HLA-G regulates the invasive properties of JEG-3 choriocarcinoma cells by controlling STAT3 activation

Placenta 34 (2013) 1044e1052

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HLA-G regulates the invasive properties of JEG-3 choriocarcinoma cells by controlling STAT3 activation X. Liu a, b,1, W. Gu a, b, *, X. Li a, b a b

Obstetrics and Gynecology Hospital, Fudan University, Shanghai, PR China Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Shanghai, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 June 2013 Received in revised form 28 July 2013 Accepted 31 July 2013

The expression of human leucocyte antigen-G (HLA-G) in trophoblasts plays a crucial role in successful embryonic implantation, and reduced HLA-G expression might contribute to adverse obstetric outcomes. In this study, we silenced HLA-G expression using RNA interference in JEG-3 cells, resulting in a notably attenuated invasion capacity of the cells in a Transwell assay; however, no alterations in cell proliferation or apoptosis were observed. The down-regulation of HLA-G dampened the activation of signal transducer and activator of transcription 3 (STAT3), whereas the up-regulation of HLA-G promoted STAT3 activation and invasion in JEG-3 cells treated with human galectin-1. Most importantly, interleukin-6 (IL-6), but not galectin-1, was shown to rescue invasion deficiency in a dose-dependent manner. Thus, we demonstrate that HLA-G is able to regulate JEG-3 cell invasion by influencing STAT3 activation, which may underlie the implantation defects accompanying HLA-G hypo-expression in pre-eclampsia. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: JEG-3 Trophoblast invasion HLA-G STAT3 IL-6 Human galectin-1

1. Introduction Normal development of the placenta and adequate trophoblast functioning are prerequisites for a successful pregnancy [1,2]. The major challenge in establishing a proper maternalefetal interface during pregnancy in mammals is that the embryo must evade the mother’s immune system [3e5]. Extra-embryonic trophoblasts invade the uterine spiral arteries and induce vascular remodelling in order to increase maternal blood flow to the placenta [6,7]. Accumulating evidence indicates that insufficient invasion of extraembryonic trophoblasts might contribute to the occurrence of some obstetric complications, such as pre-eclampsia, miscarriage and intrauterine growth retardation (IUGR) [8e10]. Human leucocyte antigens (HLAs) are the most complex polymorphism system involved in immune tolerance [11], and the HLA family can be divided into classic and non-classical HLAs. Many in vivo and in vitro studies have demonstrated that HLAs play important roles in trophoblast invasion into the decidua [12]. For example, Hemberger et al. reported that major histocompatibility complex (MHC) repertoires on the surface of trophoblasts might

* Corresponding author. Obstetrics and Gynecology Hospital, Fudan University, 419 Fangxie Road, Shanghai 200011, PR China. Fax: þ86 21 63455090. E-mail address: [email protected] (W. Gu). 1 Xueyuan Liu is the first author. 0143-4004/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.placenta.2013.07.070

affect uterine vascularisation and fetal growth [11]. Interestingly, extra-embryonic trophoblasts express non-classical molecules with low polymorphism, such as HLA-C, HLA-E and HLA-G, but not the classic polymorphic molecules, such as HLA-A and HLA-B, in order to elude recognition and attack by the maternal immune system [11]. In particular, HLA-G has been implicated in the formation of the placenta and the pathogenesis of pre-eclampsia [13,14] and seven HLA-G isoforms have been identified to date, including four membrane-bound isoforms (G1eG4) and three soluble isoforms (G5eG7) [15]. Previous studies have documented roles for the HLAG proteins in the interactions between immune cells, such as natural killer (NK) cells and trophoblasts, whereby HLA-G isoforms inhibit NK cytotoxicity and cytokine production [16]. However, it remains unclear whether HLA-G might exert an independent regulatory role on the invasive property of trophoblasts. Although the intrinsic function of HLA-G in trophoblasts has been specifically addressed, we sought to re-analyse the intrinsic regulatory effect of HLA-G due to the use of an unsuitable cell line and questionable data in previous studies [17,18]. Trophoblast invasion can be regulated through interactions of extracellular molecules, such as interleukin-6 (IL6), with their cell membrane receptors via such intracellular signalling transducers as the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) [19] and signal transducer and activator of

