Trophoblast Cells HTR-8 Research Articles

Downregulation of TCL6 protected human trophoblast cells from LPS-induced inflammation and ferroptosis.

Dysregulation of noncoding RNAs has been reported to have a close correlation with preeclampsia(PE)development. TCL6 was upregulated in patients with PE. In this study, we examined the impacts of TCL6 on modulating HTR-8/SVneo cells induced by LPS. LPS (100 and 200 ng/ml) was applied to induce inflammation in trophoblast cells HTR-8/SVneo. Cell viability, apoptosis, and transwell experiments were conducted. The ELISA methods were used for pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. MDA, GSH, and GPX kits were employed. Transfection was performed for expression regulation of TCL6, miR-485-5p, and TFRC in cells. Bioinformatic online tools were used to predict the targeting sites. Luciferase and RNA immunoprecipitation-qPCR were done to verify the interactions of TCL6, miR-485-5p, and TFRC. RNA expression levels were measured using RT-qPCR, and protein expression of TFRC and GPX4 was detected using a western blot. The free Fe (II) contents were measured. LPS decreased viability, invasion, and migration but enhanced apoptosis, ferroptosis, and inflammation. TCL6 expression was enhanced by LPS induction. The knockdown of TCL6 increased HTR-8/SVneo cell viability and invasion but inhibited cell apoptosis, inflammation, and ferroptosis while inhibition of miR-485-5p could reverse this through TFRC regulation. Moreover, miR-485-5p was sponged by TCL6 and bound to TFRC. TCL6 protected trophoblast cells from LPS-induced injury through the TFRC pathway.

Oxidative low-density lipoprotein oxLDL induces pyroptosis in trophoblast cells HTR-8/Svneo by downregulating TET2

ObjectiveTo investigate pyroptosis in the trophoblast cells HTR-8/Svneo induced by TET2 in oxidized low-density lipoprotein (oxLDL). MethodsHTR-8/Svneo cells were treated with vehicle (0 mg/L) and 50 mg/L oxLDL for 24 h. The effects of oxLDL on TET2 expression in HTR-8/Svneo cells were detected by Western blot. Knockdown of TET2 was performed in HTR-8/Svneo cells using si-TET2. CCK8, Transwell, and wound healing assays were used to detect the effect of TET2 knockdown on the proliferation, cell cycle, migration, and invasion of HTR-8/Svneo cells, respectively. Western blot was used to quantify the expression levels of the pyroptosis proteins GSDMD and caspase-1 in si-TET2 treated cells. ResultsoxLDL downregulates TET2 expression in a concentration-dependent manner. TET2 knockdown inhibits HTR-8/Svneo invasion and migration but promotes cell proliferation. Western blot results showed that TET2 knockdown upgregulated the expression of GSDMD and caspase-1. ConclusionoxLDL induces pyroptosis of HTR-8/Svneo by downregulating TET2 expression, resulting in decreased trophoblast invasion and migration. These data suggest that TET2-induced pyroptosis play a critical role in gestational pathologies, such as preeclampsia. Further studies can clarify the mechanisms of this process and elucidate potential preventive and therapeutic targets.

Transcriptional Factor Forkhead Box D1 Upregulates Sirtuin3 by Activating the Wnt/β-Catenin Pathway to Alleviate HTR-8/Svneo Trophoblast Cell Dysfunction in Preeclampsia

The proliferation, invasion, migration and apoptosis of trophoblast cells in preeclampsia are closely related to the occurrence and development of preeclampsia. Transcription factors forkhead box D1 and Sirtuin3 are abnormally expressed in preeclampsia, and Sirtuin3 plays a regulatory role in cell proliferation, invasion and apoptosis in related diseases. However, the studies on forkhead box D1 and Sirtuin3 in preeclampsia and their specific mechanisms have not been reported so far. In this study, the expression of Sirtuin3 in Human chorionic trophoblast cells HTR-8/Svneo was inhibited by cell transfection, and then the effects of Sirtuin3 expression in interfering cells on cell invasion, migration and apoptosis were detected by MTT, TUNEL, Western blot, wound healing and Transwell techniques. Subsequently, the binding between forkhead box D1 and Sirtuin3 was predicted by JASPAR website and verified by luciferase assay and ChIP assay. Finally, cell invasion, migration and apoptosis were detected after overexpression of forkhead box D1 and interference with Sirtuin3, and the Wnt/β-catenin signaling pathway was detected to explore the mechanism. We found that interfering with Sirtuin3 induced apoptosis of HTR-8/Svneo cells and inhibited cell invasion and migration. Forkhead box D1 transcriptional activation of Sirtuin3 alleviated HTR-8/SVneo cell dysfunction through activation of the Wnt/β-catenin signaling pathway. Overall, transcriptional factor forkhead box D1 can upregulate Sirtuin3 by activating the Wnt/β-catenin pathway to alleviate HTR-8/Svneo trophoblast cell dysfunction in preeclampsia.

