Osteopontin facilitates invasion in human trophoblastic cells via promoting matrix metalloproteinase-9 in vitro

Successful implantation of embryo and placentation depend on proper trophoblast proliferation and differentiated into specialized invasive trophoblast. However, little is known about the regulatory factors and mechanisms in trophoblast proliferation and differentiation. Osteopontin (OPN) is a member of the small integrin-binding ligand N-linked glycoprotein family and participates in cell adhesion and invasion. It has been identified that OPN is highly expressed in invasive trophoblasts in human placenta. In this study, we demonstrated that OPN is constitutively expressed in highly invasive phenotype of human choriocarcinoma cell lines of JAR and JEG-3 cells, and OPN could promote trophoblast proliferation and invasion, partly through promoting MMP-9 secretion. Inhibition of OPN will compromise the abilities of proliferation and invasion in JAR and JEG-3 cell lines. Our data showed that the expression of OPN in trophoblast may participate in placentation, OPN expression defects may be involved in gestational trophoblastic diseases.     

Invasion of Cytotrophoblastic (JEG-3) Cells Is Up-Regulated by Interleukin-15 In Vitro

PROBLEM: Trophoblast invasion into the uterus is controlled by many factors. Some cytokines (interleukin [IL]-1, IL-6, and IL-10) have been shown previously to play an important role in placentation. The human placenta is an important source of IL-15, although the cellular source of IL-15 in the placenta has not yet been specified. IL-15 influences cell adhesion and migration by redistributing adhesion molecules in lymphocytes and has been shown to have effects on endothelial cells and in some human tumors. METHOD OF STUDY: To study the role of IL-15 in trophoblast invasion, we investigated the effect of IL-15 (concentrations, 1–10 ng/ml) in a trophoblast invasion model (JEG-3 with matrigel-coated filters). Cell invasion was assessed using matrigel-coated filters and was expressed as the quotient of invading cells in comparison with the number of cells that had passed the control membrane. Cell migration was studied by examining the number of cells that had passed the filters without matrigel. Cell proliferation was quantified by a tetrazolium salt WST-1 cleavage assay. Matrix metalloproteinase (MMP)-1, MMP-2, and MMP-9 activities were measured by specific enzyme assays. RESULTS: IL-15 significantly (P < 0.05) increased the in vitro invasion of cytotrophoblastic (JEG-3) cells in a dose-dependent manner. There was a fourfold increase in the invasion at a concentration of 10 ng/ml of IL-15. Migration also was increased by a factor of 2.3 (P < 0.05). Cell proliferation, however, remained unchanged. The collagenolytic activity of cytotrophoblastic (JEG-3) cells was increased by IL-15 stimulation. A significant increase in MMP-1 concentration occurred after the incubation of JEG-3 cells with IL-15. No changes appeared in MMP-2, MMP-9, and tissue inhibitor of metalloproteinase-1 concentrations. CONCLUSIONS: Trophoblast invasion and migration, but not proliferation, are enhanced by IL-15. Our results suggest a role for IL-15 in the modulation of MMP-1 secretion by JEG-3 cells. Furthermore, we speculate, that IL-15 might be related to the changes of cell adhesion molecule phenotype during the process of invasion.     

MicroRNA-155 通过调节CXCR4 / PI3K / AKT 途径影响滋养细胞的侵袭与迁移

目的:以人绒毛膜滋养层细胞系JEG-3 细胞为研究对象,结合该细胞侵袭和迁移能力的变化情况,研究转染miR-155 mimics 和miR-155 inhibitor 之后CXCR4 的表达变化及其对下游PI3K/ AKT 信号通路的影响作用,从而探讨miR-155参与子痫前期发生发展的分子机制。方法:设计miR-155mimics 和miR-155 inhibitor,对JEG-3 进行转染,通过Transwell 侵袭实验、划痕实验,观察转染后细胞的侵袭和迁移能力的变化;利用Real-time PCR 检测CXCR4 mRNA 的表达;利用Western blot检测CXCR4 及下游p鄄AKT 蛋白的表达水平。结果:Real-time PCR 结果显示,miR-155 mimics 转染组CXCR4 mRNA 相对表达量(0.589依0.096)明显低于空白对照组(1.503依0.090)和阴性对照组(1.146依0.153),差异有统计学意义(P<0.05);miR-155 inhibitor 转染组CXCR4 mRNA 相对表达量(1.739依0.083)与两组对照组相比差异也有统计学意义(P<0.05)。Western blot 结果显示miR-155 mimics 转染组CXCR4 蛋白和下游p-AKT 蛋白表达水平均明显降低,而miR-155 inhibitor 转染组CXCR4 和p-AKT 蛋白水平则升高,与空白对照组和阴性对照组相比差异均有统计学意义(P<0.05)。Transwell 侵袭实验结果显示,与空白对照组(63郾46依2郾37)和阴性对照组(49.29依5.81)侵袭细胞数相比,miR-155 mimics 转染组侵袭细胞数(22.89依9.42)明显减少,差异有统计学意义(P<0.05);同时,miR-155 inhibitor 转染组侵袭细胞数(81.50依11.25)明显升高,差异有统计学意义(P<0.05)。划痕实验结果显示,转染miR-155 mimics 后,JEG-3 细胞相对迁移距离(0.159依0.058)低于空白对照组(1.080依0.045)和阴性对照组(0.823依0.201),差异有统计学意义(P<0.05);而miR-155 inhibitor 转染组,JEG-3 细胞相对迁移距离(1.640依0.078)明显增加,差异有统计学意义(P<0.05)。结论:miR-155 可能通过抑制CXCR4 的表达进而抑制其下游PI3K/AKT 信号通路的活化,从而影响滋养细胞的侵袭及迁移能力,最终导致子痫前期的发生发展。     

