparison of in vitro models of the human placenta.

Studying the human placenta through in vitro cell culture methods is necessary due to limited access and amenability of human placental tissue to certain experimental methods as well as distinct anatomical and physiological differences between animal and human placentas. Selecting an in vitro culture model of the human placenta is challenging due to representation of different trophoblast cell types with distinct biological roles and limited comparative studies that define key characteristics of these models. Therefore, the aim of this research was to create a comprehensive transcriptomic comparison of common in vitro models of the human placenta compared to bulk placental tissue from the CANDLE and GAPPS cohorts (N=1083). We performed differential gene expression analysis on publicly available RNA sequencing data from 6 common in vitro models of the human placenta (HTR-8/SVneo, BeWo, JEG-3, JAR, Primary Trophoblasts, and Villous Explants) and compared to CANDLE and GAPPS bulk placental tissue or cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast cell types derived from bulk placental tissue. All in vitro placental models had a substantial number of differentially expressed genes (DEGs, FDR<0.01) compared to the CANDLE and GAPPS placentas (Average DEGs=10,873), and the individual trophoblast cell types (Average DEGs=5,346), indicating that there are vast differences in gene expression compared to bulk and cell-type specific human placental tissue. Hierarchical clustering identified 53 gene clusters with distinct expression profiles across placental models, with 22 clusters enriched for specific KEGG pathways, 7 clusters enriched for high-expression placental genes, and 7 clusters enriched for absorption, distribution, metabolism, and excretion genes. In vitro placental models were classified by fetal sex based on expression of Y-chromosome genes that identified HTR-8/SVneo cells as being of female origin, while JEG-3, JAR, and BeWo cells are of male origin. Overall, none of the models were a close approximation of the transcriptome of bulk human placental tissue, highlighting the challenges with model selection. To enable researchers to select appropriate models, we have compiled data on differential gene expression, clustering, and fetal sex into an accessible web application: "Comparative Transcriptomic Placental Model Atlas (CTPMA)" which can be utilized by researchers to make informed decisions about their selection of in vitro placental models.

SPOP attenuates the proliferation, invasion, and migration of choriocarcinoma JAR cells by promoting KIF23 degradation

Abstract Objectives Choriocarcinoma is a highly malignant and aggressive trophoblastic tumor. In our previous study, we discovered that the speckle-type POZ protein (SPOP), which recognizes substrates of E3 ubiquitin ligase, plays a crucial role in trophoblast-derived choriocarcinoma cell lines. Therefore, we investigated the correlation between SPOP and the substrate kinesin-like protein KIF23, as well as the role of KIF23 in choriocarcinoma cells. Methods We constructed JAR cells overexpressing SPOP using lentiviral vectors and subsequently screened the related proteins through ubiquitination-modified quantitative proteomic analysis. The relationship between KIF23 and SPOP was determined using western blotting, and CCK-8, plate cloning, flow cytometry, and Transwell assays were used to investigate the effects of KIF23 and SPOP/KIF23. Results We identified the KIF23 protein and observed that SPOP promoted its degradation. The abundance of KIF23 increased after the addition of the protease inhibitor MG132. KIF23 was highly expressed in choriocarcinoma cells. Compared with JAR cells transfected with NC–small-interfering RNA (siRNA), the proliferation, invasion, migration, and percentage of G0/G1 cells in the KIF23-siRNA group were significantly lower, and the activation of the Akt/GSK3β signaling pathway was markedly attenuated. Additionally, the sh-SPOP+KIF23-siRNA group exhibited significantly inhibited JAR cell proliferation, invasion, and migration, along with clearly attenuated activation of the Akt/GSK3β signaling pathway. Conclusions SPOP attenuates the proliferation, invasion, and migration of choriocarcinoma JAR cells by promoting KIF23 degradation.