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transcription 3 (STAT3) signalling pathways [20]. It was previously demonstrated that IL-6 and its receptors are expressed on the surface of trophoblasts, with both endogenous and externally added IL6 promoting the activation of STAT3 to modulate the migration and invasion of several normal or cancerous trophoblast cell lines [21e24]. Moreover, active STAT3, the crucial downstream mediator of the IL-6 signalling pathway, has been found to colocalise with focal adhesion proteins [25], whereas STAT3 downregulation perturbed the motility and invasion of cancerous trophoblasts [26,27]. Notably, numerous works have suggested that the activation of STAT3 is essential for the invasion of primary trophoblasts and trophoblast cell lines [28]. However, although IL-6 and STAT3 activation have been shown to modulate the migration and invasion of primary trophoblasts and trophoblast cell lines [24,28,29], the possible relationship between HLA-G expression in these cells and STAT3 activation and whether IL6 can rescue the phenotype caused by the down-regulation of HLA-G have not yet been examined. In the present study, we provide evidence indicating that HLA-G regulates the invasion potential of JEG-3 cells by facilitating STAT3 activation without interaction with immune cells. Furthermore, IL6 was found to be able to rescue the defective invasion phenotype caused by HLA-G down-regulation. These results provide insight into a plausible involvement of reduced HLA-G expression in the pathogenesis of pre-eclampsia. 2. Materials and methods 2.1. Cell culture The JEG-3 cells were purchased from the Cell Source Center of the School of Basic Medicine Peking Union Medical College. The cells were cultured in RPMI 1640 medium (Gibco, Grand Island, NY, USA; A10491-01) supplemented with 10% fetal bovine serum (FBS; PAA, Piscataway, NJ, USA; A15-151) without penicillin/streptomycin and amphotericin B in a humidified atmosphere of 5% CO2 at 37  C. 2.2. Gene silencing by small interfering RNA and transfection efficiency measurement Two 25-nt small interfering RNA (siRNA) oligonucleotides targeting HLA-G at exon 5 and/or exon 6 (according to the HLA-G messenger RNA (mRNA) sequence in GenBank; NM_002127) and control siRNA oligonucleotides not targeting any sequence in the human genome were synthesised by GenePharma Co., Ltd., Shanghai, China. The sequences of the HLA-G siRNAs and control siRNA are shown in Table 1. JEG-3 cells were cultured in 35-mm dishes (Corning Incorporated, Corning, NY, USA; 430165). On reaching 50e70% confluence, the cells were washed three times with Opti-MEM medium (Invitrogen, Carlsbad, CA, USA), and 500 ml of fresh OptiMEM medium was then added to each well. The siRNA targeting HLA-G (10 nM) was mixed with 200 ml of Opti-MEM and incubated for 5 min at room temperature. In another tube, 5 ml of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA; 11668019) was mixed with 200 ml of Opti-MEM medium and incubated for 5 min at room temperature. The solutions were then mixed together, incubated for 20 min at room temperature and added to the wells; after 5 h of incubation, the solutions were replaced with 1.5 ml normal culture medium. To verify the efficiency of the siRNA molecules against HLA-G, all the experiments included no-transfection samples as blanks and samples transfected with non-genomic siRNA as controls. The transfection efficiency was determined by immunocytochemistry to measure the abundance of the green fluorescence cells relative to the total cells in the culture. The efficiency of the siRNA knockdown was verified using a Western blotting analysis

