Fresh placental tissues at 26–28 weeks of gestation were obtained from 3 healthy pregnant women (age, 24.2±2.4 years) and from 3 female patients with preeclampsia (age, 25.7±1.8 years) who were recruited from the Department of Obstetrics and Gynecology, Fujian Provincial Hospital (Fujian, China), between July 2015 and December 2015. The specimens were immediately snap-frozen, and stored in liquid nitrogen until use. The present study was conducted with the approval of the Ethics Committee of Fujian Provincial Hospital (Fuzhou, China), and informed consent was obtained from all patients.

The human trophoblast cell line HTR8/SVneo was obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma-Aldrich; Merck KGaA) at 37°C in a humidified 5% CO₂/95% air atmosphere.

The small interfering RNA against JARID2 (siRNA-JARID2; 5′-AGGAAGAGGAGGAGGACAA-3′) and control siRNA (scramble; 5′-GAGUGGGUCUGGGUCUUCCCGUAGA-3′) were chemically synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). HTR8/SVneo cells were transfected for 24 h with siRNA-JARID2 or scramble using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol.

Total RNA was extracted from frozen tissues or cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was synthesized from the extracted RNA (5 µg) using the EasyScript First-Strand cDNA Synthesis SuperMix kit (Invitrogen; Thermo Fisher Scientific, Inc.). qPCR analysis was performed using a 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) with Fast Start Universal SYBR Green Master (Roche Diagnostics, Basel, Switzerland). PCR amplification was performed using the following primers: JARID2, 5′-GACACCAAACCCAATCACCAC-3′ (sense) and 5′-GTTCAACCTGCCACTGACCTT-3′ (antisense); and β-actin, 5′-TTAGTTGCGTTACACCCTTTC-3′ (sense) and 5′-ACCTTCACCGTTCCAGTTT-3′ (antisense). The thermocycling conditions were as follows: 95°C for 4 min, followed by 40 cycles of 95°C for 25 sec, 55°C for 30 sec and 72°C for 20 sec with 2 sec for plate reading, then melting curve analysis from 65 to 95°C. Data were analyzed using the formula: R=2^{−[ΔCq sample-ΔCq control]} (14).

Total protein was extracted from frozen tissues or cells using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Protein content was determined using a bicinchoninic acid protein assay (Pierce; Thermo Fisher Scientific, Inc.). Proteins (30 µg/lane) were separated using SDS-PAGE on a 12% gel and transferred onto a nitrocellulose membrane (EMD Millipore, Billerica, MA, USA). Following blocking with 5% fat-free milk in PBS containing 0.1% (v/v) Tween-20 at room temperature for 1 h, membranes were incubated with one of the following mouse primary antibodies: anti-JARID2 (1:3,000; cat. no. SAB2105079; Sigma; Merck KGaA), anti-MMP2 (1:2,000; cat. no. sc-13594), anti-MMP9 (1:3,000; cat. no. sc-21733), anti-PI3K (1:2,000; cat. no. sc-365290), anti-p-PI3K (1:2,500; cat. no. sc-293115), anti-Akt (1:3,000; cat. no. sc-5298), anti-p-Akt (1:2,500; cat. no. sc-52940) or anti-GAPDH (1:2,000; cat. no. sc-47724; all Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight. Membranes were subsequently incubated with goat anti-mouse horseradish peroxidase (HRP)-conjugated immunoglobulin (Ig)G (1:2,500; cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) or goat anti-rabbit HRP-conjugated IgG (1:3,000; cat. no. sc-2004; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The target protein was visualized using enhanced chemiluminescence (Pierce; Thermo Fisher Scientific, Inc.) and densitometry was performed using Gel-Pro Analyzer software version 4.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Cell viability was measured using the Cell Counting Kit-8 assay (CCK-8; Dojindo Molecular Technologies, Inc. Kumamoto, Japan). HTR8/SVneo cells were plated at a density of 1×10⁴ cells/well in 96-well plates and transfected with siRNA-JARID2 or scramble. Following incubation for 24 h, the CCK-8 reagents were added and incubated with the cells for 1 h. Subsequently, the absorbance at 450 nm was measured using an ELISA plate reader.

Cell invasion assays were performed using Transwell™ chambers (Costar; Corning Incorporated, Corning, NY, USA). Transfected HTR8/SVneo cells (1×10⁴ cells/well) were plated in the top chamber of the insert, which had been precoated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA; 8 µm pore size), and 500 µl DMEM containing 10% FBS was added to the lower chamber. Following 24 h of incubation, cells transferred to the lower surface of the base membrane were stained with hematoxylin and eosin (Sigma-Aldrich; Merck KGaA), and counted per 4 high power fields under a light microscope (magnification, ×100). The migration assay was performed by the same procedure, except that the inserts were not pre-coated with Matrigel.

Data are expressed as the mean ± standard deviation. All experiments were repeated at least three times. Statistical significance was analyzed using one-way factorial analysis of variance followed by a Dunnett's post hoc test or Student's two-tailed t-test. P<0.05 was considered to indicate a statistically significant difference.

In order to investigate the role of JARID2 in preeclampsia, the expression levels of JARID2 in placental tissues were examined by RT-qPCR analysis and western blotting. As presented in Fig. 1A, JARID2 mRNA levels were significantly decreased in placentas obtained from preeclamptic patients compared with placentas from healthy subjects. Similarly, the results of the western blot analysis indicated that the protein expression of JARID2 was significantly decreased in preeclamptic placentas compared with the control group (Fig. 1B).

In order to gain further insight into the effect of JARID2 on cell viability, JARID2 was downregulated using siRNA against JARID2 in HTR8/SVneo cells. As demonstrated in Fig. 2A and B, respectively, siRNA knockdown of JARID2 significantly decreased the expression of JARID2 at the mRNA and protein levels in HTR8/SVneo cells. In addition, the effects of si-JARID2 on HTR8/SVneo cell viability were examined. The results of the CCK-8 assay demonstrated that, compared with the scramble group, knockdown of JARID2 significantly inhibited the viability of HTR8/SVneo cells (Fig. 2C).

The present study investigated the effects of JARID2 on HTR8/SVneo cell migration and invasion. The Transwell migration assay demonstrated that the knockdown of JARID2 significantly inhibited the migratory capacity of HTR8/SVneo cells, compared with the scramble group (Fig. 3A). The Matrigel assay demonstrated that the number of cells that passed through the Matrigel-coated membrane into the lower chamber was significantly decreased in si-JARID2-transfected cells compared with scramble-transfected cells (Fig. 3B). In addition, the present study analyzed the effect of JARID2 on MMP expression in HTR8/SVneo cells. The results of the western blot analysis demonstrated that knockdown of JARID2 significantly decreased the expression of MMP2 and MMP9 protein, compared with the scramble group (Fig. 3C).

In order to gain insights into the downstream signaling pathways modulated by si-JARID2 in preeclampsia inhibition, the present study investigated the effect of si-JARID2 on the activation of the PI3K/Akt signaling pathway in HTR8/SVneo cells. As presented in Fig. 4, the knockdown of JARID2 significantly decreased the levels of phosphorylated PI3K and Akt in HTR8/SVneo cells, compared with the scramble group.