358 In Vitro Uptake of Harmful Algal Bloom Toxin Microcystin-LR in Human Placental Cells

OBJECTIVES/GOALS: Harmful algal blooms (HABs) are increasing in both frequency and intensity due to climate change. HABs release the toxin microcystin-LR (MC-LR) which enters cells via organic anion-transporting polypeptides (OATPs). In this study, we sought to assess the ability of MC-LR to accumulate in trophoblasts, potentially disrupting placental functions. METHODS/STUDY POPULATION: Intracellular accumulation of MC-LR at exposure concentrations of 0.1, 1, and 10 µM over 6 hrs was evaluated in immortalized JAR placental cytotrophoblasts. Western blotting was used to evaluate protein-bound MC-LR accumulation in JAR cells. The function of OATP transporters in JAR cells was determined by pre-incubating cells with 10µM cyclosporin A, a general OATP inhibitor for 1 hr, and then incubated with 1µM OATP substrate fluorescein for up to 40 min. Fluorescence of fluorescein was measured at Ex/Em: 494nm/515nm by spectrophotometry. RESULTS/ANTICIPATED RESULTS: A concentration-dependent increase of MC-LR bound proteins in JAR cells was observed at 6 hrs with the greatest intracellular accumulation of MC-LR at 10µM. In the transporter experiments, a significant decrease of fluorescein uptake by up to 45% into JAR cells was observed following cyclosporin A inhibition of OATPs. These findings are consistent with the functional expression of OATP transporters in JAR placenta cells. Ongoing studies are evaluating whether the cyclosporin A-mediated inhibition of OATPs also inhibits the uptake of MC-LR. DISCUSSION/SIGNIFICANCE: Although MC-LR is well-known for its hepatotoxic and neurotoxic effects, there is growing interest in examining its potential adverse impacts on female reproductive health, particularly during pregnancy. Active uptake of MC-LR into the placenta could interfere with placental and fetal development.

The metabolic activation of pentachlorophenol to chloranil as a potent inhibitor of human and rat placental 3β-hydroxysteroid dehydrogenases

Pentachlorophenol (PCP) is a widely used pesticide. However, whether PCP and its metabolite chloranil have endocrine-disrupting effects by inhibiting placental 3β-hydroxysteroid dehydrogenase 1 (3β-HSD1) remains unclear. The study used in vitro assays with human and rat placental microsomes to measure 3β-HSD activity as well as human JAr cells to evaluate progesterone production. The results showed that PCP exhibited moderate inhibition of human 3β-HSD1, with an IC50 value of 29.83 μM and displayed mixed inhibition in terms of mode of action. Conversely, chloranil proved to be a potent inhibitor, demonstrating an IC50 value of 147 nM, and displaying a mixed mode of action. PCP significantly decreased progesterone production by JAr cells at 50 μM, while chloranil markedly reduced progesterone production at ≥1 μM. Interestingly, PCP and chloranil moderately inhibited rat placental homolog 3β-HSD4, with IC50 values of 27.94 and 23.42 μM, respectively. Dithiothreitol (DTT) alone significantly increased human 3β-HSD1 activity. Chloranil not PCP mediated inhibition of human 3β-HSD1 activity was completely reversed by DTT and that of rat 3β-HSD4 was partially reversed by DTT. Docking analysis revealed that both PCP and chloranil can bind to the catalytic domain of 3β-HSDs. The difference in the amino acid residue Cys83 in human 3β-HSD1 may explain why chloranil is a potent inhibitor through its interaction with the cysteine residue of human 3β-HSD1. In conclusion, PCP is metabolically activated to chloranil as a potent inhibitor of human 3β-HSD1.

Mechanisms of YAP1-mediated trophoblast ferroptosis in recurrent pregnancy loss.

The purpose of our study is to investigate the function of YAP1 in the trophoblast ferroptosis and maternal-fetal interface communication of RPL. We collected 25 villous tissues and detected the expression of YAP1. Cell counting kit-8 assay, scratch wound-healing assay, and Matrigel invasion assay were performed to observe the proliferation, migration, and invasion of HTR-8/SVneo and JAR cells. Subsequently, measured the levels of reactive oxygen species (ROS), malondialdehyde (MDA), reduced glutathione (GSH), SLC7A11, SOD2, and GPX4. Ultimately, the use of ferroptosis activator (erastin) and inhibitor (Ferrostatin-1, fer-1) further confirmed the regulation by YAP1. In addition, established an in vitro-induced cell model to study the effect of YAP1 on the decidualization process. Finally, animal models were implemented for further confirmation. We found that YAP1 was downregulated in RPL patients. Overexpression of YAP1 could significantly enhance the proliferation, migration, and invasion of trophoblasts, and inhibit ferroptosis. Knocking down YAP1 exhibited the opposite effect. Rescue experiments have shown that YAP1 could upregulate the expression of SLC7A11 and GPX4, which are key molecules in the classic pathway of ferroptosis. In addition, the decidualization was impaired when hESCs were treated with conditioned medium of YAP1 knockdown trophoblasts. Moreover, we found that Yap1, Slc7a11, and Gpx4 were downregulated in the RPL mice, along with increased MDA and decreased GSH. Downregulation of YAP1 induces ferroptosis, thereby damaging the trophoblast invasion processes, which also disturbs the communication at the maternal-fetal interface. Our study identified YAP1 as a potential key molecule in the pathogenesis of RPL.