HDAC5 inactivates CYR61-regulated CD31/mTOR axis to prevent the occurrence of preeclampsia.

Our study was to pinpoint the significance of histone deacetylase 5 (HDAC5) affecting the pathogenesis of preeclampsia (PE) via CD31/mammalian target of rapamycin (mTOR) axis by regulating cysteine-rich angiogenic inducer 61 (CYR61). Expression of HDAC5, CYR61, and CD31/mTOR in placental tissues of patients with PE and trophoblast cells HTR-8/SVneo cells was determined first followed by their interaction analysis. Following different transfection, the significance of HDAC5 in cell functions was assayed in relation to CYR61 and CD31/mTOR. An in vivo PE mouse model was constructed for further validation. The clinical tissue and in vitro cell experimentations discovered that HDAC5 was downregulated in placental tissues of PE patients and trophoblast cells, while CYR61, CD31, mTOR, and p-mTOR displayed upregulation. After overexpression of HDAC5, trophoblast cell functions were enhanced. HDAC5 reduced the acetylation enrichment of H3K27 to inhibit the expression of CYR61. Furthermore, CYR61 promoted the activation of CD31/mTOR axis, thereby inhibiting HTR-8/SVneo cell functions. The in vivo rat model confirmed the above alterations. Taken together, HDAC5 contributes to downregulation of CYR61 through histone deacetylation, inactivating CD31/mTOR axis, which prevents the occurrence and development of PE.

The role of circRNA polyribonucleotide nucleoside transferase 1 on Gestational Diabetes Mellitus.

This study aimed to focus on the mechanism of circRNA polyribonucleotide nucleoside transferase 1 (circ-PNPT1)-mediated miR-889-3p/PAK1 on gestational diabetes mellitus (GDM). Placental tissues from normal pregnancy and GDM patients were collected to detect the levels of circ-PNPT1, miR-889-3p, and PAK1. The high glucose-induced human trophoblast cells HTR-8/SVneo were adopted to stimulate the GDM model in vitro (HG group) and were transfected with lentivirus to silence circ-PNPT1 (si-circ-PNPT1 group) and mimic to overexpress miR-889-3p (miR-889-3p group). Cell proliferation, apoptosis, migration, and invasion were detected by CKK-8, flow cytometry, Transwell, and scratch assay, respectively. The results showed that the expressions of circ-PNPT1 and PAK1 in the GDM patients were up-regulated, and miR-889-3p was down-regulated (P< 0.05). Compared with cells in the control group, the circ-PNPT1 and PAK1 in the HG group were up-regulated, and miR-889-3p was down-regulated (P< 0.05). The cell proliferation, migration, and invasion abilities were weakened, and the apoptosis rate increased (P< 0.05). E-cadherin protein was elevated, and the N-cadherin and Vimentin decreased (P< 0.05). Compared with the HG group, the expressions of circ-PNPT1 and PAK1 in the other two groups decreased, and miR-889-3p increased (P< 0.05). The cell proliferation, migration, and invasion were enhanced, and the apoptosis rate decreased (P< 0.05). E-cadherin, N-cadherin, and Vimentin decreased (P< 0.05). There were targeted binding sites for miR-889-3p with circ-PNPT1 and PAK1, indicating circ-PNPT1 promoted HG-induced trophoblast dysfunction through the miR-889-3p/PAK1 axis.