hCMV induced IL-6 release in trophoblast and trophoblast like cells.

BACKGROUND: Human cytomegalovirus (hCMV) infection of the trophoblasts is a crucial event in virus transmission from mother to child, being one responsible factor for intrauterine infection of the unborn. Differences of virus replication in trophoblasts depending on time point of pregnancy and degree of differentiation of trophoblasts might influence this transmission. Furthermore, immunological reactions of the trophoblasts to hCMV infection might be important defence mechanisms too. OBJECTIVES: hCMV replication and interleukin-6 release in trophoblasts and trophoblast like cells (choriocarcinoma cells) was investigated. STUDY DESIGN: Trophoblasts from term and 1st trimester placentas were isolated and infected with hCMV. hCMV production and release to the supernatant as well as interleukin-6 release and interleukin-6 mRNA production by these infected cells was measured. Choriocarcinoma cell lines (JEG-3, JAR) were treated the same. Non-infected trophoblasts were used as controls. RESULTS: In 1st trimester trophoblast, term trophoblasts and JEG-3 permissive hCMV replication was observed, although with different kinetics and efficiency. In JAR no complete virus replication was seen. High levels of interleukin-6 were measured in the supernatants of all hCMV infected cells immediately after infection. IL-6 mRNA upregulation was seen 48 h after infection in those cell types replication of hCMV occurred (1st trimester trophoblasts, term trophoblasts, JEG-3). At that time-point hCMV immediate early proteins appeared. In JARs no virus production and no IL-6 mRNA upregulation was seen, and IL-6 levels in the supernatant of these hCMV infected cells declined significantly until day 6 after infection compared to mock infected cells. CONCLUSION: These observations show that hCMV replication is influenced by the degree of trophoblast differentiation. Interleukin-6 is upregulated by hCMV infection, but is independent of complete virus replication.     

二氢杨梅素对绒毛膜癌细胞增殖和迁移能力的影响

目的 探讨二氢杨梅素（DMY）对绒毛膜癌（绒癌）JEG-3及JAR细胞增殖和迁移能力的影响。 方法 MTT法检测不同浓度的二氢杨梅素(0 mg/L, 20 mg/L, 40 mg/L, 60 mg/L, 80 mg/L)作用一定时间后,对绒癌JEG-3和JAR细胞增殖能力的影响;细胞划痕实验和Transwell法检测不同浓度二氢杨梅素(0 mg/L, 40 mg/L, 60 mg/L, 80 mg/L)分别作用绒癌JEG-3细胞及JAR细胞一定时间后,对其迁移能力的影响;Real-time PCR和Western blotting方法分别检测不同浓度二氢杨梅素(0 mg/L, 40 mg/L, 60 mg/L, 80 mg/L)作用绒癌JEG-3及JAR细胞后,基质金属蛋白酶2（MMP-2）mRNA和蛋白表达水平的影响。 结果 不同浓度二氢杨梅素作用绒癌JEG-3和JAR细胞24 h和48 h后,随着二氢杨梅素浓度增加,对JEG-3和JAR细胞增殖抑制作用增强（P＜0.05）。二氢杨梅素作用绒癌JEG-3及JAR细胞后,显著抑制细胞迁移能力,且具有浓度依赖性（P＜0.05）。不同浓度二氢杨梅素作用JEG-3和JAR细胞后,MMP-2 的mRNA和蛋白表达水平明显下降（P＜0.05）。 结论 二氢杨梅素能够抑制JEG-3及JAR细胞的增殖能力且具有浓度依赖性,同时二氢杨梅素可能通过下调绒癌JEG-3及JAR细胞中MMP-2 mRNA和蛋白的表达,抑制绒癌细胞的侵袭迁移。     

Calreticulin associates with non-HLA-A,-B class I proteins in the human choriocarcinoma cell lines JEG-3 and BeWo.