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and reverse transcriptase-polymerase chain reaction (RT-PCR) at 48 h after transfection. For the Matrigel invasion assay (N ¼ 5), the cells were trypsinised at 48 h after transfection, as detailed below. To verify the activation of STAT3, the JEG-3 cells were transfected with siRNA; after 24 h, the medium was removed and replaced with RPMI-1640 medium without FBS. Then, the serum starved for 48 h and the total protein was extracted. 2.3. Galectin-1 treatment in vitro To increase the expression of HLA-G, the JEG-3 cells were cultured in RPMI 1640 medium without FBS and starved for 24 h. The cells were then treated with different concentrations (0, 100 or 1000 ng mle1; N ¼ 3 per treatment) of human recombinant galectin-1 (PeproTech Inc., Rocky hill, NJ, USA; 450-39) in serum-free medium, as previously described [30]. After a 48-h incubation, the RNA or protein was extracted, as detailed later. 2.4. RNA extraction and semi-quantitative RT-PCR The total RNA from JEG-3 cells was extracted using Trizol (Invitrogen, Carlsbad, CA, USA; 15596026). Reverse transcription and first-strand complementary DNA (cDNA) synthesis was performed using a PrimeScriptÔ RT reagent Kit (TaKaRa, Otsu, Shiga, Japan; RR047A). The gene expression level was quantified by semiquantitative RT-PCR using b-actin as an internal control. The specific primers for HLA-G and b-actin are shown in Table 2. The PCR amplification was performed for 4 min at 95  C, followed by 25 cycles of 30-s denaturation at 95  C and 30-s annealing at 60  C for both HLA-G and b-actin. The amplification products were confirmed by sequencing. 2.5. Western blotting Total proteins were extracted with a cell lysis buffer (Beyotime, Shanghai, P. R. China; P0013) containing two protease inhibitors (1:100, 524627; Calbiochem, Billerica, MA, USA; and 1:100, P5726; Sigma, St Louis, MO, USA). The samples were separated by 10% sodium dodecyl sulphateepolyacrylamide gel electrophoresis (SDSePAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes at a constant current of 280 mA for 90 min (Bio-Rad Laboratories, Hercules, CA, USA; 162-0177). The membranes were blocked for 1 h at room temperature in Trisbuffered saline with o.1% Triton-100 (TBST) buffer containing 5% bovine serum albumin (BSA). The membranes were then incubated with the primary antibody and diluted in blocking buffer overnight at 4  C; detailed information for the antibodies is shown in Table 3. After extensive washes in TBST, the PVDF membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (HRP; Invitrogen, Carlsbad, CA, USA; 1:5000, A10677 or G21234) for 1 h at room temperature. After three 5-min washes in TBST, the membranes were developed according to the protocol. Moreover, the illumination intensities of bands were detected by Image J software. 2.6. Matrigel invasion assay To determine the invasion capacity of the cells after the down-regulation of HLA-G, the JEG-3 cells transfected with siRNAs were trypsinised after 48 h and resuspended in culture medium. The cells were seeded at a density of 1.0  105 cells/ ml into the upper chambers of BD BioCoatÔ BD Matrigel cell culture inserts precoated with 1:5 diluted Matrigel (BD Biosciences, MA, USA, 354480). The cells were allowed to invade for 24 h in RPMI-1640 medium without FBS in a humidified 37  C incubator with 5% CO2. After fixation with 4% paraformaldehyde (PFA), the cells in the upper chamber were scraped off lightly using cotton swabs, and the cells in the lower chamber were stained with DAPI (4,6-diamino-2-phenylindole) dye (N ¼ 3 per treatment). Nine random fields of invaded cells were counted for each well in the experiment. For the galectin-1 treatment experiments, the JEG-3 cells were incubated in culture medium with different concentrations of galectin-1 (100 or 1000 ng mle1; N ¼ 3 per treatment). For the IL-6 treatment experiments, the JEG-3 cells were incubated in culture medium with different concentrations of IL-6 (0, 10, 20 or 30 ng mle1, N ¼ 3 per treatment). Following treatment, the samples were processed as described above.