Decreased expression of SEMA4D in recurrent implantation failure induces reduction of trophoblast invasion and migration via the Met/PI3K/Akt pathway

Recurrent implantation failure (RIF) associated with impaired endometrial receptivity and other factors. Disease-specific therapy has yet to be developed due to the lack of understanding of underlying mechanism(s). Herein we investigated the key factors of endometrial receptivity in RIF patients by transcriptomic sequencing. In vitro cellular model was used to delineate the molecular mechanism of key factors on proliferation, invasion and migration of trophoblast cells. SEMA4D was identified as the key factors of endometrial receptivity with significantly lower expression in the mid-secretory endometrium of RIF patients compared with those from normal fertile women. The binding of SEMA4D to its receptor Plexin-B1 on human trophoblast cells HTR-8/SVneo resulted in the activation of Met/PI3K/Akt signaling, which promotes trophoblast cell invasion and migration by enhancing MMP-2 expression. Moreover, the effect of SEMA4D on HTR-8/SVneo could be blocked by knocking down Met with specific siRNA or treating with LY294002. Collectively, our data indicate that decreased expression of SEMA4D in endometrium impair the process of trophoblast invasion and migration through Met/PI3K/Akt pathway, which provides insights into this essential physiological process in the development of RIF.

LncRNA DANCR promotes the migration an invasion and of trophoblast cells through microRNA-214-5p in preeclampsia

ABSTRACT Studies have shown that lncRNA DANCR is down-regulated in placental tissues of patients with preeclampsia (PE). The aim of this study was to explore the effect of lncRNA DANCR on trophoblast cells as well as its acting mechanism. We disrupted or overexpressed lncRNA DANCR in trophoblast cells HTR-8/SVneo and JEG-3 and detected the associated cellular functional changes by MTT, flow cytometry, Transwell experiment, and scratch experiment. The results showed that overexpression of lncRNA DANCR significantly increased the proliferation, invasion, migration, and EMT process of trophoblast cells. Interfering with lncRNA DANCR showed the opposite result. Further, the targeted interaction between lncRNA DANCR and miR-214-5p was confirmed by the dual-luciferase reporter gene assay. In addition, the expression of PI3K/AKT signaling pathway-related proteins was analyzed by Western blot. Overexpression of lncRNA DANCR can increase the phosphorylation of PI3K/AKT protein and activate this signaling pathway. In conclusion, the enforcing of lncRNA DANCR activates the activation of the PI3K/AKT pathway by down-regulating miR-214-5p, and promotes the migration and invasion of chorionic trophoblast cells. This provides a potential new target for PE therapy.

AUTOPHAGY IN THE HTR-8/SVNEO CELL OXIDATIVE STRESS MODEL IS ASSOCIATED WITH NLRP1 INFLAMMASOME ACTIVATION

Based on the establishment of an in vitro model of trophoblast oxidative damage in human placental trophoblast cell HTR-8/SVneo, this study investigated whether there were activation of NLRP1 inflammasomes and excessive autophagy in oxidative stress damage. And further demonstrate whether there is a cascade relationship between the activation of NLRP1 inflammasomes and the phenomenon of excessive autophagy.

Silencing of ANGPTL8 Alleviates Insulin Resistance in Trophoblast Cells.

This study aims to investigate the effect of angiopoietin like 8 (ANGPTL8) on gestational diabetes mellitus (GDM) and insulin resistance (IR). The GDM model was induced by high fat diet in mice, and IR was observed. The expression and secretion of ANGPTL8 were promoted in placenta of GDM mice. IR was induced in trophoblast cell HTR-8/SVneo by treatment of high concentration of insulin, and the expression levels of ANGPTL8 were increased. Silencing of ANGPTL8 alleviated IR and decreased glucose uptake in HTR-8/SVneo cells. However, the inflammation and oxidative stress in IR cells were not restrained by ANGPTL8 knockdown. In addition, c-Jun N-terminal kinase (JNK) signaling was activated by IR, which was inhibited by silencing of ANGPTL8. The effect of ANGPTL8 knockdown on IR was attenuated by JNK antagonist, and aggravated by JNK agonist, suggesting that ANGPTL8 affected IR by regulating JNK signaling. In conclusion, we demonstrated that the silencing of ANGPTL8 ameliorated IR by inhibiting JNK signaling in trophoblast cells. These findings may provide novel insights for diagnosis and treatment of GDM in clinic.

