Endometrial carcinoma cell lines Ishikawa and KLE were obtained from the European Collection of Cell Cultures (ECACC, Wiltshire, UK) and the American Type Culture Collection (ATCC, Manassas, USA). It is well known that Ishikawa cells express α and β receptors while KLE cells only express ERβ. Ishikawa cells were grown in MEM medium supplemented with 5% fetal bovine serum (FBS; Gibco, USA). KLE cells were maintained in DMEM-F12 supplemented with 10% FBS. All cells were grown in an incubator at 37°C in a 5% CO₂ environment.

The ERα and ERβ expression vectors pEGFP-C1-ERα and pEGFP-C1-ERβ were a gift from Dr Michael Mancini (Addgene plasmid 28230 and 28237). pEGFP-C1 empty plasmid was preserved by the Key Laboratory of Gynecologic Oncology of Qilu Hospital. Ishikawa and KLE cells were grown routinely in medium with 10% FBS and seeded at a density of 1×10⁵ cells/well in 6-well plates. When grown to 80% confluence, cells switched to serum-free medium 2 h prior to transfection. For plasmid DNA transfection, 4 μg of plasmid DNA was incubated with 8 μl of Lipofectamine 2000 (Invitrogen, USA), and then added to the cells. The medium was changed into complete medium with 10% FBS after 5 h. After 24 h incubation, transfection efficiency was detected under a fluorescence microscope, followed by cell proliferation, migration and invasion assays. For RNA and protein analyses, cells were harvested at 48 h after transfection.

Total RNA was isolated from Ishikawa and KLE cells with TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions and as previously described (15). RNA quantity and quality were assessed with NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, USA). Total RNA (1 μg) was used to prepare cDNA. cDNA synthesis was performed using Prime Script RT reagent kit (Takara, Japan) according to the manufacturer’s protocol. Briefly, RNA was incubated for 5 min at 65°C and cooled immediately on ice; reverse transcription master premix was added to a total volume of 20 μl and cDNA was systhesized for 30 min at 42°C, followed by inactivation of the enzyme for 5 min at 95°C, then cooled at 4°C. PCR was performed using Prime Script RT-PCR kit (Takara) and carried out in a final volume of 50 μl containing 5 μl 10X PCR buffer, 2 μl of dNTP mixture, 0.5 μl of each primer, 0.5 μl of Takara Ex Taq HS and 5 μl cDNA. The PCR conditions were: 30 sec denaturation at 94°C, 30 sec annealing at 60°C, 1 min extension at 72°C for 40 cycles. Amiplified products were electrophoresed in 2% agarose gels, and the amount in each band was quantitatively analyzed using the Image J software. Each band was normalized relative to the β-actin band in the same sample. Specific primers were: sense: 5′-CGA CAT GCT GCT GGC TAC ATC-3′, antisense: 5′-AGA CTT CAG GGT GCT GGA CAGA-3′ for ERα; sense: 5′-AGA GTC CCG GTG TGA AGC AAGA-3′, antisense: 5′-TGC AGA CAG CGC AGA AGT GA-3′ for ERβ; sense: 5′-CAC ACA GGG GAG GTG ATA GC-3′, antisense: 5′-GAC CAA AAG CCT TCA TAC ATC TCA-3′ for β-actin.

Real-time PCR analyses were carried out using the Light Cycler System (Roche Diagnostics GmbH, Mannheim, Germany) as previously described(16), and were performed using SYBR Premix Ex Taq (Takara) according to the manufacturer’s protocol. Analysis of mRNA expression determined by real-time PCR was defined by 2^−ΔΔCt measurements.

Cells were lysed in RIPA buffer (Beyotime Biotechnology, China), with the addition of protease inhibitors mixture (1 mM EDTA, 1 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin) for 20 min on ice. Then, lysates were cleared by centrifugation at 12,000 rpm for 20 min at 4°C. Protein concentrations were measured with the BCA Protein Assay kit (Beyotime Biotechnology). Western blot analysis was carried out as previously reported (17). Total proteins (30 μg) were separated by SDS-PAGE on 10% gel and then transferred onto PVDF membranes (Millipore, USA) at 200 mA for 1.5 h. PVDF membranes were then placed in blocking buffer (5% non-fat milk in TBS-T) for 1 h and subsequently incubated with primary antibodies overnight at 4°C. After washing in TBS-T three times, membranes were incubated with secondary antibodies for 1 h at room temperature. Protein expression was detected using ECL (Millipore) and bands were scanned using Image Quant LAS 4000 system (GE Healthcare). The mean density of the band was quantified using Image J. Relative target protein expression was normalized to β-actin. Primary antibodies used in this study were: ERα (1:100, ab37438, Abcam), ERβ (1:1,000, ab3576, Abcam), p-PI3K p85α (1:500, AP0153, Bioworld), AKT (1:500, AP0059, Bioworld), p-AKT (1:500, BS4006, Bioworld), mTOR (1:500 BS3611, Bioworld), p-mTOR (1:500, BS4706, Bioworld) and β-actin (1:3,000, AP0060, Bioworld). The secondary antibody was goat anti-rabbit IgG-HRP antibodies (1:20,000, BS10350, Bioworld).

