Regulates Trophoblast Proliferation Research Articles

GPR124 promotes trophoblast proliferation, migration, and invasion and inhibits trophoblast cell apoptosis and inflammation via JNK and P38 MAPK pathways.

Early-onset preeclampsia, which occurrs before 34 weeks of gestation, is the most dangerous classification of preeclampsia, which is a pregnancy-specific disease that causes 1% of maternal deaths. G protein-coupled receptor 124 (GPR124) is significantly expressed at various stages of the human reproductive process, particularly during embryogenesis and angiogenesis. Our prior investigation demonstrated a notable decrease in GPR124 expression in the placentas of patients with early-onset preeclampsia compared to that in normal pregnancy placentas. However, there is a lack of extensive investigation into the molecular processes that contribute to the role of GPR124 in placenta development. This study aimed to examine the mechanisms by which GPR124 affects the occurrence of early-onset preeclampsia and its function in trophoblast. Proliferative, invasive, migratory, apoptotic, and inflammatory processes were identified in GPR124 knockdown, GPR124 overexpression, and normal HTR8/SVneo cells. The mechanism of GPR124-mediated cell function in GPR124 knockdown HTR8/SVneo cells was examined using inhibitors of the JNK or P38 MAPK pathway. Downregulation of GPR124 was found to significantly inhibit proliferation, invasion and migration, and promote apoptosis of HTR8/SVneo cells when compared to the control and GPR124 overexpression groups. This observation is consistent with the pathological characteristics of preeclampsia. In addition, GPR124 overexpression inhibits the secretion of pro-inflammatory cytokines interleukin (IL)-8 and interferon-γ (IFN-γ) while enhancing the secretion of the anti-inflammatory cytokine interleukin (IL)-4. Furthermore, GPR124 suppresses the activation of P-JNK and P-P38 within the JNK/P38 MAPK pathway. The invasion, apoptosis, and inflammation mediated by GPR124 were partially restored by suppressing the JNK and P38 MAPK pathways in HTR8/SVneo cells. GPR124 plays a crucial role in regulating trophoblast proliferation, invasion, migration, apoptosis, and inflammation via the JNK and P38 MAPK pathways. Furthermore, the effect of GPR124 on trophoblast suggests its involvement in the pathogenesis of early-onset preeclampsia.

CircRNA_0088196 Regulates Trophoblast Proliferation and Apoptosis in Preeclampsia Through the miR-379-5p/HSPA5 Axis.

Existing research has confirmed the dysregulation of circular RNA (circRNA) in a wide variety of human diseases. Thus, in this study, we explored the potential mechanism of circRNA_0088196 in preeclampsia (PE). We performed quantitative real-time PCR to examine circRNA_0088196 expression and verified the function of circRNA_0088196 in vitro using CCK-8, TUNEL, flow cytometry, and Western blotting analyses. Additionally, we studied the mechanism using dual-luciferase reporter gene experiments. The results of our research revealed the up-regulation of circRNA_0088196 in PE patients' placentas and Heat Shock 70 kDa Protein 5 (HSPA5)-stimulated trophoblast (HTR-8/SVneo) cells. An investigation of the mechanism also showed that there was a binding between miR-379-5p and circRNA_0088196. Additionally, circRNA_0088196 inhibited HTR-8/SVneo cell proliferation and promoted cell apoptosis via the miR-337-3p/HSPA5 axis, thereby facilitating PE. In vivo experiments indicated that circRNA_0088196 regulated HTR-8/SVneo cell production through miR-379-5p. Overall, the findings of this study illustrate that circRNA_0088196 interference promotes cell apoptosis and inhibits HTR-8/SVneo proliferation via the miR-379-5p/HSPA5 axis, thereby accelerating the development of PE.