MiR-212-3p promotes proliferation and migration of trophoblast in fetal growth restriction by targeting placental growth factor

ABSTRACT The purpose of this study was to evaluate the function and possible mechanism of miR-212-3p in fetal growth restriction (FGR) and to demonstrate the relationship between miR-212-3p and placental growth factor (PGF). First, we used qRT-PCR to detect the expression of miR-212-3p and PGF in placental tissues of normal delivery (HC group) and FGR, as well as in human trophoblast cell HTR-8/Svneo. The results revealed that miR-212-3p expression was significantly upregulated and PGF was significantly downregulated in placental tissue in the FGR group compared with the HC group. In addition, interference with miR-212-3p expression increased the proliferation, invasion, and migration of HTR-8/SVneo cells and decreased apoptosis of cells. Meanwhile, Western blot results showed that miR-212-3p expression downregulation promoted the phosphorylated protein expression of Phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT), which in turn activated the PI3K/AKT signaling pathway. And the results of dual luciferase reporter further showed that miR-212-3p could target PGF, and the expression of both was negatively correlated in FGR group tissues. In addition, downregulation of miR-212-3p expression reversed the inhibitory effect of PGF downregulation on HTR-8/SVneo cells. In conclusion, miR-212-3p can target and inhibit the PGF expression and regulate the PI3K/AKT signaling pathway to regulate trophoblast cell invasion, migration, proliferation and cell apoptosis. This provides a potential biomarker for the development of FGR.

Downregulating miR-96-5p promotes proliferation, migration, and invasion, and inhibits apoptosis in human trophoblast cells via targeting DDAH1

Several microRNAs (miRs) have been found to have modulating effects on trophoblast functions, yet the biological role and function of miR-96-5p and its interaction with Dimethylarginine Dimethylaminohydrolase 1 (DDAH1) remained poorly understood.After lentivirus transfection, the proliferation, migration, invasion and apoptosis of human trophoblast cells HTR-8/SVneo and SGHPL-4 were determined by Cell Counting Kit-8 (CCK-8) assay, scratch assay, Transwell, and flow cytometry, respectively. Relative expressions of miR-96-5p, DDAH1, and apoptosis-related proteins (B-cell lymphoma 2, Bcl-2; Bcl-2-associated X protein, Bax; cleaved (C) caspase-3) were detected via quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot as needed. The target gene of miR-96-5p and their potential binding sites were predicted using TargetScan V7.2 and confirmed by dual-luciferase reporter assay.MiR-96-5p downregulation promoted proliferation, migration and invasion, suppressed apoptosis, and decreased miR-96-5p expression in trophoblast cells in vitro, while miR-96-5p upregulation had the opposite effects. DDAH1 was recognized as a target gene of miR-96-5p, and silencing DDAH1 reversed the effects of miR-96-5p downregulation on the proliferation, migration, invasion and apoptosis of trophoblast cells as well as the expressions of apoptosis-related proteins.MiR-96-5p downregulation promotes proliferation, migration, and invasion, and suppresses apoptosis in human trophoblast cells in vitro via targeting DDAH1, which provides evidence for the implication of miR-96-5p in the functional modulation of trophoblasts.

HDAC2-mediated proliferation of trophoblast cells requires the miR-183/FOXA1/IL-8 signaling pathway.

Pre-eclampsia (PE) is a major cause of maternal and perinatal death. Previous research has indicated the role of histone deacetylase 2 (HDAC2) in the pathogenesis of PE but the relevant molecular mechanisms are unknown. However, there is hitherto little information concerning the molecular mechanism behind HDAC2 in PE. Herein, we hypothesized that HDAC2 promotes trophoblast cell proliferation and this requires the involvement of microRNA-183 (miR-183), forkhead box protein A1 (FOXA1), and interleukin 8 (IL-8). We collected placental specimens from 30 PE affected and 30 normal pregnant women. HDAC2 and FOXA1 were poorly expressed while miR-183 and IL-8 were highly expressed in placental tissues in PE. In vitro, HDAC2 overexpression enhanced the proliferation, migration, and invasion of human trophoblast cells HTR-8/SVNEO. HDAC2 inhibited the expression of miR-183 by diminishing H4 acetylation in the miR-183 promoter region. miR-183 inhibition by its specific inhibitor increased the expression of FOXA1 and thus enhanced HTR-8/SVNEO cell proliferation, migration, and invasion. FOXA1, a transcriptional factor, enhanced HTR-8/SVNEO cell proliferation, migration, and invasion by inhibiting the transcription of IL-8. We also observed HDAC2 knockdown was lost upon FOXA1 overexpression, suggesting that HDAC2 could promote HTR-8/SVNEO proliferation, migration, and invasion through the miR-183/FOXA1/IL-8 pathway. In summary, the results highlighted the role of the HDAC2/miR-183/FOXA1/IL-8 pathway in PE pathogenesis and thus suggest a novel molecular target for PE.