Table 1 Oligonucleotides sequences of HLA-G specific RNA interference. Name

Sequences of oligonucleotides

Target nucleotide site

Control

50 -UUCUCCGAACGUGUCACGUTT-30 50 -ACGUGACACGUUCGGAGAATT-30 50 -UUUCUCCACAGCACAGCAGCGACCG-30 50 -CGGUCGCUGCUGUGCUGUGGAGAAA-30 50 -AUGGUGGGCAGGGAAGACUGCUUCC-30 50 -GGAAGCAGUCUUCCCUGCCCACCAU-30

None

Si1 Si2

Table 2 Primers applied in this paper. Name of primers

1155e1179 (exon6)

HLA-G

1071e1095 (exon5-6)

Actin

Sequences of primers 0

Length of products 0

5 -CTGACCCTGACCGAGACCTGG-3 50 -GTCGCAGCCAATCATCCACTGGAG-30 50 -CGTTGACATCCGTAAAGAC-30 50 -TGGAAGGTGGACAGTGAG-30

338 200

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Table 3 Primary and secondary antibodies used in this paper. Name of primary antibody

Species, dilution

Source.

GFP STAT3 phospho-STAT3 (Y705) a-Tubulin Activated cleavage caspase3 phospho-Histone 3 Brdu HLA-G 546-Alexa goat anti-rabbit 546-Alexa goat anti-mouse HRP-goat anti-mouse HRP-goat anti-rabbit

Rabbit, 1:1000 Mouse, 1:1000 Rabbit, 1:500 Mouse, 1:5000 Rabbit, 1:200 Mouse, 1:500 Mouse, 1:200 Mouse, 1:200 Goat, 1:1000 Goat, 1:1000 Mouse, 1:1000 Rabbit, 1:1000

Invitrogen, A11122 CST, 9139S CST, 9145P Sigma, T6074 CST, 9661 Upstate, 06-570 Sigma, B2531 Novus biological, NB500314 Invitrogen, A11010 Invitrogen, A11003 Invitrogen, A10668 Invitrogen, G21234

medium) incorporation assay, the fixed cells were incubated with 2 N HCl (1:4 mixture of hydrochloric acid:ddH2O) at 37  C and washed three times with PBS for 10 min each. Cells were then incubated with primary antibodies overnight at 4  C; detailed information about the primary antibodies and the working concentrations is shown in Table 3. After three 5-min washes in PBS, the cells were incubated with a 1:1000 dilution of secondary antibody conjugated to Alexa 546 for 1 h at room temperature. The nuclei were stained with DAPI dye (Sigma, St Louis, MO, USA; D8417) for 30 min, and the coverslips were mounted with 90% glycerol. The images were acquired using an Olympus confocal microscope. The Brduþ, PH3þ and activated cleavage caspaseþ cells were counted using Image J software. 2.8. Statistical analyses Statistical calculations were performed using the GraphPad.Prism5 software and are presented as the mean  the standard error of the mean (SEM). Statistical significance was determined using a one-way analysis of variance (ANOVA) and a P value <0.05 was considered statistically significant.

2.7. Immunocytochemistry To determine the proliferation and apoptosis rates in the JEG-3 cells after HLA-G down-regulation, the cells transfected with siRNA were cultured on 18  18-mm glass coverslips. After 2 days, the cells were fixed with 4% PFA for 10 min at room temperature and washed three times in phosphate-buffered saline (PBS). The cells were then permeabilised and blocked with phosphate-buffered saline with 0.2% Triton-100 (PBST) containing 10% BSA for 1 h at room temperature. For the 4-h bromodeoxyuridine (BrdU; final concentration of 1 mg mle1 in the culture

3. Results 3.1. Down-regulation of HLA-G in JEG-3 cells via siRNA interference To explore the function of HLA-G in JEG-3 cells, we designed and synthesised two pairs of specific siRNA oligonucleotides targeting

Fig. 1. HLA-G siRNA reduces HLA-G mRNA and protein expression levels in JEG-3 cells. All of the data shown were obtained from three independent experiments. (A) Representative transfection efficiency images of JEG-3 cells. Scale bar, 20 mm. (B) HLA-G mRNA expression in JEG-3 cells was detected using RT-PCR; actin was used as an internal control. (C) HLA-G mRNA expression levels relative to the blank (non-transfected group) and control (non-genomic siRNA transfected group). (D) HLA-G expression in JEG-3 cells was detected using a Western blotting analysis; a-tubulin was used as an internal control. (E) HLA-G expression levels relative to the blank (non-transfected group) and control (non-genomic siRNA transfected group). The significance of the results was determined using a one-way ANOVA. The data are presented as the mean  SEM; ***P < 0.001.