Hsa_circ_0002348 regulates trophoblast proliferation and apoptosis through miR-126-3p/BAK1 axis in preeclampsia

BackgroundPreeclampsia is a common pregnancy complication characterized by high blood pressure and damage to organs. Abnormal placenta and vascular function can lead to preeclampsia. Accumulating evidence has suggested a potential link between circular RNAs (circRNAs) and preeclampsia. As a placenta and endothelial-expressed circRNA, hsa_circ_0002348, may be promising to be the novel molecular target for preeclampsia. However, the function and mechanism of hsa_circ_0002348 in preeclampsia has not been elucidated.Materials and methodsAn overlap analysis of two circRNA profiles from placenta and endothelial cells was used to identify a functionally unknown circRNA, hsa_circ_0002348. Quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH) were used to detect its expression in the trophoblast cells and placental tissues. The mouse model of lipopolysaccharide (LPS)-induced preeclampsia was established to determine the in vivo role of hsa_circ_0002348. RNA immunoprecipitation (RIP), Luciferase reporter assay, qRT-PCR, western blot, gain- and loss-of-function and rescue experiments were conducted to uncover the role of hsa_circ_0002348 and its interaction with miR-126-3p and BAK1 in regulating trophoblast proliferation and apoptosis. Fluorescence in situ hybridization (FISH) and Immunohistochemistry (IHC) were performed to examine the expression of miR-126-3p and BAK1 in mice and human placentas, respectively.ResultsHsa_circ_0002348 was significantly increased in the preeclampsia placentas, and positively correlated with the severity of preeclampsia patients’ clinical manifestations. Its overexpression exacerbated preeclampsia-like features in the mouse model of LPS-induced preeclampsia. Functionally, hsa_circ_0002348 was found to inhibit trophoblast proliferation and promote trophoblast apoptosis. Mechanistically, hsa_circ_0002348, as an endogenous miR-126-3p sponge, upregulated the expression of BAK1. Additionally, both hsa_circ_0002348 knockdown and miR-126-3p overexpression enhanced the mammalian target of rapamycin (mTOR) and ERK1/2 signaling pathway.ConclusionsHsa_circ_0002348 might be a novel regulator of trophoblast proliferation and apoptosis through miR-126-3p/BAK1 axis in preeclampsia, which may serve as a potential target for detecting and treating preeclampsia.

USP7 prevents recurrent spontaneous abortion by targeting EZH2 to regulate trophoblast proliferation, apoptosis, migration and invasion through the Wnt/β-catenin pathway†.

Trophoblasts are significant components of the placenta and play crucial roles in maternal-fetal crosstalk. Adequate trophoblast migration and invasion are essential for embryo implantation and a healthy pregnancy. Ubiquitin-specific protease 7 (USP7), a member of the deubiquitinating enzyme family, regulates the processes of migration and invasion in multiple tumor cells. However, the effects of USP7 on trophoblasts and its possible mechanism in the development of recurrent spontaneous abortion (RSA) are still unclear. In this study, we analyzed the expression of USP7 in villous tissues obtained from RSA patients and healthy controls, and then GNE-6776 (a USP7-specific inhibitor) and USP7 siRNA were used in a trophoblast cell line, HTR-8/SVneo, to further assess the effect of USP7 on the biological function of trophoblasts. Our results provide convincing evidence that USP7 is downregulated in the placental villous tissues of RSA patients. USP7 was found to have a crucial role in the proliferation, apoptosis, migration, invasion and epithelial-mesenchymal transition (EMT) process of trophoblast cells. Further experiments revealed that USP7 directly interacted with enhancer of zeste homolog 2 (EZH2) and regulated the Wnt/β-catenin signaling pathway in trophoblasts. Taken together, these findings indicate the vital role of USP7 in regulating trophoblast proliferation, migration and invasion, thus affecting the pathogenesis of RSA, providing new insights into the important role of USP7 in the maternal-fetal interface.

Long noncoding RNA WT1-AS regulates trophoblast proliferation, migration, and invasion via the microRNA-186-5p/CADM2 axis.

