The breast adenocarcinoma MCF-7 cell line was purchased from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). The cells were cultured in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal calf serum (Invitrogen; Thermo Fisher Scientific, Inc.), streptomycin (100 U/ml; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml; Thermo Fisher Scientific, Inc.) at 37°C in 5% CO₂. When the cells had reached 80% confluence, they were cultured with hypoxia-mimetic agent, cobalt chloride (CoCl₂; Sigma-Aldrich, St. Louis, MO, USA) at various concentrations (50, 100 and 200 µmol/l) for at 37°C in 5% CO₂ for 24 h. A maximum HIF-2α accumulation was reached at 100 µmol/l CoCl₂; therefore, 100 µmol/l CoCl₂ was selected for use as the standard concentration for subsequent experiments (Fig. 1).

The HIF-2α siRNAs were designed according to the study by Meade et al (22) and were synthesized by Guangzhou Ruibo Biological Technology Co., Ltd. (Guangzhou, China) as follows: siRNA-1, sense 5′-GCAAAUGUACCCAAUGAUADTDT-3′ and antisense 5′-UAUCAUUGGGUACAUUUGCDTDT-3′; siRNA-2, sense 5′-CAGCAUCUUUGAUAGCAGUDTDT-3′ and antisense 5′-ACUGCUAUCAAAGAUGCUGDTDT-3′; negative control siRNA, sense 5′-CAGCAGGGUUGAUAGCAUGDTDT-3′ and antisense 5′-ACUGCCCCCAAAGAUGCUGDTDT-3æ. The MCF-7 cells were transfected with the siRNAs. Briefly, the cells were seeded into 24-well plates and cultured in RPMI-1640 medium for 24 h (at 37°C in 5% CO₂) to reach 50–70% confluence. HIF-2α siRNA or the negative control siRNA were transfected into the cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The cells were placed in a normoxic or hypoxic atmosphere for 24 h following transfection. The experimental groups were as follows: Parental cell group, MCF-7 cells without CoCl₂ treatment; control group, MCF-7 cells treated with 100 µmol/l CoCl₂; hypoxia + RNAi 1 group, HIF-2α siRNA1-transfected MCF-7 cells treated with 100 µmol/l CoCl₂; hypoxia + RNAi 2 group, HIF-2α siRNA2-transfected MCF-7 cells treated with 100 µmol/l CoCl₂.

Total RNA was extracted from the cells using TRIzol reagent (Gibco^®; Thermo Fisher Scientific, Inc.). The cDNA was synthesized from the RNA using the Reverse Transcription System (Promega Corporation, Madison, WI, USA), according to the manufacturer's protocol. The expressions of HIF-2α and MMP-2 were detected using the following primers: HIF-2α, forward 5′-TGAAAACAGAGTCCGAAGCC-3′ and reverse 5′-GTGGCTGACTTGAGGTTGA-3′; MMP-2, forward 5′-TTCAAGGACCGGTTCATTTGGCGGACTGTG-3′ and reverse 5′-TTCCAAACTTCACGCTCTTCAGACTTTGGTT-3′. The following glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were also used: forward, 5′-ATTCATCTCTCCTCTCCCA-3′ and reverse, 5′-GTTGGTGGTTGGTACTGT-3′. Primers were designed using the Primer Premier Software version 5 (Premier Biosoft International, Palo Alto, CA, USA) and synthesized by Invitrogen (Thermo Fisher Scientific, Inc.). PCR was performed using the SYBR Premix Ex Taq II kit (Takara Biotechnology Co., Ltd., Dalian, China) and the ABI 9700 PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.), and under the following conditions: 94°C for 45 sec and 55°C for 1 min for 35 cycles, with an initial denaturation step at 72°C for 10 min. The PCR products were subjected to 1.5% agarose gel electrophoresis, with GAPDH (580 bp) as an internal control. PCR products were quantified with a TotalLab Quant Phoretix 1D Pro software software (TotalLab Ltd., Newcastle Upon Tyne, UK).

Protein was extracted from the cells using NP-40 Lysis Buffer (Beyotime Institute of Biotechnology, Haimen, China). Protein concentration was determined using a bicinchoninic acid assay (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Total protein of breast carcinoma cells was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were displaced to polyvinylidene difluoride (PVDF) membranes by Pharmacia Phast gel electrophoresis system (Roche Diagnostics, Indianapolis, IN, USA). The PVDF membrane was blocked with 5% skim milk blocking buffer (Beyotime Institute of Biotechnology) for 1 h. The immunoblots were incubated with the following primary antibodies: Rabbit polyclonal anti-HIF-2α (dilution, 1:150; catalog no., ab73895; Abcam, Cambridge, UK); mouse monoclonal anti-MMP-2 (dilution, 1:500; catalog no., ab3158; Abcam); and mouse monoclonal anti-β-actin (dilution, 1:500; catalog no., ab6276; Abcam). Subsequently, the membranes were incubated at 4°C for 24 h with gentle agitation. After washing twice with phosphate-buffered saline, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit (dilution, 1:1,000; catalog no., BA1054-1; BosterBio, Wuhan, China) and goat anti-mouse (dilution, 1:1,000; catalog no., BA1051; BosterBio) IgG secondary antibodies for 1 h at room temperature. After washing with PBS, the immuno-blotting signal was detected using a chemiluminescence system (ECL; Thermo Fisher Scientific, Inc.). The results were quantified using TotalLab version 2.0 software (Total Lab, Newcastle upon Tyne, UK). β-actin protein was used as an internal control.