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the regions common to all HLA-G isoforms. The efficiency of the HLA-G knockdown was confirmed using RT-PCR and Western blotting analysis, and the transfection efficiency was approximately 70% (Fig. 1A). We observed a marked decrease in the levels of HLA-G messenger RNA (mRNA; Fig. 1B and C) and protein (Fig. 1D and E) by either siRNA1 or siRNA2 compared to the blanks (non-transfected group) and the controls (non-genomic siRNA-transfected group), thereby demonstrating that these two HLA-G siRNAs were suitable for use in the ensuing functional assays.

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3.2. Invasiveness of JEG-3 cells after HLA-G down- or up-regulation Because reduced implantation efficiency has been documented in pregnant women whose trophoblasts exhibit HLA-G deficiency [31], we investigated whether the invasiveness of JEG-3 cells was affected by HLA-G down-regulation by applying the Transwell method to monitor JEG-3 cell invasiveness. Using non-transfected control, siRNA-transfected and HLA-G siRNA-transfected cells seeded at the same cell density in Matrigel-coated inserts, we

Fig. 2. HLA-G regulates the invasion capacity of JEG-3 cells. All of the data shown were obtained from five independent experiments. (A) Representative images of transfected cells on the bottom side of the chamber; at 24 h after seeding, the cells were fixed and stained with DAPI dye (green). Scale bar, 50 mm. (B) The average number of cells invading the gel was quantified for each HLA-G down-regulation experiment. (C) JEG-3 cells were incubated with increasing concentrations of human galectin-1 for 48 h, and HLA-G expression was analysed using RT-PCR. (D) The results show HLA-G expression in the JEG-3 cells relative to the non-treated controls. (E) Representative images of galectin-1 treated cells on the bottom side of the chamber; at 24 h after seeding, the cells were fixed and stained with DAPI dye (green). Scale bar, 50 mm. (F) The average number of cells that invaded the gel was quantified for each galectin-1 treatment experiment. The significance of the results was determined using a one-way ANOVA. The data are presented as the mean  SEM; ***P < 0.001.

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observed a significant reduction in the invasion potential of the JEG-3 cells after HLA-G knockdown (P < 0.01) (Fig. 2A and B). To further explore the function of HLA-G in trophoblast invasion, we treated the JEG-3 cells with human galectin-1 expressed from different trophoblast types, such as extravillous cytotrophoblasts (EVCTs) [32], which have been shown to induce HLA-G expression in the HIPEC65 cell line [30]. Firstly, we found that the level of HLAG mRNA was up-regulated when the JEG-3 cells were treated with 1000 ng mle1 exogenous human galectin-1 but not when treated with 100 ng mle1 galectin-1 (Fig. 2C and D). Consistently, we found a slight but significant increase in the invasion potential of the JEG3 cells after treatment with 1000 ng mle1 human galectin-1 (Fig. 2E and F). 3.3. Effects of HLA-G down-regulation on JEG-3 cell proliferation and survival We next investigated whether the retention of the HLA-G knockdown JEG-3 cells in the Transwell insert was due to change in proliferation. Firstly, we analysed acute BrdU incorporation and found that the percentage of BrdUþDAPIþ cells was not significantly