MiR-124–3p promotes trophoblast cell HTR-8/SVneo pyroptosis by targeting placental growth factor

IntroductionMiR-124–3p is one of the aberrantly expressed miRNAs in the placentas of patients with preeclampsia (PE), a severe obstetric complication characterised by hypertension and proteinuria. This study aimed to investigate the role of miR-124–3p in the invasion, migration and death of trophoblast cells and explore the potential mechanisms. MethodsMiR-124–3p expression in placental tissues was compared with that in normal placenta. HTR8/SVneo cells were then transfected with miR-124–3p mimics to examine cellular apoptosis, migration and invasion. Furthermore, the expression of pyroptosis-related molecular NLRP3, Pro-caspase1, caspase1, IL-1β and GSDMD was examined with Western blot. Dual luciferase reporter assay was performed to confirm that placental growth factor (PLGF) is a direct target of miR-124–3p, and HTR-8/SVneo cells were transfected with small interfering RNA PLGF (siPLGF) to determine whether PLGF knockdown promotes HTR-8/SVneo pyroptosis. Finally, intracellular ROS was diminished with N‐acetyl‐l‐cysteine (NAC) to observe whether the pro-pyroptosis effect of PLGF knockdown is alleviated. ResultsResults in this study showed that miR-124–3p expression was remarkably increased in the placenta of patients with PE. Moreover, the transfection of miR-124–3p mimics in trophoblastic cells significantly decreased cell migration and invasion but increased cell apoptosis and the expression of NLRP3, pro-caspase1, caspase1, IL-1β and GSDMD. Therefore, PLGF was confirmed as a direct target of miR-124–3p. Finally, siPLGF transfection can mimic the effects of miR-124–3p, and NAC can inhibit this effect. ConclusionIn summary, miR-124–3p is upregulated in PE, and in vitro functional analysis revealed that this mRNA inhibits trophoblast invasion and migration but promotes cell pyroptosis partly via the PLGF-ROS pathway.

Elevated miR-23a impairs trophoblast migration and invasiveness through HDAC2 inhibition and NF-κB activation

Preeclampsia (PE) is a pregnancy-specific disorder characterized by the onset of hypertension and proteinuria with onset after the 20th week of gestation. The pathogenesis of PE is attributed to increased trophoblast cell death and poor trophoblast migration/invasiveness. This study investigates the function of microRNA-23a (miR-23a) in PE and its effects on migration and invasion of trophoblast cells HTR-8/SVneo. We found higher expression of miR-23a in placental tissue samples from PE pregnant women compared to samples from normal pregnant women. Enhancing miR-23a expression by its specific mimic reduced HTR-8/SVneo cell migration and invasion and increased HTR-8/SVneo cell apoptosis. The dual-luciferase reporter gene assay revealed miR-23a binding with HDAC2. We found that HDAC2 was poorly expressed in placental tissue samples from PE pregnant women, and its expression correlated inversely with miR-23a expression. HTR-8/SVneo cells showed diminished HDAC2 expression upon miR-23a elevation and increased HDAC2 expression upon miR-23a inhibition. Lentivirus-mediated HDAC2 knockdown mimicked the effects of miR-23a on HTR-8/SVneo cells and led to NF-κB activation. Similarly, HDAC2 overexpression and NF-κB inhibition both abrogated the effects of miR-23a on HTR-8/SVneo cells, suggesting that miR-23a reduced HTR-8/SVneo cell migration and invasion and increased HTR-8/SVneo cell apoptosis by HDAC2 inhibition and NF-κB activation. In summary, these results support a novel role of miR-23b in invasion and apoptosis of trophoblast cells, and imply that targeting miR-23b may be a new avenue for treating PE.

MiR-93 Inhibits Trophoblast Cell Proliferation and Promotes Cell Apoptosis by Targeting BCL2L2 in Recurrent Spontaneous Abortion.

