30 PE patients were recruited from the Department of Obstetrics at the Nantong Women and Children Health Care Hospital between October 2015 and January 2016 (mean age 27.8±2.5 years; mean gestational age 34.1±2.3 weeks). Then 30 healthy pregnant women (mean age 27.7±3.1 years; mean gestational age 33.7±1.1 weeks) were recruited simultaneously as a control group. There were no significant differences between the two groups in terms of maternal and gestational ages. PE diagnosis was defined as severe gestational hypertension (systolic blood pressure of at least 140 mmHg and/or diastolic blood pressure of at least 90 mmHg on 2 occasions at least 4 h apart) and proteinuria (>30 mg/dl in at least 2 random urine specimens) after 20 weeks' gestation. Patients had no other obstetric or medical complications or histories of autoimmune disorders. All experimental procedures using human samples were performed with the approval of the Nantong Women and Children Health Care Hospital Ethics Committee and all the participants provided informed consent.

Blood samples (10 ml) were collected from each participant, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation at 2,000 × g for 20 min at 4°C (GE Healthcare Life Sciences, Little Chalfont, UK) and CD4⁺ T cells were sorted by immune-magnetic beads coated with anti-human CD4 antibodies (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). CD4⁺ T cells were washed twice and resuspended in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), placed in 24-well plates in the presence of anti-CD3 (cat. no. 14-0037-82) monoclonal antibodies (mAbs) 20 µl (diluted 1:10; eBioscience; Thermo Fisher Scientific, Inc.) and anti-CD28 mAbs (cat. no. 16-0288-81) 10 µl (diluted 1:2; eBioscience; Thermo Fisher Scientific, Inc.), and cultured at 37°C in 5% CO₂ to induce cell differentiation. On day 3, 500,000 U/l IL-2 (Changsheng Gene Pharmaceutical Co., Ltd., Changchun, China), was added to maintain cell growth and half of the medium was renewed. On day 6, suspended cells were collected.

siRNAs were designed using Primer 3 and the specificity was tested using the Primer-BLAST program (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). Then, 3 pairs of RORγt or T-bet specific double-stranded siRNAs were designed to target distinct sets of RNA sequences and the siRNAs were chemically synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). Details are presented in Table I.

To establish a stable transfection and evaluate transfection efficiency, 6-FAM-siRNA (Shanghai GenePharma Co., Ltd.) was transfected into cells simultaneously to measure the efficiency of transfection. In all, 3 conditions were tested on 2.5×10⁴ cells in each well of a 24-well plate with 30 pmol siRNA and 1, 2 or 3 µl DMRIE-C (Life Technologies; Thermo Fisher Scientific, Inc.); following 6 h transfection, efficiency was ascertained by fluorescence microscopy.

The medium was removed and cells were washed with Optimem^® I (Gibco; Thermo Fisher Scientific, Inc.) 1 day prior to transfection. Cells were then suspended at a concentration of 5×10⁴/ml and 2.5×10⁴ cells were seeded in a 24-well plate and incubated at 37°C in 5% CO₂ in a humidified incubator. After 24 h and at 80% confluence the cells were transfected with siRNA using DMRIE-C (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. There were 5 groups: Blank control (BC) transfected with blank liposome, negative control (NC) transfected with scrambled-siRNA, group 1 transfected with T-bet-siRNA, group 2 transfected with RORγt-siRNA and group 3 transfected with T-bet-siRNA and RORγt-siRNA. DMRIE-C reagent was diluted in Optimem^® I medium in a 0.5 ml Eppendorf tube, mixed at room temperature for 10 min and then diluted siRNA was added to the mixture and incubated for 30 min to form the siRNA/transfection reagent complex. The complex was then added to the cells and cultured in serum-free medium. Following transfection for 4–5 h at 37°C in 5% CO₂, the medium in each well was replaced with complete RPMI 1640 without antibiotics and incubated for 48 h at 37°C prior to subsequent experiments.

Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Inc.) and complementary DNA (cDNA) was synthesized from 1 µg RNA using the RevertAid™ First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.). RT-qPCR was conducted with the aid of the Rotor-gene Q Realtime PCR Platform and SYBR Green PCR Master Mix (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Oligonucleotide primer pairs used for the analysis were as follows: T-bet, sense AAT GTG ACC CAG ATG ATT GTG C and anti-sense ATG CTG GTG TCA ACA GAT GTG TAC; GAT A3, sense ACC GGC TTC GGA TGC AA and anti-sense TGC TCT CCT GGC TGC AGA C; RORγt, sense TAA CCA AAA ATG GAT GGG ATG and anti-sense, AAG CTG TGG CCT CAA GGA T; FOXP3, sense GAG AAG GAG AAG CTG AGT GCC AT and anti-sense AGC AGG AGC CCT TGT CGG AT; GAP DH, sense ACG GCA AATTC AAC GGG ACA GTC A and anti-sense TGG GG GCA TCG GCA GAA GG. Primers were synthesized by Shanghai GenePharma Co., Ltd. The amplifications were conducted in a 36-well plate in a 20 µl reaction volume containing 1X Real-time PCR Master Mix, 0.2 µM primer, 1 U rTaq polymerase, 2 µl cDNA samples and RNase-free water. The samples were run in triplicate for the target and for house-keeping genes using the following conditions: Initial denaturation for 3 min at 95°C, followed by a total of 40 cycles (denaturation 12 sec at 95°C and annealing 40 sec at 62°C) and extension for 30 sec at 72°C. Statistical analysis of the mean value as the representative of each investigated gene and the 2^−ΔΔCq method were used to analyze relative changes in gene expression (18).

Cells were homogenized in lysate buffer containing 10 mM Tris-HCl (pH 7.4), 1% Triton X-100, 1% sodium deoxycholate, 5 mM EDTA, 1 mM PMSF, 10 mg/l aprotinin and 50 mg/l leupeptin. The homogenate was then centrifuged at 12,000 × g for 10 min at 4°C to collect the supernatant. Protein concentration was determined using a BCA protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The supernatant was diluted in 5X SDS loading buffer and was boiled. Then 20 µg of protein were subjected to SDS polyacrylamide gel electrophoresis (5–10% gradient gels) and transferred to polyvinylidine diflouride filter membranes. The membrane was then blocked with 5% nonfat milk for 2 h at room temperature, and probed with the following monoclonal antibodies: Anti-T-bet mAbs, cat. no. sc21749; anti-GATA3-mAbs, cat. no. 268×; anti-RORγt mAbs, cat. no. Sc6062; anti-FOXP3 mAbs, cat. no. sc-166212, at a dilution of 1:100. Membranes were washed in Tris-buffered saline containing 0.5% Tween-20 and incubated with 5% nonfat dry milk overnight at 4°C. Following incubation with the appropriate horseradish peroxidase conjugated anti-human IgG monoclonal anti-body (diluted 1:100, cat. no. sc-2453), All monoclonal antibodies were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Proteins were detected with an enhanced chemiluminescent system (Pierce, Thermo Fisher Scientific, Inc.).

For intracellular cytokine staining, lymphocytes were collected and cultured in 96-well plates (200 µl and 2×10⁹ cells per well). 500 ng/ml Ionomycin (Invitrogen; Thermo Fisher Scientific, Inc.) and 10 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) were added and cultured at 37°C in 5% CO₂ for 3 h, then treated with 2.5 µmol monensin for 2 h. Cells were collected and the supernatant was removed by centrifugation at 1,500 × g for 5 min at room temperature; surface staining was performed by incubation with the corresponding fluorescently labelled antibodies on ice for 15 min in the dark. Mouse monoclonal antibodies (mAb) were used at a dilution of 1:20 and provided by eBioscience (Thermo Fisher Scientific, Inc.). The antibodies were as follows: fluoresein isothiocyanate (FITC)-labelled anti-human IL-17A (IgG1, cat. no. 11-7179), anti-human interferon-γ (IgG1, cat no. 53-7319), anti-human FOXP3 (IgG2a, cat no. 11-4776-41), phycoerythrin (PE)-labelled IL-10 (IgG1, cat no. 53-7108-41), perCy5.5-labeled anti-human CD8 (IgG1, cat no. 45-0088-41). Cells were washed twice with staining buffer and fixed in 500 µl of 40 g/l paraformaldehyde (PFA) at 4°C for 30 min. Cells were washed twice using staining buffer, then intracellular staining was performed with the Cytofix-Cytoperm buffer kit (BD Biosciences, Franklin Lakes, NJ, USA). Following cell membrane permeation for 30 min at 4°C, the supernatant was removed and incubated with fluorescently labeled antibodies (BD Biosciences) against intracellular cytokines. Following a final wash, 300 µl of 40 g/l PFA was added and flow cytometric measures were performed on a FACSCalibur flow cytometer (and analyzed using FlowJo software version 7.6.1; FlowJo, LLC., Ashland, OR, USA).

Statistical analysis was performed using GraphPad Prism v.5.0a (GraphPad Software, Inc. La Jolla, CA, USA). All data were expressed as the mean ± standard error. Comparison between two groups were performed by paired or unpaired Student's t-test, as appropriate. One-way analysis of variance followed by Dunnett's test were used for comparisons among multiple treatment groups. P<0.05 was considered to indicate a statistically significant difference.