Human Seminal Extracellular Vesicles Enhance Endometrial Receptivity Through Leukemia Inhibitory Factor.

Seminal extracellular vesicles (EVs) contain different subgroups that have diverse effects on sperm function. However, the effect of seminal EVs-especially their subgroups-on endometrial receptivity is largely unknown. Here, we found that seminal EVs could be divided into high-density EVs (EV-H), medium density EVs, and low-density EVs after purification using iodixanol. We demonstrated that EV-H could promote the expression and secretion of leukemia inhibitor factor (LIF) in human endometrial cells. In EV-H-treated endometrial cells, we identified 1274 differentially expressed genes (DEGs). DEGs were enriched in cell adhesion and AKT and STAT3 pathways. Therefore, we illustrated that EV-H enhanced the adhesion of human choriocarcinoma JAr cell spheroids to endometrial cells through the LIF-STAT3 pathway. Collectively, our findings indicated that seminal EV-H could regulate endometrial receptivity through the LIF pathway, which could provide novel insights into male fertility.

Loss of KLF15 impairs endometrial receptivity by inhibiting EMT in endometriosis.

The impaired endometrial receptivity is a major factor contributing to infertility in patients with endometriosis (EM), but the underlying mechanism remains unclear. Our study aimed to investigate the role of Kruppel-like factor 15 (KLF15) in endometrial receptivity and its regulation in EM. We observed a significant decrease in KLF15 expression in the mid-secretory epithelial endometrial cells of EM patients compared to normal females without EM. To confirm the role of KLF15 in endometrial receptivity, we found a significantly reduced KLF15 expression and a significant decrease in embryo implantation number in the rat model via uterine horn infection with siRNA. This highlights the importance of KLF15 as a regulator receptivity. Furthermore, through ChIP-qPCR, we discovered that the progesterone receptor (PR) directly binds to KLF15 promoter regions, indicating that progesterone resistance may mediate the decrease in KLF15 expression in EM patients. Additionally, we found that the mid-secretory endometrium of EM patients exhibited impaired epithelial-mesenchymal transition (EMT). Knockdown of KLF15 upregulated E-cadherin and downregulated vimentin expression, leading to inhibited invasiveness and migration of Ishikawa cells. Overexpression KLF15 promotes EMT, invasiveness, and migration ability, and increases the attachment rate of JAR cells to Ishikawa cells. Through RNA-seq analysis, we identified TWIST2 as a downstream gene of KLF15. We confirmed that KLF15 directly binds to the promoter region of TWIST2 via ChIP-qPCR, promoting epithelial cell EMT during the establishment of endometrial receptivity. Our study reveals the involvement of KLF15 in the regulation of endometrial receptivity and its downstream effects on EMT. These findings provide valuable insights into potential therapeutic approaches for treating non-receptive endometrium in patients with EM.

N-linked α2,6-sialylation of integrin β1 by the sialyltransferase ST6Gal1 promotes cell proliferation and stemness in gestational trophoblastic disease

IntroductionGestational trophoblastic disease (GTD) encompasses a spectrum of rare pre-malignant and malignant entities originating from trophoblastic tissue, including partial hydatidiform mole, complete hydatidiform mole and choriocarcinoma. β-galactoside α2,6 sialyltransferase 1 (ST6Gal1), the primary sialyltransferase responsible for the addition of α2,6 sialic acids, is strongly associated with the occurrence and development of several tumor types. However, the role of ST6Gal1/α2,6 -sialylation of trophoblast cells in GTD is still not well understood. MethodsThe expression of ST6Gal1 was investigated in GTD and human immortalized trophoblastic HTR-8/SVneo cells and human gestational choriocarcinoma JAR cells. We evaluated the effect of ST6Gal1 on proliferation and stemness of trophoblastic cells. We also examined the effect of internal miR-199a-5p on ST6Gal1 expression. The role of ST6Gal1 in regulating α2,6-sialylated integrin β1 and its significance in the activation of integrin β1/focal adhesion kinase (FAK) signaling pathway were also explored. ResultsST6Gal1 was observed to be highly expressed in GTD. Overexpression of ST6Gal1 promoted the proliferation and stemness of HTR-8/SVneo cells, whereas knockdown of ST6Gal1 suppressed the viability and stemness of JAR cells. MiR-199a-5p targeted and inhibited the expression of ST6Gal1 in trophoblastic cells. In addition, we revealed integrin β1 was highly α2,6-sialylated in JAR cells. Inhibition of ST6Gal1 reduced α2,6-sialylation on integrin β1 and suppressed the integrin β1/FAK pathway in JAR cells, thereby affecting its biological functions. DiscussionThis study demonstrated that ST6Gal1 plays important roles in promoting proliferation and stemness through the integrin β1 signaling pathway in GTD. Therefore, ST6Gal1 may have a potential role in the occurrence and development of GTD.