This study aimed to determine the role of long noncoding RNA (lncRNA) WT1 antisense RNA (WT1-AS) in the occurrence and progression of preeclampsia (PE) and to determine the underlying molecular mechanisms. The associations between WT1-AS and microRNA (miR)-186-5p, and miR-186-5p and cell adhesion molecule 2 (CADM2) were predicted using StarBase software and verified via dual-luciferase assays. To explore the role of the human chorionic trophoblast line HTR-8/SVneo, gene (WT1-AS/miR-186-5p) gain/loss of function experiments were performed. Qualitative reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to evaluate transfection efficiency. Cell proliferation, apoptosis, cell migration, and invasion were assessed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), flow cytometry, and transwell analysis, respectively. Moreover, CADM2 protein expression was measured by western blotting. The results indicated that overexpression of WT1-AS inhibited cell viability, migration, and invasion, and induced apoptosis in HTR-8/SVneo cells. We observed that miR-186a-5p directly targeted WT1-AS, and miR-186a-5p knockdown reversed the effects of WT1-AS knockdown in HTR-8/SVneo cells. Binding sites were found between miR-186-5p and CADM2, and CADM2-overexpression reversed the influence of miR-186-5p mimic on HTR-8/SVneo cells. In summary, our findings demonstrated that lncRNA WT1-AS participates in PE by regulating the proliferation and invasion of placental trophoblasts, through the miR-186-5p/CADM2 axis.

Peroxiredoxin2 regulates trophoblast proliferation and migration through SPIB-HDAC2 pathway

Adequate proliferation and migration of placental trophoblasts is the prerequisite of a successful pregnancy. Peroxiredoxin2 (Prdx2) is a multi-functional gene involved in various signal events to maintain essential biological functions and normal cellular homeostasis. In this study, substantially lower Prdx2 levels were found in the first trimester cytotrophoblasts of women who suffered from recurrent miscarriage (RM). Prdx2 downregulation inhibited trophoblast proliferation and migration. We demonstrated that histone deacetylase2 (HDAC2) acts downstream of Prdx2 in regulating trophoblast proliferation and migration. HDAC2 deacetylates histone-3-lysine-9 in E-cadherin (E-cad) promoter and reduces the transcription of E-cad epigenetically, whereas it promotes the expression of Slug and Snail genes. These molecular changes may contribute to the trophoblast epithelial-mesenchymal transition. We further verified whether Prdx2 modulated the expression of HDAC2 through SPIB. SPIB could bind to the HDAC2 promoter PU-box region and induce HDAC2 expression. In RM, down-regulated Prdx2 suppresses SPIB-HDAC2 pathway, leading to increased E-cad and decreased Slug and Snail, and eventually restrains trophoblast proliferation and migration. Our study unveils the role of Prdx2-regulated SPIB-HDAC2 pathway in the pathology of RM and provides diagnostic and therapeutic targets for RM as well as other “great obstetrical syndromes” including preeclampsia and intrauterine growth restriction.

The Involvement of Cell Adhesion Molecules, Tight Junctions, and Gap Junctions in Human Placentation.

Placentation is a major determinant of the success of pregnancy. It is regulated by several factors such as cell adhesion molecules, tight junctions, and gap junctions. The cell adhesion molecules are integrins, cadherins, immunoglobulins, nectins, and selectins. The tight junctions are composed of claudins, occludin, and junction adhesion molecule proteins while the gap junctions are composed of connexins of varying molecular weights. During placentation, some of these molecules regulate trophoblast proliferation, trophoblast fusion, trophoblast migration, trophoblast invasion, trophoblast-endothelium adhesion, glandular remodeling, and spiral artery remodeling. There is a dysregulated placental expression of some of these molecules during obstetric complications. We have, hereby, indicated the expression patterns of the subunits of each of these molecules in the various trophoblast subtypes and in the decidua, and have highlighted their involvement in physiological and pathological placentation. The available evidence points to the relevance of these molecules as distinguishing markers of the various trophoblast lineages and as potential therapeutic targets in the management of malplacentation-mediated diseases.

The Role of circRNA-SETD2/miR-519a/PTEN Axis in Fetal Birth Weight through Regulating Trophoblast Proliferation.

