Human NESCs and ESCs were kindly donated by the School of Medicine, Shanghai Jiao Tong University (Shanghai, China). The ESCs and NESCs were cultured in DMEM/F12 (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA) in an atmosphere of 5% CO₂ at 37°C. In the monolayer culture, and as previously described, the cells were identified by immunofluorescence staining using vimentin, cytokeratin (24) and prolactin (PRL) (25).

After the third passage and when the ESCs reached approximately 80% confluence, they were fixed in 4% paraformaldehyde on ice. They were then washed 3 times with phosphate-buffered saline (PBS) and blocked with 10% horse serum. Subsequently, the samples were incubated overnight with primary antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), including mouse monoclonal vimentin (sc-6260), mouse monoclonal cytokeratin (sc-57004) and goat polyclonal PRL (sc-7805) at 4°C. After the cells had been again washed 3 times, the cell slides were incubated at room temperature with the goat anti-mouse (A-11017) and rabbit anti-goat (A-11078) secondary antibodies (Invitrogen, Carlsbad, CA, USA) for 30 min. Images were captured using a laser confocal microscope (FV1000; Olympus, Tokyo, Japan).

To enhance the acetylation of histones, the ESCs were digested and plated in 10-cm dishes (Corning, Inc., Tewksbury, MA, USA), followed by incubation for 24 h with VPA (8 mM; Sigma-Aldrich). Untreated cells served as controls.

ChIP assay was performed using a Pierce Agarose ChIP kit (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer's instructions. The immunoprecipitated DNA was subjected to PCR analysis of the promoter regions of the CYP19 gene. The sequences of the primers for the ChIP assay were as follows: CYP19 sense, 5′-AGT AA CACA GAA CAG TTG CA-3′ and antisense, 5′-TCC AGA CTC GCA TGA ATT CTC CGT A-3′, and the product was 188 bp. The PCR conditions were as follows: 94°C for 10 min, followed by 35 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec, with a final extension at 72°C for 10 min, using a PCR amplification kit (Takara Biotechnology, Dalian, China). The PCR products were analyzed by 2% agarose gel electrophoresis.

The siRNA was synthesized by GenaPharma Co. (Shanghai, China). Briefly, 5 µl of siRNA (CYP19 siRNA, sc-41498; Santa Cruz Biotechnology, Inc.), 10 µl of Lipofectamine 2000 (Invitrogen) and 245 µl of non-serum DMEM were mixed, followed by incubation for 30 min at room temperature. The mixtures were then equally distributed into the 6-well cultured cells followed by incubation at 37°C for transfection into the cells. Cells transfected with the control siRNA (sc-37007; Santa Cruz Biotechnology, Inc.) instead of the CYP19 siRNA were used as the negative control.

Cell viability was evaluated by MTT assay. Briefly, the cells were digested and re-seeded into evenly 96-well plates. Subsequently, 20 µl MTT solution (5 mg/ml) were added to the medium, and the cells were incubated for 4 h at 37°C. The mixtures were then centrifuged at 8,000 rpm for 15 min and the supernatant was discarded. The formazan crystals were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich). The absorbance of the samples was measured at 490 nm using an EnVision^® Multilabel Reader (PerkinElmer, Waltham, MA, USA).

The proliferation of the ESCs was investigated by BrdU staining using BrdU enzyme-linked immunosorbent assay (ELISA) kit (colorimetric; Roche Diagnostics GmbH, Penzberg, Germany). Briefly, 5×10³ cells/well were seeded into 96-well plates. When the cells grew to a confluence of 50%, the medium was discarded and fresh BrdU-medium was added. Subsequently, the cells were incubated in 5% CO₂ at 37°C for 60 min. After being washed with PBS and fixed with 70% ethanol, the cells were incubated with mouse monoclonal BrdU antibody (sc-32323 FITC; Santa Cruz Biotechnology, Inc.). The cells were observed and counted in randomly selected fields under a fluorescence microscope (Olympus). Data were presented as percentages of BrdU-positive cells vs. the total cell percentage.

For Hoechst staining, 1 ml of Hoechst 33258 staining solution (10 µg/ml; Beyotime, Nanjing, China) was added to the medium, and the cells were incubated at room temperature for 5 min. Subsequently, after the Hoechst medium was discarded and the cells were washed with PBS, fluorescence mounting liquid (Beyotime) was added. The apoptotic rate was calculated as the number of apoptotic cells/total number of cells x100%. For the TUNEL, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature and permeabilized with ice-cold 0.1% Triton X-100 for 10 min. Cell apoptosis was evaluated using a TUNEL Apoptosis Detection kit (Merck Millipore, Billerica, MA, USA) following the manufacturer's instructions. All cells were observed under a fluorescence microscope (Olympus). The positive cells were counted in randomly selected fields, and the number was averaged for further analysis.

Total RNA was isolated using TRIzol reagent (Invitrogen). Reverse transcription of the RNA (1 mg) into complementary DNA (cDNA) was conducted using the QuantiTect Reverse Transcription kit (Qiagen, Courtaboeuf, France). Subsequently, quantitative PCR (qPCR_ was performed using the QuantiTect SYBR-Green RT-PCR kit (Qiagen) with the StepOnePlus™ Real-Time PCR system (Life Technologies, Rockville, MD, USA). The CYP19 mRNA levels were measured by RT-qPCR. β-actin served as a reference gene. All samples were tested in triplicate. Data were analyzed using the comparative threshold cycle method (2^ΔΔCt).