The expression levels of T-bet and RORγt mRNA in patients with PE were significantly higher compared with normal pregnancies (P<0.05; Table II), while no difference was observed in GATA3 and FOXP3 mRNA levels among the groups. The ratio of T-bet: GATA3 and RORγt:FOXP3 mRNA in PBMC of patients with PE were significantly higher compared with normal pregnancy patients (P<0.05), indicating a Th1 and Th17-shift.

Activated CD4⁺ T cells transfected with 6-FAM-siRNA were observed using fluorescence microscopy. Cells emitting a green fluorescent signal were considered to be transfected successfully. The optimum transfection conditions involved mixing 30 pmol siRNA with 2 µl DMRIE-C, giving a transfection efficiency of ~73.6%.

RT-qPCR and western blot analysis were performed to screen for the most effective siRNAs. Gene and protein expression were normalized to GAPDH. As demonstrated in Fig. 1, in all the T-bet-specific siRNA treated groups, no statistically significant differences were observed in the levels of T-bet mRNA between the blank control and nonspecific siRNA control (P>0.05). In the siT-bet-1837/1859 treated group, T-bet mRNA knockdown efficiency decreased by 75% and the relative ratio was 0.250±0.068; western blot analysis demonstrated that the relative density of T-bet protein was 0.250±0.068, significantly lower compared with the blank and nonspecific siRNA controls (P<0.01; Fig. 1A-C).

Of all RORγt-specific siRNAs treated groups, the siRORγt-629/651 group demonstrated over 70% knockdown efficiency and the relative ratio was 0.293±0.091; western blot analysis demonstrated that the level of RORγt protein expression was 0.049±0.013, significantly lower compared with the blank and nonspecific siRNA controls (P<0.01; Fig. 1D-F), while no significant difference was observed between the blank and nonspecific siRNA controls.

These data suggested that siRORγt-629/651 and siT-bet-1837/1859 were the most efficient and specific siRNAs to downregulate mRNA levels, silence genes and inhibit protein expression.

It was next investigated whether siRORγt-629/651 or siT-bet-1837/1859 alone or in combination affected T lymphocyte subsets. As demonstrated in Fig. 2, T-bet mRNA levels were decreased by 70% and western blot analysis demonstrated a clear decrease in protein expression (25–40%) in the T-bet knockdown and T-bet/RORγt double knockdown groups compared with the BC and NC groups. Comparison of mRNA and protein levels of the transcription factor GATA3 demonstrated no significant differences among the 5 groups. A decreased ratio of T-bet: GATA3 mRNA was observed in the T-bet and T-bet/RORγt double knockdown groups compared with the BC and NC groups. Knockdown of T-bet or RORγt alone led to a significant decrease (30 and 60%, respectively) in RORγt gene expression along with a significant increase (40%) in FOXP3 gene expression under the same experimental conditions. The T-bet/RORγt double knockdown demonstrated a marked decrease (75%) in RORγt gene expression and a marked increase (50%) in FOXP3 gene expression compared with the BC and NC groups. The ratio of RORγt: FOXP3 mRNA notably increased in each siRNA group. To confirm these results, T lymphocyte cell expression of transcription factors was analyzed by western blot analysis and it was determined that T-bet knockdown assays led to a decrease in RORγt and an increase in FOXP3 protein expression. This change was more evident in the T-bet/RORγt double knockdown group. It was also confirmed that treatment with siRNA did not alter the expression of GATA3.

To investigate whether siRORγt-629/651 and siT-bet-1837/1859 would influence the expression of cytokines by T lymphocytes, flow cytometry was employed to identify IFN-γ⁺ (Th1), IL-4⁺ (Th2), IL-17⁺ (Th17) and FOXP3⁺ IL-10⁺ (Treg); all of which are cytokines secreted by subsets of CD4⁺ T cells. Multi-cytokine analysis demonstrated that compared with the control group and scrambled-siRNA, knockdown of T-bet alone demonstrated a marked decrease in IFN-γ and IL-17 levels and an increase in IL-10 levels. Knockdown of RORγt alone led to a marked decrease in IL-17 and an increase in IL-10. In the T-bet/RORγt double knockdown group, a marked decrease of IFN-γ and IL-17 and a significant increase in IL-10 was observed. Notably, no significant differences in IL-4 were detected among the 5 groups (Fig. 3).

The ratio of IFN-γ:IL-4 and IL-17:IL-10 was analyzed in each group and it was identified that the ratio of IFN-γ:IL-4 was significantly decreased in the T-bet knockdown and T-bet/RORγt double knockdown groups compared with the control and scramble-siRNA groups. The ratio of IL-17:IL-10 was significantly lower in the siRORγt, siT-bet and combination groups compared with the control and scramble-siRNA groups (Fig. 3).