Recurrent spontaneous abortion (RSA) is a common health problem that affects 1-5% of women in reproductive age. Plenty of studies have indicated that microRNAs (miRNAs) are involved in the occurrence of miscarriage. MiR-93 has a wide range of functions in mammalian tissues and plays an important role in many diseases especially for cancers. However, it remains unknown whether miR-93 is associated with human RSA. In this report, clinical samples revealed that miR-93 expression was significantly elevated in the villi tissues of RSA patients. Upregulation of miR-93 inhibited human trophoblast cells HTR-8/SVneo cell proliferation, migration, and invasiveness, but promoted cell apoptosis in vitro. Conversely, the downregulation of miR-93 reversed these effects. Bcl-2 like protein 2 (BCL2L2), a potential target gene of miR-93, was inversely correlated with miR-93 expression in the villi of clinical samples. Furthermore, the luciferase reporter system demonstrated that miR-93 directly downregulated the expression of BCL2L2 by binding a specific sequence of its 3'-untranslated region (3'UTR). Collectively, these data strongly suggest that miR-93 regulates trophoblast cell proliferation, migration, invasive, and apoptosis by targeting BCL2L2 expression and is involved in the pathogenesis of RSA.

MiR-132 serves as a diagnostic biomarker in gestational diabetes mellitus and its regulatory effect on trophoblast cell viability

BackgroundGestational diabetes mellitus (GDM) leads to poor pregnancy outcomes. Strategies that improve trophoblast cell function are important methods for GDM treatment. This study aimed to investigate the expression and diagnostic potential of microRNA-132 (miR-132) in GDM patients, and further analyzed the effects of miR-132 on HTR-8/SVneo cell proliferation.MethodsQuantitative real-time PCR was applied to estimate the expression of miR-132. A receiver operating characteristics curve (ROC) analysis was performed to evaluate the diagnostic value of serum miR-132 in GDM patients. In vitro regulation of miR-132 in trophoblast cell HTR-8/SVneo was achieved by cell transfection, and the effects of miR-132 on cell proliferation were assessed using CCK-8 assay.ResultsExpression of miR-132 was decreased in serum and placenta tissues in GDM patients compared with the healthy women. A negative correlation was found between the serum miR-132 levels and fasting blood glucose of the GDM patients. A ROC curve shown the serum miR-132 had considerable diagnostic accuracy with an area under the curve (AUC) of 0.898. High glucose (HG) treatment induced an inhibition in HTR-8/SVneo cell proliferation and the expression of miR-132. The overexpression of miR-132 in HTR-8/SVneo cells could markedly rescued the HG - induced suppressed cell proliferation.ConclusionAll the data of this study revealed the reduced expression of miR-132 in serum and placenta tissues of GDM, and serum miR-132 serves a candidate biomarker in the diagnosis of GDM. miR-132 may act a protective role against GDM via enhancing the trophoblast cell proliferation.

Disordered p53-MALAT1 pathway is associated with recurrent miscarriage.

Recurrent miscarriage (RM) affects about 1% of couples; however, the etiologies of half of the cases remain unknown. P53, a negative cell cycle regulator, has been found to modulate the expression of several long noncoding RNAs (lncRNAs) and overexpressed p53 has been observed in RM patients. To investigate the relationship between p53 and lncRNAs in the pathogenesis of RM, we detected the expression of p53 and six candidate lncRNAs in the villous from 27 RM patients and paired healthy controls. We found the level of NEAT1 and MALAT1 was reduced significantly and only the MALAT1 level negatively correlated with p53 protein level. By luciferase assay, we confirmed that p53 repress MALAT1 expression through directly binding to the promoter region. Functional study by using human trophoblast cell HTR-8/SVneo, we observed that p53 overexpression lead to decreased cells proliferation, migration, invasion and increased apoptosis. Meanwhile, MALAT1 overexpression partially restored these function of p53 overexpression.

ANXA4 promotes trophoblast invasion via the PI3K/Akt/eNOS pathway in preeclampsia.