different between the groups (Fig. 3A and D). To further verify this result, we investigated the percentage of cells in the mitotic phase using phosphorylated histone H3 immunostaining. As anticipated, the percentage of PH3þDAPIþ cells was not significantly different between the groups (Fig. 3B and E). Next, we used immunocytochemistry to determine the percentage of apoptotic cells after HLA-G knockdown to evaluate whether the aberrant invasiveness caused by the down-regulation of HLA-G originated from the induction of apoptosis. Again, we found that the percentage of activated cleavage caspase3þDAPIþ cells was not significantly different between the groups (Fig. 3C and F). Taken together, we demonstrated that the down-regulation of HLA-G did not disturb the proliferation of or induce apoptosis in the JEG-3 cells, indicating that the defect in invasiveness was not the result of altered proliferation or survival. 3.4. Levels of STAT3 activation after HLA-G down- or up-regulation The Janus kinase-signal transducer and activator of transcription 3 (JAK-STAT3) signalling pathway is an important pathway involved

Fig. 3. HLA-G down-regulation does not affect the proliferation and survival of JEG-3 cells. All of the data shown were obtained from four independent experiments. (A) Representative images of cells after 4 h of BrdU incorporation. Two days after siRNA transfection, the cells were fixed and stained with DAPI dye (green) and an anti-BrdU antibody (red). (B) Representative images of cells showing the percentage of cells in the mitotic phase. Two days after transfection, the cells were fixed and stained with DAPI dye (green) and an antibody against phosphorylated histone 3 (PH3) (red). (C) Representative images of cells showing the percentage of apoptotic cell. Two days after transfection, the cells were fixed and stained with DAPI dye (green) and an antibody against activated cleaved caspase3 (red). Scale bar, 30 mm. (D) The average percentage of transfected cells that were BrdUþDAPIþ. (E) The average percentage of transfected cells that were PH3þDAPIþ. (F) The average percentage of transfected cells that were activated cleaved caspase3þDAPIþ. NS, P > 0.05.

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in the migration and invasion of a variety of cell types, and the phosphorylation of STAT3 at Y705 represents a key step in this pathway [21]. Thus, we investigated the possibility that HLA-G may modulate STAT3 activation through the phosphorylation of Y705 to mediate and/or regulate the invasion potential of trophoblasts. We measured the level of total STAT3 and activated STAT3 using a Western blot analysis after altering HLA-G expression and found reduced STAT3 activation in the knockdown group in comparison to the non-transfected cells or cells transfected with non-genomic siRNA (P < 0.05; Fig. 4A and C). However, the total STAT3 levels did not change (Fig. 4B and D). Thus, we found that reducing the expression of HLA-G inhibited STAT3 activation. We then investigated whether STAT3 could be activated with galectin-1 treatment. Interestingly, we found that the level of activated STAT3 increased after the cells were treated with 1000 ng mle1 human galectin-1, though the total STAT3 levels were unchanged (Fig. 4EeH). To explore whether restoring STAT3 activation could rescue the invasion deficiency caused by the down-regulation of HLA-G, we treated the siRNA-transfected cells and non-genomic siRNAtransfected cells with different concentrations of human galectin-1

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or IL-6 e a well-documented agonist of STAT3 activation [33] e and monitored the invasiveness of the JEG-3 cells. Firstly, we found that galectin-1 could not rescue the invasion deficiency after HLA-G down-regulation (Fig. 6A and C). However, we did find that treatment with 10 or 20 ng mle1 IL-6 could partially elevate the average level of activated STAT3 from 51  1.3% to 64  2.8% or 97  8.5%, respectively (Fig. 5A and B) and that the average number of invaded cells increased from 144.3  20.84 to 392.0  25.73 or 784.0  14.76, respectively (Fig. 6A and B). When the IL-6 concentration was increased to 30 ng mle1, the level of activated STAT3 was restored to normal (Fig. 5A and B) and the average number of invaded cells was also restored to normal (Fig. 6A and B). As described above, we again did not detect any changes in the level of total STAT3 (Fig. 5C and D). 4. Discussion It has been consistently observed that the abnormal expression of HLA-G in trophoblasts is associated with placental dysfunction [34]. In a previous study, the down-regulation of HLA-G via short