Differential expression of tsRNAs and miRNAs in embryo culture medium: potential impact on embryo implantation.

Can small RNA derived from embryos in conditioned embryo culture medium (ECM) influence embryo implantation? We employed small RNA sequencing to investigate the expression profiles of transfer RNA-derived small RNA (tsRNA) and microRNA (miRNA) in ECM from high-quality and low-quality embryos. Quantitative real-time PCR was employed to validate the findings of small RNA sequencing. Additionally, we conducted bioinformatics analysis to predict the potential functions of these small RNAs in embryo implantation. To establish the role of tiRNA-1:35-Leu-TAG-2 in embryonic trophoblast cell adhesion, we utilized co-culture systems involving JAR and Ishikawa cells. Our analysis revealed upregulation of nine tsRNAs and four miRNAs in ECM derived from high-quality embryos, whereas 37 tsRNAs and 12 miRNAs exhibited upregulation in ECM from low-quality embryos. The bioinformatics analysis of tsRNA, miRNA, and mRNA pathways indicated that their respective target genes may play pivotal roles in both embryo development and endometrial receptivity. Utilizing tiRNA mimics, we demonstrated that the prominently expressed tiRNA-1:35-Leu-TAG-2 in the low-quality ECM group can be internalized by Ishikawa cells. Notably, transfection of tiRNA-1:35-Leu-TAG-2 into Ishikawa cells reduced the attachment rate of JAR spheroids. Our investigation uncovers significant variation in the expression profiles of tsRNAs and miRNAs between ECM derived from high- and low-quality embryos. Intriguingly, the release of tiRNA-1:35-Leu-TAG-2 by low-quality embryos detrimentally affects embryo implantation and endometrial receptivity. These findings provide fresh insights into understanding the molecular foundations of embryo-endometrial communication.

In vitro effects of heparin-binding epidermal growth factor on adhesion stage of implantation.

A member of the epidermal growth factor (EGF) family, the heparin-binding EGF (HB-EGF) is expressed in the uteri of both humans and mice during the implantation process. To study the effects of HB-EGF on adhesion stage, we developed an in vitro implantation model employing Ishikawa cell line and JAR cell line, which may attach to Ishikawa cells. For 1, 6, 12, and 24 hours, co-cultures of JAR spheroids grown on Ishikawa monolayers were treated with 1, 10, and 100 ng∕mL doses of HB-EGF. Using immunocytochemistry and Western blot analysis, the effects of HB-EGF on the protein expressions of E-cadherin, Erb-B2 receptor tyrosine kinase 4 (ErbB4), and integrin ανβ3 in Ishikawa and JAR cells were examined semi-quantitatively and quantitatively. Ultrastructural changes of in vitro implantation model were investigated by transmission electron microscopy. We revealed that HB-EGF influenced trophoblast cell adhesion to endometrial cells by upregulating the expression of the proteins ErbB4 and trophoblastic integrin ανβ3. Decrease in trophoblastic E-cadherin expression and increase in endometrial E-cadherin expression were demonstrated accompanying morphological variations in cells required for the invasion. We discovered ultrastructurally that Ishikawa cells acquired uterodome-like appearance, including the organelles, when 10 and 100 ng∕mL dosages of HB-EGF were administered for 12 and 24 hours. However, following additional hours of adhesion and invasion, their intercellular spaces enlarged. The trafficking of vesicular transport was enhanced by JAR spheroids. We therefore discovered that in this implantation paradigm, HB-EGF may enhance the receptivity of Ishikawa cells and the adherence of JAR cells.

Twist-1 Stimulates the Malignant Behaviors of Hydatidiform Mole via the PI3K/AKT Pathway.