Abnormal birth weight is the one of the major causes of adulthood diseases such as obesity, metabolic syndrome, cardiovascular disease, type 2 diabetes, and hypertension. Accumulating evidence has suggested that the placental trophoblast is one of the most important reasons that influence birth weight. Our previous study showed that miR-519a are correlated with low fetal birth weight through regulating trophoblast proliferation. To further clarify the detailed mechanisms on how it is regulated, we screened the placental-specific circular RNAs (circRNAs) via microarray assay. The result identified that circ-SETD2 was highly expressed in the placenta of the patients with fetal macrosomia compared with healthy donors. Furthermore, bioinformatic analyses and the luciferase reporter assay revealed that miR-519a possessing the binding sites for both circ-SETD2 and phosphate and tensin homolog was deleted on chromosome 10 (PTEN). Interestingly, upregulation of circ-SETD2 enhanced the proliferation and invasion of the human trophoblast-like cell line HTR8/SVneo cell. A parallel study performed by Western blotting showed that overexpression of circ-SETD2 reduced miR-519a levels and increased PTEN levels in HTR8/SVneo cells. Importantly, the enhancement of HTR8/SVneo cell activity by circ-SETD2 overexpression was nullified when the cells were cotransfected by circ-SETD2 and miR-519a, suggesting the involvement of the circ-SETD2/miR-519a/PTEN axis in trophoblast activity. Taken together, we illustrate the role of circ-SETD2, as an upstream signaling of miR-519a/PTEN, in placenta development via regulating trophoblast proliferation and invasion. These findings improve our understanding of the mechanisms of progression of fetal macrosomia and will guide future development of therapeutic strategies against the disease by targeting the circ-SETD2/miR-519a/PTEN axis.

HAI-1 regulates placental folds development by influencing trophoblast cell proliferation and invasion in pigs.

Fetal development is critically dependent on the efficiency of the placenta. Porcine trophoblast cell proliferation and invasion have crucial roles in placental fold development, which is one of the essential events determining placental efficiency. The membrane serine proteinase inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1) can regulate cellular invasion and motility in different types of epithelial cells, including trophoblast cells in mice. This work used quantitative polymerase chain reaction (qPCR) and immunohistochemistry to compare the expression level and location of HAI-1 in the placenta on gestational days 26, 50, and 95 in Yorkshire and Meishan pigs. The role of HAI-1 in porcine trophoblast cell (PTr2) proliferation, invasion, and migration in vitro was investigated by analyzing the effects of HAI-1 gene silencing or overexpression. Polymorphism in the HAI-1gene was detected to determine associations between the genotype and piglet birth weight in 400 healthy pure-bred Yorkshire piglets. qPCR results showed that HAI-1 mRNA levels significantly increased (P<0.01) between gestational days 26 and 50 and then decreased (P<0.01) between days 50 and 95 in both Meishan and Yorkshire pigs. Immunohistochemical analysis showed that HAI-1 protein was strongly expressed by the high columnar trophoblast cells located at the top of the placental folds with low proliferative and invasion capacities. However, it was expressed at very low levels in cuboidal trophoblast cells located at the side and base of the placental folds with high proliferative and invasion capacities. In vitro experiments indicated that HAI-1 had the ability to reduce the proliferation, invasion and migration of trophoblast cells. In addition, one single-nucleotide polymorphism (SNP) of HAI-1 showed a significant association (P<0.05) with piglet birth weight. These results revealed that HAI-1 could be a vital molecule in placental folds development by regulating trophoblast proliferation and invasion in pigs.

SPRY4 regulates trophoblast proliferation and apoptosis via regulating IFN-γ-induced STAT1 expression and activation in recurrent miscarriage.

The dysregulation of trophoblast functions is one of the leading causes of recurrent miscarriage (RM), which frustrates 1%-5% of couples of childbearing ages. Sprouty 4 (SPRY4) is considered as a tumour suppressor and exerts a negative role in cell viability. However, its role in regulating trophoblast behaviors at the maternal-fetal interface remains largely unknown. First-trimester villous samples were collected from RM patients and healthy controls (HCs) to determine the SPRY4 expression in human placenta during early pregnancy. The HTR8/SVneo cell line was introduced to clarify trophoblast cell functions via transfecting with specific short interfering RNA against SPRY4 or SPRY4-overexpressing lentivirus in vitro. In addition, gene expression microarray analysis was performed to explore the downstream molecules and pathways. Our results revealed that SPRY4 expression was significantly increased in the first-trimester cytotrophoblasts of RM patients compared with HCs. Furthermore, SPRY4 overexpression inhibited trophoblast proliferation and accelerated apoptosis in vitro, while SPRY4 knockdown reversed these effects. Mechanistically, IFN-γ -induced STAT1 expression and activation were involved in the regulation of trophoblast proliferation and apoptosis by SPRY4, and IFN-γ promoted SPRY4 expression and STAT1 phosphorylation through PI3K/AKT pathway. Additionally, both STAT1 and phosphorylated STAT (p-STAT) levels were also upregulated in trophoblasts from RM patients and positively correlated with SPRY4 expression. Our findings indicate that SPRY4 may act as a negative regulator of trophoblast functions through upregulating IFN-γ/PI3K/AKT-induced STAT1 activation. High levels of SPRY4 and STAT1 may contribute to RM development and progression, and blocking of either target could be a novel therapeutic strategy for RM patients.