Human placental trophoblast expresses as unusual repertoire of major histocompatibility complex (MHC) class I products that appears to reflect the unique role of this epithelium in mediating feto-maternal relations during pregnancy. Trophoblast is devoid of human leucocyte antigen (HLA)-A,-B antigens but can express one or more non-HLA-A,-B class I proteins. The human choriocarcinoma cell lines JEG-3, BeWo and JAR are widely used as models to study trophoblast. During attempts to isolate non-HLA-A,-B class I from JEG-3 and BeWo by immunoaffinity chromatography using a monoclonal antibody to beta 2-microglobulin we observed a 55,000 MW protein co-purifying with class I. N-terminal amino acid sequencing and immunoblotting using a specific antiserum identified this product as calreticulin, a molecule recently shown to be involved in the assembly of classical class I in human B-lymphoblastoid cells. In our hands JEG-3 and BeWo were found to express 45,000 MW non-HLA-A,-B class I proteins while the 40,000 MW HLA-G product was identified only in JEG-3. Our data suggest that calreticulin associates with non-HLA-A,-B class I heterodimers and with free 45,000 MW non-HLA-A,-B class I H chains in JEG-3. JAR was found to be devoid of detectable class I H chains but contained beta 2-microglobulin and calreticulin. However, calreticulin-beta 2-microglobulin complexes were not detected in JAR. Calreticulin and class I were apparently co-localized within the endoplasmic reticulum of JEG-3 cells whereas only class I was expressed at the cell surface. These studies demonstrate that calreticulin is associated with non-HLA-A,-B class I products in human choriocarcinoma cells.     

siRNA抑制PPARγ表达对绒癌细胞JEG-3侵袭力的影响

目的: 采用小分子干扰RNA(siRNA)技术特异性地抑制绒癌细胞株JEG-3中过氧化物酶体增殖物激活受体γ(PPARγ)的表达,研究PPARγ影响JEG-3细胞侵袭力的机制。方法: 将实验设为实验组(转染 PPARγ 基因的特异性siRNA)、阴性对照组(转染阴性对照siRNA)和空白对照组(不转染任何siRNA片段,其它试剂与另两组相同),采用实时定量PCR技术从mRNA水平检测PPARγ及黏蛋白-1(MUC1)的表达;利用Transwell侵袭实验观察siRNA转染JEG-3细胞后细胞侵袭力的变化。结果: siRNA使PPARγ mRNA的表达水平下调了(75.0±0.8)% (P<0.05),MUC1mRNA的表达水平下调了(65.0±1.3)% (P<0.05),JEG-3细胞侵袭Matrigel的能力显著增强(P<0.05)。结论: PPARγ表达水平的下降能相应下调MUC1的表达水平,并增强滋养细胞的侵袭能力,推测PPARγ可能通过调节 MUC1 基因影响滋养细胞的侵袭能力,从而参与了子痫前期等病理妊娠的发生。     

The use of Alamar Blue assay for quantitative analysis of viability, migration and invasion of choriocarcinoma cells

BACKGROUND: The current techniques for quantifying trophoblast viability, migration and invasion are mainly limited by the need to sacrifice the cells during the test procedure. In this study, the vital dye AB (AB) was used to quantify cell number and viability of BeWo and JEG-3 choriocarcinoma cells, as well as their migration and invasion through fibronectin-coated filters. METHODS :AB was directly added to culture medium of incubated test and control cells. At various time intervals, the redox reaction, in which AB is reduced by the cells, was measured by absorbance readings at 540 and 630 nm. For cell migration and invasion, cells were cultured onto uncoated or fibronectin-coated inserts, respectively. AB reduction of migrated cells was normalized to that of control cells to calculate percentages of migration. This model was also tested in the presence of a reported inhibitor, transforming growth factor (TGF) beta. RESULTS: The curve of %AB reduction versus cell number was linear, with intra- and inter-assay Coefficient of Variations of 1.88%and 2.94%, respectively. AB reduction increased with both seeding concentrations and incubation time with AB. TGFbeta treatment caused a modest decrease in AB reduction in both JEG-3 and BeWo cells. TGFbeta treatment also decreased migration in BeWo, but not in JEG-3, cells. CONCLUSIONS: AB assay is a simple and reliable method for quantifying trophoblast viability, migration and invasion.     