The cells were digested using 1% trypsin-ethylenediaminetetraacetic acid (EDTA) and harvested. Subsequently, the cell lysates were centrifuged, and then mixed with radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich). The mixture was occasionally shaken on ice to release the protein. The protein concentration was determined using Bio-Rad Protein Analysis kits (Bio-Rad, Hercules, CA, USA). Target proteins were isolated and transferred onto polyvinylidene difluoride (PVDF) membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The transferred proteins were incubated with primary antibodies (all from Santa Cruz Biotechnology, Inc.), including goat polyclonal acetylated histone H3 (Lys9, sc-8655; diluted at 1:1,000, v/v), rabbit polyclonal acetylated histone H4 (Lys12, sc-8661-R; 1:1,000), goat polyclonal CYP19 (sc-14245; 1:500) and rabbit polyclonal β-actin (sc-130656; 1:1,000) antibodies. The PVDF membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (diluted at 1:5,000, v/v; Santa Cruz Biotechnology, Inc.), followed by treatment with chemiluminescence substrate (Pierce Biotechnology). The blots were observed and photographed using an ImageQuant LAS 4000 biomolecular imager (GE Healthcare, Piscataway, NJ, USA), and densitometric analysis was carried out using Quantity One software (Bio-Rad).

Data are presented as the means ± SD of at least 3 independent experiments. The differences between 2 groups were examined using the Student's t-test, while the differences between 3 or more groups were compared by one-way analysis of variance (ANOVA). Values of P<0.05 and P<0.01 were considered to indicate statistically significant differences.

As ESCs are characterized by the positive expression of vimentin, cytokeratin and PRL (26), both the ESCs and NESCs were subjected to immunofluorescence staining for the detection of the expression of these markers. As shown in Fig. 1, fluorescence staining for vimentin, cytokeratin and PRL was visible in the cytoplasm of the ESCs, but was absent in the NESCs (Fig. 1).

The differences in the expression of acetylated histones between the ESCs and NESCs were further examined by western blot analysis. The results revealed that the ESCs had a significantly lower expression of acetylated histone H3 and H4 compared with the NESCs (Fig. 2A). The expression of acetylated histone H3 and H4 in the NESCs was 7.9-fold (Fig. 2B) and 18.2-fold (Fig. 2C) greater, respectively, compared with that in the ESCs. These results confirm the differences between ESCs and NESCs, and the following experiments were conducted using the ESC model.

To investigate whether VPA alters the acetylation of histones in ESCs, we evaluated the global acetylation of histone H3 and H4 by western blot analysis. Low levels of acetylated histone H3 and H4 were detected in the control group (Fig. 3). By contrast, the levels of acetylated histone H3 and acetylated histone H4 in the ESCs were significantly increased by treatment with VPA. The levels of acetylated histone H3 and H4 in the VPA-treated ESCs were 8.7-fold (Fig. 3B) and 6.9-fold (Fig. 3C) greater, respectively, compared with those of the control group, thus demonstrating that VPA promotes histone acetylation.

To examine the effects of VPA on the acetylation of the promoter region of the CYP19 gene in ESCs, ChIP assay with antibodies against acetylated histone H3 and acetylated histone H4 was performed. The immunoprecipitated DNA from the untreated and VPA-treated ESCs was isolated and subjected to PCR using primers for the promoter regions of the CYP19 gene, and a 188-bp fragment was amplified (Fig. 4). VPA significantly decreased the amounts of PCR product, suggesting that the acetylation of histone H3 and H4 was reduced in the promoter region of the CYP19 gene in the VPA-treated ESCs (Fig. 4).

To investigate the expression of CYP19 at the mRNA and protein level following treatment with VPA, RT-qPCR and western blot analysis were performed. The relative mRNA expression of CYP19 in the VPA-treated ESCs (1.42±0.38) was significantly decreased compared with that of the control group (8.83±1.02; Fig. 5A). Similarly, at the protein level, CYP19 expression was inhibited by VPA (Fig. 5B) and a 6.1-fold decrease was noted, compared with the control group (Fig. 5C). These results confirm that VPA inhibits CYP19 expression.

The effects of VPA and CYP19 on the viability and proliferation of the ESCs were investigated by MTT assay and BrdU assay, respectively. The viability of the VPA-treated ESCs was significantly decreased compared with that of the control group (Fig. 6A). VPA significantly inhibited BrdU incorporation into the ESCs. BrdU incorporation decreased to 20.7±2.52% relative to the control group following treatment with VPA at 8 mM (Fig. 6B). Furthermore, we also noted that CYP19 siRNA inhibited the viability (Fig. 6A) and proliferation (Fig. 6B) of the ESCs compared with the control siRNA group and even enhanced the inhibitory effects of VPA.

To investigate the effects of VPA and CYP19 siRNA on ESC apoptosis, Hoechst staining and TUNEL assay were performed. The percentage of apoptotic cells in the VPA-treated group increased to 34.4±2.89%, and in the CYP19 siRNA group, it increased to 43.5±1.49%, which was a significant increase compared with the control and control siRNA groups, respectively (Fig. 7A). Similarly, the results from TUNEL assay results also revealed a sudden increase in the number of TUNEL-positive cells in both the VPA- and CYP19 siRNA-treated groups (Fig. 7B).