Regulation of placentation by the transforming growth factor beta superfamily†.

During pregnancy, there is increased expression of some cytokines at the fetal-maternal interface; and the clarification of their roles in trophoblast-endometrium interactions is crucial to understanding the mechanism of placentation. This review addresses the up-to-date reported mechanisms by which the members of the transforming growth factor beta superfamily regulate trophoblast proliferation, differentiation, and invasion of the decidua, which are the main phases of placentation. The available information shows that these cytokines regulate placentation in somehow a synergistic and an antagonistic manner; and that dysregulation of their levels can lead to aberrant placentation. Nevertheless, prospective studies are needed to reconcile some conflicting reports; and identify some unknown mediators involved in the actions of these cytokines before their detailed mechanistic regulation of human placentation could be fully characterized. The TGF beta superfamily are expressed in the placenta, and regulate the process of placentation through the activation of several signaling pathways.

ANXA7 regulates trophoblast proliferation and apoptosis in preeclampsia.

Preeclampsia (PE) is a unique gestational disorder leading to maternal and neonatal morbidity and mortality. AnnexinA7 (ANXA7) is a calcium-dependent phospholipid-binding protein that promotes membrane fusion during exocytosis. However, the function of ANXA7 in placental trophoblast is poorly understood. The present study aimed to investigate a possible association between ANXA7 and human trophoblast apoptosis. We collected human placental tissues from patients with PE and normal pregnant women to elucidate the expression level of ANXA7. The ANXA7-knockdown and ANXA7-overexpressing HTR8/SVneo cells were utilized for studying the function of ANXA7 in trophoblast. The proliferation and apoptosis levels of trophoblast were examined with Western blot assay, flow cytometry, Cell Counting Kit-8 assay, and immunohistochemistry. ANXA7 expression was significantly lower in placentas from patients with PE patients compared with that in from normal pregnant controls. Knockdown of ANXA7 induced cell apoptosis and inhibited cell proliferation in HTR-8 via by downregulating BCL2 protein levels. Overexpression of ANXA7 reduced apoptosis and promoted HTR8 proliferation. Further analyses showed that ANXA7 knockdown inhibited the activation of the JAK1/STAT3 pathway in HTR-8 cells. Our findings revealed a new regulatory pathway of ANXA7/JAK1/STAT3 in trophoblast apoptosis in preeclampsia, suggesting that ANXA7 is a potential therapeutic target for preeclampsia.

MiRNA-210-3p regulates trophoblast proliferation and invasiveness through fibroblast growth factor 1 in selective intrauterine growth restriction.

Selective intrauterine growth restriction (sIUGR), which affects approximately 10%‐15% of monochorionic (MC) twin pregnancies, is highly associated with intrauterine foetal death and neurological impairment in both twins. Data suggest that unequal sharing of the single placenta is the main contributor to birth weight discordance. While MC twins and their placenta derive from a single zygote and harbour almost identical genetic material, the underlying mechanisms of phenotypic discrepancies in MC twins remain unclear. MicroRNAs are small non‐coding RNA molecules that regulate gene expression but do not change the DNA sequence. Our preliminary study showed that microRNA‐210‐3p (miR‐210‐3p) was significantly upregulated in the placental share of the smaller sIUGR twin. Here, we investigate the potential role of miR‐210‐3p in placental dysplasia, which generally results from dysfunction of trophoblast cells. Functional analysis revealed that miR‐210‐3p, induced by hypoxia‐inducible factor 1α (HIF1α) under hypoxic conditions, suppressed the proliferation and invasiveness of trophoblast cell lines. Further RNA sequencing analysis and luciferase reporter assays were performed, revealing that fibroblast growth factor 1 (FGF1) is an influential target gene of miR‐210‐3p. Moreover, correlations among miR‐210‐3p levels, HIF1α and FGF1 expression and the smaller placental share were validated in sIUGR specimens. These findings suggest that upregulation of miR‐210‐3p may contribute to impaired placentation of the smaller twin by decreasing FGF1 expression in sIUGR.