The inadequate trophoblast invasion is associated with the development of preeclampsia (PE). Considering that annexin A4 (ANXA4) enhances tumor invasion, we aimed to explore the functional role of ANXA4 in trophoblast cells and to examine the underlying mechanism. ANXA4 expression in PE placentas was analyzed using immunohistochemistry and Western blotting. Cell proliferation, invasion, and apoptosis were determined using a MTT assay, Transwell assay, and flow cytometry, respectively. The expression levels of matrix metalloproteinase (MMP)-2, MMP-9, phosphoinositide 3-kinase (PI3K), Akt, phosphorylated (p)-Akt, and phosphorylated endothelial nitric oxide synthase (p-eNOS) were detected by Western blotting. Placentas were prepared for pathological examination using hematoxylin and eosin staining and apoptosis determination using the TUNEL method. Expression of ANXA4, PI3K, p-Akt and p-eNOS was downregulated in human PE placentas and PE placenta-derived extravillous cytotrophoblasts (EVCTs). Furthermore, ANXA4 overexpression promoted cell proliferation and invasion, inhibited cell apoptosis, and upregulated protein expression of PI3K, p-Akt, and p-eNOS in human trophoblast cells HTR-8/SVneo and JEG-3. By contrast, ANXA4 knockdown exerted the opposite effects. Furthermore, inhibition of the PI3K/Akt pathway by LY294002 abrogated the ANXA4 overexpression-mediated effects on trophoblast behavior. Furthermore, eNOS knockdown abrogated the ANXA4 overexpression-induced promotion of cell invasion and MMP2/9 expression. Additionally, in N-nitro-l-arginine methyl ester (l-NAME)-induced PE rats, ANXA4 overexpression alleviated PE progression, accompanied by an increase in expression of PI3K, p-Akt, and p-eNOS in rat placentas. Our findings demonstrate that ANXA4 expression is downregulated in PE. ANXA4 may promote trophoblast invasion via the PI3K/Akt/eNOS pathway.

The long noncoding RNA uc.294 is upregulated in early-onset pre-eclampsia and inhibits proliferation, invasion of trophoblast cells (HTR-8/SVneo).

Recently, a large number of long noncoding RNAs (lncRNAs) have been reported in human diseases that are evolutionarily conserved and are likely to play a role in many biological events including pre-eclampsia. In our previous research, we selected thousands of lncRNAs for their relationship with early-onset pre-eclampsia. Among these lncRNAs, a lncRNA named uc.294 attracted our attention, was once reported to specifically be expressed at a high level in the early-onset of pre-eclampsia. This study aims to investigate the function of uc.294 in early-onset pre-eclampsia and the possible mechanism. The uc.294 expression level in early-onset pre-eclampsia or in normal placenta tissues was evaluated by quantitative real-time polymerase chain reaction. To detect the proliferation, invasion, and apoptosis capacity of the trophoblast cells, we performed the Cell Counting Kit-8 assay, transwell assay, and flow cytometry, respectively. Here we report, for the first time, that uc.294 inhibits proliferation, invasion, and promotes apoptosis of trophoblast cells HTR-8/SVneo by working in key aspects of biological behaviors. However, how uc.294 acts to regulate gene functions in early-onset pre-eclampsia needs further exploration.

N-cadherin knockdown leads to disruption of trophoblastic and endothelial cell interaction in a 3D cell culture model – New insights in trophoblast invasion failure

ABSTRACTIntroduction: Trophoblast homing to maternal spiral arteries is mandatory for successful placentation. Cell-cell adhesion molecules regulate this process and adhesion molecule expression is altered in impaired placentation. We hypothesize that, similar to immune cell recruitment, trophoblast cell adherence and rolling are primarily mediated by adhesion molecules like, cadherins, immunoglobulins, selectins and their partnering ligands. Here, the interdependence of adhesion molecule expression in trophoblastic cell lines of diverse origin was investigated in relation to their interaction with endothelial cell networks on Matrigel® co-cultures and the effect of specific adhesion molecule knockdown analyzed. Methods: Trophoblastic cells were labeled in red and co-cultured with green HUVEC networks on Matrigel®. Association was quantified after collection of fluorescence microscopy pictures using Wimasis® internet platform and software. Expression of adhesion molecules was analyzed by PCR and Western blot, immuno-fluorescence and flow cytometry. The impact of adhesion molecules on trophoblast-endothelial-cell interaction was investigated using siRNA technique. Results: N-cadherin and CD162 were specifically expressed in the trophoblast cell line HTR-8/SVneo, which closely adhere to and actively migrate toward HUVEC networks on Matrigel®. Suppression of N-cadherin led to a significant alteration in trophoblast-endothelial cell interaction. Expression of VE-cadherin in closely interacting trophoblast cells was not confirmed in vitro. Discussion: We identified N-cadherin to mediate specific interaction between HUVEC and the migrating trophoblast cells HTR-8/SVneo in a Matrigel® co-culture model. VE-cadherin contribution could not be confirmed in vitro. Our results support the hypothesis that impaired N-cadherin but not VE-cadherin expression is involved in trophoblast recruitment to the maternal endothelium.