Fig. 4. Activation of STAT3 positively correlates with the expression of HLA-G. All of the data shown were obtained from five independent experiments. (A) Representative Western blotting showing the levels of activated STAT3 following the down-regulation of HLA-G in JEG-3 cells; a-tubulin was used as an internal control. (B) Representative Western blotting for total STAT3 after the down-regulation of HLA-G in JEG-3 cells. a-Tubulin was used as an internal control. (C) Activated STAT3 expression in JEG-3 cells relative to the blank (nontransfected group) and control (non-genomic siRNA transfected group). (D) Total STAT3 expression in JEG-3 cells relative to the blank (non-transfected group) and control (nongenomic siRNA transfected group). (E) Representative Western blotting showing the levels of activated STAT3 expression following human galectin-1 stimulation. (F) Representative Western blotting showing the total STAT3 levels following human galectin-1 stimulation. (G) Activated STAT3 expression in JEG-3 cells relative to the non-treated controls treated with increasing human galectin-1 concentrations. (H) Total STAT3 expression in JEG-3 cells relative to the non-treated controls treated with increasing human galectin-1 concentrations. In the graphs, the data are presented as the mean  SEM; NS, P > 0.05; ***P < 0.001, as analysed using a one-way ANOVA.

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Fig. 5. IL-6 can rescue the levels of activated STAT3 in JEG-3 cells in a dose-dependent manner. All of the data shown were obtained from five independent experiments. (A) Representative Western blotting showing the levels of activated STAT3 following HLA-G down-regulation and IL-6 stimulation in JEG-3 cells. (B) Activated STAT3 expression in JEG-3 cells. (C) Representative Western blotting showing the total STAT3 level following HLA-G down-regulation and IL-6 stimulation in JEG-3 cells. (D) Total STAT3 expression levels in JEG-3 cells. The significance of the results was determined using a one-way ANOVA. The data are presented as the mean  SEM; NS, P > 0.05; **P < 0.01.

hairpin RNA (shRNA) interference in trophoblast-derived cell lines increased their sensitivity to the cytotoxic effects of immune cells [16]. Additionally, extensive studies have demonstrated that immune cells, such as NKs, in the decidua direct trophoblast invasiveness [35]. These results indicate that HLA-G interacts with receptors on the surface of immune cells to affect the biological behaviour of trophoblasts. The present study sought to investigate whether an intrinsic mechanism exists that accounts for this association between HLA-G and trophoblast invasion. Our in vitro results clearly showed that a reduction in HLA-G expression decreased the invasion potential of JEG-3 cells, whereas the cell proliferation and apoptosis rates were not affected. Therefore, the observed invasion defect in the HLA-G-knockdown cells could not be interpreted as an alteration in either cell growth or survival. Furthermore, we found that treating JEG-3 cells with exogenous human galectin-1 improved the expression of HLA-G and increased the invasion capacity of the cells. This observation is in agreement with previous results that showed that the human galectin-1/HLAG axis is a crucial component of the human trophoblast-invasion machinery [30,36]. Intriguingly, a previous study demonstrated that exogenous galectin-1 stimulation can increase the levels of the membrane-bound HLA-G1 and HLA-G2 isoforms but not the levels of the soluble isoforms, HLA-G5 and HLA-G6, in a human EVCTderived cell line. Moreover, it has been shown that the overexpression of soluble HLA-G inhibits the trophoblast invasion induced by epidermal growth factor (EGF) and hepatocyte growth factor (HGF) [37]. Ian et al. reported that membrane-bound HLA-G might be the predominant mRNA isoform expressed in JEG-3 cells [38]. Taken together, although we could not clearly distinguish which HLA-G isoforms are involved in the regulation of the invasiveness of the trophoblast-derived cell lines, these observations support the hypothesis that membrane-bound HLA-G may play a more important role in trophoblast invasion in vitro. Conversely, the mechanism by which galectin-1 increases HLA-G expression remains unknown. In light of Sandra’s report that silencing endogenous galectin-1 decreased the phosphorylation of Src and Tex family proteins, galectin-1 may positively affect HLA-G transcription by increasing the phosphorylation of these proteins [30].