Hydatidiform mole (HM) is a common pregnancy disease among women of gestational age. Twist-related protein 1 (Twist-1) is involved in the development of various tumors, but its role in HM is poorly defined. This study aimed to explore Twist-1 expression and its biological function in HM cells. Twist-1 expression in HM was detected by immunohistochemistry and quantitative real-time polymerase chain reaction (qRT-PCR). The effects of silencing Twist-1 on choriocarcinoma (CCA) cell proliferation were detected by cell counting kit-8 (CCK-8) and clone formation assays. CCA cell migration and invasion were detected through transwell assay. Western blot was used to detect epithelial-mesenchymal transition (EMT) and the expression of phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway-related proteins. Twist-1 expression was upregulated in HM tissues (p < 0.001) and CCA cells (p < 0.01). Twist-1 silencing inhibited proliferation of BeWo and JAR cells (p < 0.01, p < 0.05) as shown by CCK-8 assay (p < 0.01) and clone formation assays (p < 0.01, p < 0.05). Twist-1 silencing inhibited the migration (p < 0.01) and invasion activity (p < 0.01, p < 0.05) of BeWo and JAR cells. Western blot results showed that Twist-1 silencing promoted E-cadherin (p < 0.01) expression, and inhibited N-cadherin (p < 0.01, p < 0.05) and vimentin (p < 0.01, p < 0.05) expression in BeWo and JAR cells. Twist-1 downregulation decreased protein levels of p-PI3K (p < 0.01) and p-AKT (p < 0.01, p < 0.05) in BeWo and JAR cells. Silencing Twist-1 inhibits the malignant behavior of CCA cells, which may play a part by inhibiting the EMT process and the PI3K/AKT pathway.

Comparative analysis of galectin-1, galectin-3, and galectin-8 signatures in normal and choriocarcinoma trophoblast cell lines

Galectins are found in the placenta during both normal and pathological conditions. Specifically, galectins -1, -3, and -8 have been identified in extravillous trophoblast cells, with galectins -1, and -3 playing notable roles in trophoblast cell function. Altered expression profiles of galectins are linked to various cancers. This study explores the expression and localization of galectins -1, -3, and - 8 in a normal extravillous HTR-8/SVneo cell line and two choriocarcinoma cell lines, JAR and JEG-3. Expression levels of these galectins at both mRNA and protein levels were evaluated using PCR, qPCR, and Western blotting, while their cellular localization and distribution were assessed through immunocytochemistry, subcellular fractionation, and Western blotting. Galectins -1, -3, and -8 were expressed at lower levels in JAR and JEG-3 cells compared to HTR-8/SVneo cells. Additionally, different splice variants of galectin-8 were detected across all cell lines. Galectin-1 was predominantly localized at the plasma membrane. Notable differences in galectin-3 localization and distribution were observed between HTR-8/SVneo cells and choriocarcinoma cells. These findings highlight variations in the expression, staining patterns, and subcellular distribution of galectins -1, -3, and -8, suggesting their potential involvement in the pathology of choriocarcinoma.

Freshwater algae Cladophora glomerata and Vaucheria sp. from Serbia as sources of bioactive compounds: Chemical analysis and biological activities

We examined potential biological activities of two taxa of freshwater algae, Cladophora glomerata and Vaucheria sp., from Serbia. The total phenolic and pigment contents, antioxidant potential, antimicrobial, antibiofilm activities, and cytotoxicity of the ethanol and acetone extracts were evaluated. The extracts were also subjected to Fourier transform infrared spectroscopy (FTIR) analysis. The levels of total phenolic compounds, chlorophylls a and b, and carotenoids varied based on both the algal taxa and the type of extracts. FTIR analysis showed the presence of lipids, unsaturated fatty acids, protein, carbohydrates, and phenols in the algal extracts. The extracts had moderate DPPH radical scavenging activity and lower reducing power compared with ascorbic acid. The antimicrobial activity expressed as minimum inhibitory concentrations ranged from 0.31 mg/mL to 10 mg/mL. The strains of Staphylococcus aureus and Bacillus cereus isolated from food samples, as well as S. aureus ATCC 25923, were the most sensitive. For the first time, the antibiofilm activity test revealed 98.7% inhibition of S. aureus biofilm formation. The extracts exhibited cytotoxic effects on choriocarcinoma JAR cells but without selectivity on normal fetal lung fibroblast MRC-5 cells. This is the first report on the biological activities of freshwater macroalgae from Serbia.