LncRNA HOXD-AS1 promotes preeclampsia progression via MAPK pathway.

To investigate the role of HOXD-AS1 in preeclampsia and its underlying mechanism. A total of 50 preeclampsia primiparas and 34 normal pregnancies admitted in our hospital from July 2015 to July 2017 were selected as the study group and control group, respectively. Age, body weight, blood pressure, 24-h urinary protein and neonatal weight were compared between the two groups. HOXD-AS1 expression in the placenta tissues was detected by quantitative Real-time PCR (qRT-PCR). Preeclampsia patients were further assigned into high and low expression group according to their HOXD-AS1 expressions. The relationship between HOXD-AS1 expression and blood pressure, 24-h urinary protein and neonatal weight in preeclampsia patients were analyzed. For in vitro experiments, transfection efficacy of pcDNA-HOXD-AS1 and si-HOXD-AS1 were detected by qRT-PCR. Proliferative and colony formation abilities in BeWo and Wish cells were detected by CCK-8 and colony formation assay, respectively. Moreover, protein expressions of p-p38, p-JNK, and p-ERK were detected by Western blot. The systolic blood pressure, diastolic blood pressure and urinary protein in preeclampsia patients were higher than those of normal pregnancies. However, neonatal weight in preeclampsia patients was lower than that of normal pregnancies. HOXD-AS1 expressions were gradually increased in normal pregnancies, patients with late onset preeclampsia and patients with early onset preeclampsia sequentially. Additionally, higher levels of systolic pressure, diastolic pressure and 24-h urinary protein, as well as lower neonatal weight, were observed in preeclampsia patients with high expression of HOXD-AS1 than those with low expression. In vitro results demonstrated that proliferative and colony formation abilities in trophoblasts were elevated after HOXD-AS1 knockdown. Western blot data illustrated that protein expressions of p-p38 and p-JNK were decreased, while p-ERK expression was increased after overexpression of HOXD-AS1 in trophoblasts. HOXD-AS1 participates in the development and progression of preeclampsia by regulating trophoblast proliferation via the MAPK pathway.

IGF2-derived miR-483-3p contributes to macrosomia through regulating trophoblast proliferation by targeting RB1CC1