The invasion ability of the cells was detected using a Boyden chamber (BD Biosciences, Franklin Lakes, NJ, USA. For invasion assays, polycarbonate filters with 8-µm pore size (EMD Millipore, Billerica, MA, USA) were covered with Matrigel (Sigma-Aldrich), which was diluted to 1:20 with serum-free RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc.). The polycarbonate filters were dried at room temperature for 24 h. In total, ~5×10⁶ cells in 500 µl RPMI-1640 media were placed in the upper chamber and 400 µl RPMI-1640 medium was placed in the lower chamber. The cells were incubated at 37°C in 5% CO₂ for 24 h. The cells on the bottom surface of the polycarbonate filter were removed and fixed in 4% glutaraldehyde solution and stained with hematoxylin and eosin (Enzyme-linked Biotechnology Co., Ltd., Shanghai, China). Invasive cells were counted using a microscope (magnification, ×400; BX51; Olympus Corp., Tokyo, Japan). The invasion index and the cell invasion inhibition rate were calculated as previously described (23). Briefly, the invasion index was defined as the number of cells that migrated through the 8-µm pores of the filter in the experimental group divided by the number of cells that migrated through the filter in the control group × 100. The cell invasion inhibition rate was equal to the number of cells that migrated through the filter in the control group minus the number of cells that migrated through the filter in the experimental group divided by the number of cells that migrated through the filter in the control group × 100.

The data are presented as the mean ± standard deviation. Experiments were performed in triplicate. All statistical analyses were performed using SPSS version 13.0 software (SPSS, Inc., Chicago, IL, USA). Independent sample t-test was used to determine the statistical significance between the means. P<0.05 was considered to indicate a statistically significant difference.

To investigate whether the expression of HIF-2α is regulated by a hypoxic environment, the mRNA levels of HIF-2α were compared in cells that were cultured under normoxic (20% O₂) and hypoxia-mimetic (CoCl₂) conditions. After a 24 h incubation, HIF-2α mRNA levels clearly increased in the cells that were treated with CoCl₂ (100 µmol/l). It has been established that CoCl₂ treatment can prevent oxygen signaling in cells, leading to chemical hypoxia. The results revealed that the hypoxic effect increased in severity as CoCl₂ dose increased from 50 to 100 µmol/l. This indicates that CoCl₂ induces hypoxia in a dose-dependent manner. In addition, the expression of HIF-2α also increased in a dose-dependent manner. Notably, the toxicity of CoCl₂ increased in a dose-dependent manner and thus at high doses this led to severe hypoxia and subsequent cell death. As a result, the expression of HIF-2α was decreased. By contrast, normoxic conditions (20% O₂) had only a slight effect on HIF-2α transcription (Fig. 1). De novo transcription of GAPDH was similar among treatment groups. These results suggest that exposure of CoCl₂ (100 µmol/l) promoted HIF-2α transcription, evidenced by the increase in HIF-2α mRNA levels. A CoCl₂ concentration of 100 µmol/l was used in all subsequent experiments.

The association between HIF-2α expression and its target gene MMP-2 was analyzed in MCF-7 cells using siRNAs against HIF-2α. As expected, HIF-2α siRNA markedly reduced the mRNA and protein levels of the targeted HIF-2α subunit (Fig. 2). Subsequently, the effects of HIF-2α knockdown on CoCl₂-mediated induction of MMP-2 expression was analyzed. As shown in Fig. 3, MMP-2 protein and mRNA levels were downregulated significantly in MCF-7 cells transfected with HIF-2α siRNA compared with the control group (P=0.033). Overall, these data demonstrated that hypoxia induces HIF-2α expression at mRNA and protein levels in MCF-7 cells, and HIF-2α siRNA inhibited MMP-2 expression in MCF-7 cells.

The effects of HIF-2α siRNAs on cell migration were assessed using a Boyden chamber assay, which has been modified. As shown in Fig. 4, HIF-2α siRNA significantly inhibited cell migration compared with the control group (hypoxia + RNAi 1, P<0.006; hypoxia + RNAi 2, P=0.004). By contrast, the migratory ability of the control group cells were significantly increased compared with the hypoxia + RNAi groups (hypoxia + RNAi 1, P<0.006; hypoxia + RNAi 2, P=0.004) (Fig. 4). There was no difference on the migratory capabilities of the hypoxia + RNAi groups compared with the parental cell group (P=0.176). These data suggest that HIF-2α was a positive regulator for cell invasion and HIF-2α siRNA may inhibit the invasive capacity of MCF-7 cells (Table I).