#136 : Osteopontin Facilitates Embryo Growth, Implantation, and Cellular Metabolism via Interacting with αvβ3 and RGS2 Pathway: Evidence from Mouse Embryos and a 3D In-Vitro Implantation Model

Background and Aims: We conducted a study on mice to investigate how osteopontin, a mediator of cell-cell and cell-extracellular matrix (ECM) communication and invasion, influences embryonic development, metabolism, and implantation potential, with confirmation by a 3D in-vitro implantation model. Method: Embryos were collected from female mice and cultured under different conditions: HTF only, HTF supplemented with OPN at different doses, and OPN-supplemented HTF with anti-[Formula: see text]v[Formula: see text]3 Ab or RGS2 inhibitors. Embryo morphology, development, and implantation rates were evaluated. Microarrays were used to evaluate gene expression profiling. Choriocarcinoma JAR and epithelial RL95-2 cell lines were adopted for the in-vitro implantation model. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were used to measure cellular metabolism levels. Results: The supplementation of OPN best enhanced the rate of embryo hatching-out and implantation onto the matrigel at 100 nM, but a higher degeneration rate was seen at a higher concentration ([Formula: see text]200 nM). In OPN-treated embryos, microarray analysis showed 51 significantly down-regulated genes and 60 up-regulated genes, including RGS2. Inhibition of [Formula: see text]v[Formula: see text]3 integrin decreased embryo implantation rates and down-regulated RGS2. Validation of the 3D implantation model using JAR spheroids and RL95-2 monolayer showed a similar effect. OPN also increased OCR and ECAR of the JAR cells, and migration assays revealed increased migration ability in OPN-overexpressed JAR cells, which was then inhibited by [Formula: see text]v[Formula: see text]3 and RGS2 inhibitors. Conclusion: Osteopontin, at a reasonable concentration, could enhance embryo development and implantation by interacting with [Formula: see text]v[Formula: see text]3 integrin, subsequently facilitating the RGS2 pathway. OPN has the potential to be a supplement during in vitro embryo culture in ART intervention.

MiR-150-5p contributes to unexplained recurrent spontaneous abortion by targeting VEGFA and downregulating the PI3K/AKT/mTOR signaling pathway

PurposeThe purpose of this study is to investigate the function of miR-150-5p in URSA. MethodTwenty-six chorionic villous tissues were collected to examine the expression of miR-150-5p and VEGFA by using quantitative polymerase chain reaction (qPCR) and western blot assay, respectively. Transwell assay was conducted to assess the migration and invasion ability of trophoblast cells. The dual-luciferase reporter assay was applied to determine the relationship between miR-150-5p and VEGFA in vitro. Relevant signaling pathway protein expression level was measured via western blot assay. Signaling transduction inhibitor LY294002 was used to block PI3K/AKT/mTOR signaling pathway. Finally, in vivo the effect of miR-150-5p on embryonic absorption rate was evaluated in mice.ResultsClinical samples revealed that miR-150-5p expression was significantly elevated in the villous tissues and serum of URSA patients. Moreover, the overexpressing of miR-150-5p could inhibit both HTR-8/SVneo cell and JAR cell migration, invasion, and restrained PI3K/AKT/mTOR signaling pathway by targeting VEGFA in vitro. This inhibitory effect of miR-150-5p could be reversed by overexpressing the gene of vascular epithelial growth factor A (VEGFA). In contrary, inhibition of miR-150-5p significantly enhanced migration, invasion ability of both HTR-8/SVneo and JAR cells, and also could stimulate PI3K/AKT/mTOR signaling pathway. This promoting effect of miR-150-5p could be ameliorated by LY294002 (PI3K inhibitor). Finally, after miR-150-5p overexpression in vivo, the embryo resorption rate in pregnant mice was increased significantly.ConclusionsOverall, these findings imply that miR-150-5p is among the key factors that regulate the pathogenesis of URSA.

Sphingomyelin synthase 1 supports two steps of rubella virus life cycle

Our knowledge of the regulatory mechanisms that govern the replication of the rubella virus (RV) in human cells is limited. To gain insight into the host-pathogen interaction, we conducted a loss-of-function screening using the CRISPR-Cas9 system in the human placenta-derived JAR cells. We identified sphingomyelin synthase 1 (SGMS1 or SMS1) as a susceptibility factor for RV infection. Genetic knockout of SGMS1 rendered JAR cells resistant to infection by RV. The re-introduction of SGMS1 restored cellular susceptibility to RV infection. The restricted step of RV infection was post-endocytosis processes associated with the endosomal acidification. In the late phase of the RV replication cycle, the maintenance of viral persistence was disrupted, partly due to the attenuated viral gene expression. Our results shed light on the unique regulation of RV replication by a host factor during the early and late phases of viral life cycle.