For Transwell migration assays, cells were seeded at a density of 1×10⁵ per well in 100 μl FBS-free medium in the upper chambers (24-well Transwell chambers, Corning Costar, Life Sciences, MA, USA) with 8.0-μm pores. For invasion assays, briefly, Transwell inserts were coated with Matrigel (BD Biosciences, USA), and 2×10⁵ cells were plated in the upper chamber. Lower wells were filled with 500 μl medium supplemented with 20% FBS to induce cell migration. After incubation for 24 h, cells that did not migrate or invade through the pores were removed by a cotton swab. The cells on the filter surface were fixed with 90% ethanol, stained with 0.1% crystal violet, and examined under a microscope. Five random ×200 magnification microscopic fields were photographed, and the number of cells in each field was counted.

Cell proliferation was performed by Cell Counting Kit-8 (Dojindo Co., Shanghai, China) according to the manufacturer’s protocol. Briefly, 24 h after transfection, 5×10³ cells/well (100 μl/well) were seeded for five duplicates in a 96-well plate, grown in a humidified incubator at 37°C, 5% C0₂ for proper time; after adding 10 μl WST-8 dye to each well, cells were incubated for another 2 h and the absorbance was finally measured at 450 nm using a microplate reader (Thermo Fisher Scientific).

Results are expressed as the means ± standard deviation of at least three independent experiments. Two group comparisons were performed with Student’s t-test and multiple group comparisons were performed using one-way ANOVA. Differences in P-values of <0.05 were considered statistically significant. All calculations were performed using SPSS 17.0 software.

After transfection with a vector carrying the gene encoding green fluorescence protein (GFP) for 24 h, cell transfection efficiency was observed using the fluorescence microscope (Fig. 1). Five random microscopic fields were photographed, and transfection efficiency (Table I) was evaluated by comparing the GFP positive cells to the total quantity cells of each field.

To determine gene expression changes after transfection, we performed RT-PCR and real-time PCR assays. As expected, in Ishikawa and KLE cell lines, the ERα and ERβ gene expression levels increased significantly after transfection with ERα or ERβ expression vector compared with cells transfected with empty vector. There were no significant differences between the non-transfection group and the empty vector transfection group (Fig. 2A). This finding was confirmed with real-time PCR analysis (Fig. 2B and C). We also demonstrated that ERα and ERβ were both expressed in Ishikawa cells, whereas only ERβ was expressed in KLE cells, as previously reported (18,19).

Although ERα and ERβ are co-expressed in mammary tissues and endometrial carcinomas, the two receptors are differentially influenced by growth factor signaling (20). To investigate the specificity effects of ERα and ERβ on PI3K p85α expression, we used Ishikawa cells (ERα and ERβ positive) and KLE cells (only ERβ positive) in this study. Cells were transfected with ERα or ERβ expression vector for 24 h, followed by western blot analysis to detect p-p85α protein levels. Previous studies reported a role for ER/PI3K crosstalk in breast cancer cells and revealed that ERα interacted with the p85 regulatory subunit of PI3K (21,22). As there were few studies on endometrial carcinoma, our observations showed that the overexpression of ERα in Ishikawa cell lines enhanced the PI3K activity. p-p85α protein levels were strongly associated with ERα protein levels (Fig. 3A). To confirm the results, we examined ERα negative KLE cells, after transfection with ERα expression vector and results obtained were similar to those observed in Ishikawa cells (Fig. 3C). We also investigated a possible association of ERβ and PI3K p85α. However, no similar effects were observed between ERβ and PI3K activity.

To assess the influence of ERα/p85α on the PI3K/AKT/mTOR transduction cascade, we performed western blot assay to evaluate the AKT, p-AKT, mTOR and p-mTOR protein levels. As shown in our data, the phosphorylation levels of key proteins in the PI3K signaling pathway were activated after transfection with ERα expression vector; however, the levels of these proteins were unaffected in non-transfected cells and in cells transfected with empty vector or ERβ expression vector (Fig. 4A and B). Our findings of pathway analysis suggested that the overexpression of ERα in endometrial carcinoma cells activated the PI3K/AKT/mTOR signaling pathway.

Given that the expression of ERs (ERα and ERβ) was closely correlated with oncogenesis and development of endometrial carcinoma (23), we considered whether ERs possess an important role in endometrial carcinoma cell migration and invasion. Transwell migration and Matrigel invasion assays demonstrated that ERα and ERβ both significantly increased the migration and invasion capacity of Ishikawa and KLE cells (Fig. 5). As the oncogene AKT is critical for cell survival, proliferation, and promotes cell migration and invasion, and as ERα can activate the AKT pathway in endometrial carcinoma cell lines (shown in Fig. 4), we considered that ERα induced cell migration and invasiveness partly through targeting the PI3K/AKT/mTOR pathway. However, the roles of ERβ in the development and metastasis of endometrial carcinoma have not been completely elucidated. Our findings demonstrated that the overexpression of ERβ enhanced cell migration and invasion. This finding needs to be validated in other datasets. The possible mechanism for overexpression of ERs increasing the ability of cell invasion and migration remains largely unclear, and requires further studies to be completely understood.

To determine the biological function of ERs in the progression of endometrial carcinoma, we sought to determine whether ERs may also affect the proliferation of endometrial carcinoma cells. As shown in Fig. 6, upregulation of ERα resulted in an observable increase of cell proliferative activity compared with the control group. Contrary to some previous studies (24,25), we also found the promoted proliferation effect exerted by ERβ. Moreover, enhanced effect of cell growth was more significant after ERα transfection, as compared with cells transfected with ERβ expression vector. As AKT, a molecule related to cell cycle and proliferation, was found to be activated after ERα transfection, and no such effect was observed after ERβ transfection, it may provide an explanation for the observed results. Furthermore, these results indicated that the AKT pathway was both a molecular and a biological functional target for ERα.