What is the role of insulin-like growth factor 2 (IGF2)-derived miR-483-3p in macrosomia? IGF2-derived intronic miR-483-3p is overexpressed in macrosomia placentas, and miR-483-3p prompts HTR-8/SVneo extravillous trophoblast cell line proliferation through down-regulation of its target RB1 inducible coiled-coil 1 (RB1CC1). Macrosomia is a common pregnancy-associated disease and causes a number of adverse maternal and perinatal outcomes. The development of macrosomia is reportedly attributable to over proliferation of the placental cells. MicroRNAs (miRNAs) play an important role in the development of fetal and placenta by regulating their target genes. Here, we investigated the role of IGF2-derived intronic miR-483-3p in macrosomia. The expression of IGF2, miR-483-3p and its target gene in placental tissues from 30 pregnant women who had macrosomia was compared to those of 30 gestation-matched healthy pregnant controls. For in vitro studies, the human first trimester extravillous trophoblast cell line, HTR-8/SVneo cell was used. Placenta tissues were collected from pregnant women who had macrosomia without diabetes or other complications (n = 30) and healthy pregnant controls (n = 30). HTR-8/SVneo cells were transfected with specific miRNA mimics or inhibitors. MiRNA and mRNA isolated from placenta tissues or cells were measured by quantitative real-time PCR. Protein was measured by western blot. Cell proliferation was assayed using a colorimetric proliferation assay method. Cell cycle and apoptosis were analyzed by flow cytometry. The putative targets of miR-483-3p were predicted using the TargetScan, miRanda, miRDB and DIANA algorithms. Dual luciferase reporter assay was used to measure the relationship of miR-483-3p and RB1CC1. IGF2-derived miR-483-3p was overexpressed in macrosomia placentas. miR-483-3p promoted proliferation in HTR-8/SVneo cells and had a positive relationship with its host gene IGF2. Subsequently, RB1CC1 was confirmed as a direct target of miR-483-3p, which may be an important mediator of cell growth regulation for miR-483-3p. N/A. The level of IGF2 and its intronic miR-483-3p in the serum of these participants was not investigated. Further studies are required to understand the mechanisms underlying the cause of the increase of IGF2 and miR-483-3p in macrosomia. These findings give a new insight into the role of intronic miRNA and its host gene in the development of macrosomia. Furthermore, it may offer a new target for prognostic and therapeutic intervention for macrosomia. This work was supported by awards from National Natural Science Foundation of China (Nos. 81401213, 81673217, 81703260), Jiangsu Provincial Medical Youth Talent (No. QNRC2016110), Jiangsu Overseas Visiting Scholar Program for University Prominent Young & Middle-aged Teachers and Presidents, the Priority Academic Program for the Development of Jiangsu Higher Education Institutions (Public Health and Preventive Medicine), the Education Department of Jiangsu Province (No. 16KJB330010), the Science and Technology Department of Jiangsu Province (No. BK20160227), the China Postdoctoral Science Foundation funded project (No. 2016M601892). The authors declare no competing financial interests.

TROPHOBLAST INVASION AND UTERIOIMPLANTATION GROWTH IN EARLY RAT GESTATION REQUISITE ADRENOMEDULLIN

Objective: Adrenomedullin (ADM) is a currently exposed hypotensive peptide that is articulated in a variety of cell and tissue types. The existence of ADM has been immunohistochemically demonstrated in pathologic uterioimplantation regions, but no systematic study of ADM expression in early pregnancy in uterioimplantation region has been reported.Methods: Rats on the gestational day 2 were implanted subcutaneously with osmotic (Alzet) minipumps delivering 125 and 250 μg rat/day/of AM22–52 and were killed on the gestational day 9. We have ascertained the hypothesis that ADM, a multiregulatory ubiquitous peptide hormone, works as a trophoblast pro-invasion factor.Results: We confirmed ADM significance in in vitro assay using ACH-3P cell line, which is first-trimester trophoblast cell line.Conclusion: Our data support the conception that ADM is involved in the human implantation process through regulating trophoblast proliferation and differentiation.

MEF2 transcription factors in human placenta and involvement in cytotrophoblast invasion and differentiation.

Development of the human placenta and its trophoblast cell types is critical for a successful pregnancy. Defects in trophoblast invasion and differentiation are associated with adverse pregnancy outcomes, including preeclampsia. The members of myocyte enhancer factor-2 (MEF2) family of transcription factors are key regulators of cellular proliferation, differentiation, and invasion in various cell types and tissues and might play a similarly important role in regulating trophoblast proliferation, invasion, and differentiation during human placental development. In the present study, using human cytotrophoblast cell lines (HTR8/SVneo and BeWo) and primary human cytotrophoblasts (CTBs), we show that members of the MEF2 family are differentially expressed in human placental CTBs, with MEF2B and MEF2D being highly expressed in first trimester extravillous CTBs. Overexpression of MEF2D results in cytotrophoblast proliferation and enhances the invasion and migration of extravillous-like HTR8/SVneo cells. This invasive property is blocked by overexpression of a dominant negative MEF2 (dnMEF2). In contrast, MEF2A is the principal MEF2 isoform expressed in term CTBs, MEF2C and MEF2D being expressed more weakly, and MEF2B expression being undetected. Overexpression of MEF2A induces cytotrophoblast differentiation and syncytium formation in BeWo cells. During in vitro differentiation of primary CTBs, MEF2A expression is associated with CTB differentiation into syncytiotrophoblast. Additionally, the course of p38 MAPK and ERK5 activities parallels the increase in MEF2A expression. These findings suggest individual members of MEF2 family distinctively regulate cytotrophoblast proliferation, invasion, and differentiation. Dysregulation of expression of MEF2 family or of their upstream signaling pathways may be associated with placenta-related pregnancy disorders.