Expression profile of trophoblast invasion-associated genes in the pre-eclamptic placenta

The primary pathology of pre-eclampsia is thought be a defect in placentation due to failure of trophoblast invasion. Here, we aim to identify the expression profile of invasion-associated genes in the pre-eclamptic placenta. Messenger RNA (mRNA) expression levels of extracellular matrix molecule-related genes in five pre-eclamptic placentas and in five strictly matched normal placentas were assayed using complementary DNA (cDNA) microarrays representing over 220 human cytokine-associated or hormone-associated genes. Results demonstrated greater than two-fold higher expression of 18 extracellular matrix molecule genes, including cadherin, collagen, integrin and selectin, in the pre-eclamptic placenta. Extracellular matrix molecule degradation-related genes, including matrix metalloproteinase (MMP)-10, MMP-13, MMP-15, tissue inhibitor of metalloproteinase (TIMP)-2, TIMP-3, plasminogen and plasminogen activator, were also highly expressed in the pre-eclamptic placenta, compared to the normal placenta. Results suggest that the abnormal expression profiles of extracellular matrix molecules and degrading proteinases might be associated with the pathogenesis of pre-eclampsia.     

基质金属蛋白酶-9及其组织抑制物-1在人胎盘的表达及意义

目的研究基质金属蛋白酶-9(matrix metalloproteinases,MMP-9)及其组织抑制物-1(tissue inhibitor of metalloproteinases,TIMP-1)在不同孕期胎盘中的表达及与滋养层细胞侵蚀、子宫-胎儿血管系统建立的关系.方法应用原位杂交法检测56例胎盘(早孕16 例、中孕20 例、晚孕20例)中MMP-9及TIMP-1mRNA的表达.结果 MMP-9及TIMP-1mRNA主要表达于滋养细胞、绒毛间质血管壁及蜕膜组织中;MMP-9mRNA的表达早、中孕组明显强于晚孕组,TIMP-1mRNA的表达晚孕组明显强于早、中孕组,差异均有极显著性(P<0.01).结论 MMP-9及TIMP-1协同表达可能在滋养层细胞侵蚀、孕卵着床、血管重建过程中发挥一定的作用.     

Simvastatin has deleterious effects on human first trimester placental explants

BACKGROUND: Statins inhibit 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMG-CoA reductase), the rate-limiting enzyme of the mevalonate pathway, and have been used successfully in the treatment of hypercholesterolaemia. Animal models have provided evidence for the teratogenic effects of statins on pregnancy outcome. Thus statins are contraindicated during pregnancy. However, conflicting data are available from inadvertent use of statins in human pregnancy. Therefore we decided to explore the effects of simvastatin on the placenta in an in vitro human placental model. METHODS: Human first trimester placental explants that were grown on matrigel were exposed to medium supplemented with simvastatin. Migration of extravillous trophoblast cells was assessed by visual observation. Proliferative and apoptotic events of the trophoblast cells were assesed by immunohistochemical examination using anti-Ki67 and anti-activated caspase-3 antibodies respectively. Hormone levels were measured. RESULTS: Simvastatin sharply inhibited migration of extravillous trophoblast cells from the villi to the matrigel (P < 0.05). Moreover, simvastatin inhibited half of the proliferative events in the villi (P < 0.05) and increased apoptosis of cytotrophoblast cells compared to control. Moreover, simvastatin significantly decreased secretion of progesterone from the placental explants (P < 0.01). CONCLUSION: Simvastatin adversely affects human first trimester trophoblast.     

Increased cystatin C expression in the pre-eclamptic placenta

Trophoblast invasion is regulated by proteinases and their inhibitors. Cystatin C inhibits cysteine proteinases. The serum concentration of cystatin C is increased in late pregnancy and pre-eclampsia. We aimed to investigate whether the expression of cystatin C is increased in the pre-eclamptic placenta and to investigate the expression pattern of cystatin C mRNA and protein in placental tissue. Tissue samples from the central part of the placenta from 13 normal and 22 pre-eclamptic pregnancies were included. We used real-time polymerase chain reaction (RT-PCR) and in situ hybridization for mRNA expression analysis and immunohistochemistry and Western blotting for protein expression analysis. RT-PCR showed a significantly higher expression of cystatin C mRNA in pre-eclampsia than in normal pregnancy, with the highest expression in cases with severe pre-eclampsia. In situ hybridization revealed a distinct pattern of high expression in the extravillous trophoblast cells of the basal plate and low expression in the syncytiotrophoblast covering villi. The cystatin C protein distribution matched the mRNA expression pattern. Western blot analysis revealed an increased protein expression in cases with severe pre-eclampsia and confirmed the presence of cystatin C in amniotic fluid samples. The high expression of cystatin C mRNA in the extravillous trophoblast cells of the basal plate suggests a role for cystatin C in the regulation of proteases in placentation. Placental expression and secretion of cystatin C could contribute to the elevated maternal plasma levels seen in pre-eclampsia.