In summary, our results showed that HLA-G directly affects the invasion of trophoblasts without interacting with immune cells. In addition, our experiments revealed a positive relationship between HLA-G expression and STAT3 activation. We found that the down-regulation of HLA-G led to a decrease in STAT3 activation but did not affect total STAT3 expression; by contrast, the upregulation of HLA-G following treatment with human galectin-1 led to an increase in STAT3 activation but also did not change total STAT3 expression. Furthermore, we found that the restoration of STAT3 activation by IL-6 stimulation was positively correlated with the restoration of invasiveness after HLA-G down-regulation in JEG-3 cells. These results indicate that HLA-G regulates the invasiveness of JEG-3 cells via the activation of STAT3. Indeed, recent tissue-microarray research has found that the expression and activation of STAT3 are lower in the tissues of pre-eclampsia patients, which correlates with their HLA-G deficiency [39]. Previous studies have demonstrated that the activation of STAT3 is essential for the proliferation, survival and migration of various cancers and normal developmental tissues, including the placenta [40,41]. In particular, the activation of STAT3 is high during the early postimplantation stage and is indispensable in murine pregnancy: mice deficient in STAT3 or gp130, a STAT3-activating subunit, exhibit severe defects in implantation or early embryo death [28,42]. Because many investigations have indicated that STAT3 activation is associated with cell growth, particularly tumour growth [43], our results lead to the question of why cell growth and death were not perturbed when STAT3 was hypo-activated. One reasonable explanation is that a low residual amount of STAT3 activation is sufficient to maintain the proliferation and survival of JEG-3 cells. Indeed, the work of some authors appears to support this scenario. For example, when STAT3 expression was reduced in zebrafish using morpholinos, the interruption of gastrulation and the disturbance in cell movement following the loss of STAT3 activity were not accompanied by any changes in early cell-fate specification, proliferation or apoptosis [44]. Although it remains unknown how HLA-G regulates STAT3 activation to modulate the invasion of JEG-3 cells, the diverse HLA-

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Fig. 6. IL-6 but not galectin-1 can rescue the invasion capacity of JEG-3 cells in a dose-dependent manner. The IL-6 data shown were obtained from five independent experiments. The galectin-1 data shown were obtained from three independent experiments. (A) Representative images of cells on the bottom side of the chamber. At 24 h after seeding and the addition of IL-6 or 1000 ng ml1 galectin-1 to the culture medium, the cells were fixed and stained with DAPI dye (green). Scale bar, 50 mm. (B) The average number of cells invaded the gel was quantified for experiment. (C) The average number of invaded cell was quantified for galectin1 treatment experiment. The significance of the results was determined using a one-way ANOVA. The data are presented as the mean  SEM; NS, P > 0.05; ***P < 0.001.

G isoforms may form complexes to influence STAT3 activation, either indirectly through the regulation of IL-6 expression or directly through the recruitment of other critical proteins involved in the STAT3 pathway. Overall, we revealed that HLA-G regulates the invasiveness of JEG-3 cells via the regulation of STAT3 activity but does not affect cell proliferation or apoptosis. These results reveal a new activity of HLA-G in JEG-3 cells, directly explaining the clinical association between HLA-G expression and invasion deficiency in preeclampsia without immune cell interaction. Moreover, our findings suggest that certain drugs or agents that increase the levels of activated STAT3 in trophoblasts may be helpful in the treatment of pre-eclampsia patients who have associated HLA-G hypo-expression and STAT3 activation deficiency.

Funding This project was supported by Shanghai Municipal Natural Science Foundation (10ZRl404900), Health industry special funds for Public Benefit Research Foundation from the Ministry of Health, People’s Republic of China (201002013), the National Science Fund of China (81270712), Program of Shanghai Subject Chief Scientist (12XD1401300), Program of Shanghai Leading Talent (2012),

Shanghai Municipal Health Bureau (12GWZX0301) and National Key Basic Research Plan of China (973 Plan) (2010CB529500).

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