Nik-related kinase regulates trophoblast proliferation and placental development by modulating AKT phosphorylation.

Nik-related kinase (Nrk) is a Ser/Thr kinase and was initially discovered as a molecule that was predominantly detected in skeletal muscles during development. A recent study using Nrk-null mice suggested the importance of Nrk in proper placental development; however, the molecular mechanism remains unknown. In this study, we demonstrated that differentiated trophoblasts from murine embryonic stem cells (ESCs) endogenously expressed Nrk and that Nrk disruption led to the enhanced proliferation of differentiated trophoblasts. This phenomenon may reflect the overproliferation of trophoblasts that has been reported in enlarged placentas of Nrk-null mice. Furthermore, we demonstrated that AKT phosphorylation at Ser473 was upregulated in Nrk-null trophoblasts and that inhibition of AKT phosphorylation cancelled the enhanced proliferation observed in differentiated Nrk-null trophoblasts. These results indicated that the upregulation of AKT phosphorylation was the possible cause of enhanced proliferation observed in Nrk-null trophoblasts. The upregulation of AKT phosphorylation was also confirmed in enlarged Nrk-null placentas in vivo, suggesting that proper regulation of AKT by Nrk was important for normal placental development. In addition, our detailed analysis on phosphorylation status of AKT isoforms in newly established trophoblast stem cells (TSCs) revealed that different levels of upregulation of AKT phosphorylation were occurred in Nrk-null TSCs depending on AKT isoforms. These results further support the importance of Nrk in proper development of trophoblast lineage cells and indicate the possible application of TSCs for the analysis of differently regulated activation mechanisms of AKT isoforms.

Notch signalling in placental development and gestational diseases

Activation of Notch signalling upon cell-cell contact of neighbouring cells controls a plethora of cellular processes such as stem cell maintenance, cell lineage determination, cell proliferation, and survival. Accumulating evidence suggests that the pathway also critically regulates these events during placental development and differentiation. Herein, we summarize our present knowledge about Notch signalling in murine and human placentation and discuss its potential role in the pathophysiology of gestational disorders. Studies in mice suggest that Notch controls trophectoderm formation, decidualization, placental branching morphogenesis and endovascular trophoblast invasion. In humans, the particular signalling cascade promotes formation of the extravillous trophoblast lineage and regulates trophoblast proliferation, survival and differentiation. Expression patterns as well as functional analyses indicate distinct roles of Notch receptors in different trophoblast subtypes. Altered effects of Notch signalling have been detected in choriocarcinoma cells, consistent with its role in cancer development and progression. Moreover, deregulation of Notch signalling components were observed in pregnancy disorders such as preeclampsia and fetal growth restriction. In summary, Notch plays fundamental roles in different developmental processes of the placenta. Abnormal signalling through this pathway could contribute to the pathogenesis of gestational diseases with aberrant placentation and trophoblast function.

The emerging roles of placenta‐specific microRNAs in regulating trophoblast proliferation during the first trimester

In the human placenta, eight 'placenta-specific' microRNAs (miRNAs) are exclusively expressed and are associated with high cloning frequencies. The expression of placenta-specific miRNAs is known to be involved in preeclampsia, but the understanding of these miRNAs is still limited. The goal of this study was to investigate the levels and localisations of eight placenta-specific miRNAs in placental villi with different proliferative abilities during the first trimester. Immunohistochemical analyses indicated that placental trophoblast proliferation ability was associated with the weight of villi in the same gestational week during the first trimester. Of the eight placenta-specific miRNAs, quantitative real-time RT-PCR demonstrated that the expression of miR-517b and miR-1283 was increased in the lightest villi and decreased in the heaviest one, the expression of miR-519a was increased in the heaviest villi and decreased in the lightest one. In situ hybridisation analysis showed that miR-517b and miR-519a were located primarily in the trophoblast layer, while miR-1283 were expressed not only in the villous trophoblasts but also in some villous stroma cells. These findings suggest that miR-517b and miR-519a may play an important role in trophoblast proliferation during the first trimester.