Human RCC cell lines 786-O and 769-P were purchased from the China Center of Type Culture Collection (Wuhan, China) and maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Hangzhou Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou, China) at 37°C with 5% CO₂ in a humidified incubator. Rabbit anti-human STAT3, phosphorylated STAT3 (Tyr⁷⁰⁵) and MMP-2 antibodies were purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA). For inactivating STAT3, cells were treated with 20 µmol/l Stattic (Merck KGaA, Darmstadt, Germany) for 1 h at 37°C.

EZH2-overexpressing vector and siRNA targeting EZH2 and STAT3 were designed and synthesized by Invitrogen (Thermo Fisher Scientific, Inc.). The following siRNA sequences were used: siRNA EZH2 5′-AAGACTCTGAATGCAGTTGCT-3′; siRNA STAT3 5′-GAAGCAGCAGAUGGAGCTT-3′. Transfection using Lipofectamine^® 2000 reagent (Thermo Fisher Scientific, Inc.) was performed according to the manufacturer's protocol. When the cells reached a confluence of 70%, the cells were transfected with EZH2-overexpression plasmid (14 µg in 250 µl RPMI-1640 medium without serum), siRNA targeting EZH3 or STAT3 (20 pmol in 50 µl RPMI-1640 medium without serum), empty vector or control siRNA, respectively. Following 4 h, the plasmid DNA/siRNA lipid complex was replaced with normal medium. Stable EZH2-overexpression cell clones 769-P/EZH2 were selected in complete growth medium containing 3.0 µg/ml blasticidin (Invitrogen; Thermo Fisher Scientific, Inc.). Resistant clones were further verified by western blot analysis, as described below. The studies described here were performed using representative cell clones; similar results were observed with other randomly picked clones.

Tumor cells were seeded at 2×10⁴ cells/well in 96-well plates. The cell growth rate was slowed down by overnight incubation at 37°C (24 h) in serum free medium. A total of 10 mM BrdU was added for 8 h and then the medium was changed for the remainder of the 24 h incubation at 37°C. The cells were subsequently washed with PBS and fixed in 70% ethanol for 25 min at 4°C. The amount of incorporated BrdU was determined using a monoclonal antibody directed against BrdU (Zymed; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The cells were stained with tetramethylbenzidine at 37°C (30 min) for color development. The absorbance [optical density (OD)] was measured at a wavelength of 450 nm using a Microplate Autoreader (Bio-Tek Instruments, Inc., Winooski, VT, USA). Each independent experiment was performed in triplicate.

Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) was diluted with serum-free RPMI 1640 medium at 1:20 and the bottom of 96-well plate was coated. A total of 5×10³ cells were harvested and seeded in a 96-well plate with serum-free medium and allowed to attach to the Matrigel. Following 6 h, medium with unattached cells was removed and MTT was added. Following 4 h of incubation, dimethyl sulfoxide was added to solubilize the formazan crystals and the absorbance (OD) was measured at 590 nm.

The effect of altered EZH2 expression on RCC cells was determined using a Transwell assay. A total of 50 µl Matrigel (BD Biosciences) diluted with serum-free RPMI 1640 medium (1:5) was applied to 8-µm-pore-size polycarbonate membrane filters and the bottom chamber of the apparatus, containing RMPI 1640 medium with 20% FBS. Cells were harvested and resuspended in serum-free RPMI 1640 medium. A total of 5×10³ cells were seeded in a Boyden chamber (Corning Incorporated, Corning, NY, USA) and incubated for 24 h. Following incubation, cells on the upper surface were removed and the invading cells attached to the lower surface of the membrane were fixed with 4% paraformaldehyde for 5 min at room temperature and stained with Giemsa for 8 min at room temperature. Cell numbers were counted in three randomly light microscopic fields (magnification, ×20) per membrane. Three independent experiments were repeated.

The whole cell lysate was obtained using radioimmunoprecipitation assay buffer (Cell Signaling Technology, Inc.) containing proteinase inhibitors. A total of 30 µg protein was separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were initially blocked with 5% skim milk in TBS for 1 h at room temperature. For Western blotting, the primary antibodies included: EZH2 (cat. no. 5246), STAT3 (cat. no. 4904), phosphorylated STAT3 (Tyr⁷⁰⁵; cat. no. 9145), MMP-2 (cat. no. 87809), and GAPDH (cat. no. 5174) all at 1:1,000. Anti-rabbit IgG, horseradish peroxidase (HRP)-conjugated antibody (cat. no. 7074; 1:5,000) was used as the secondary antibody. All antibodies used in the present study were purchased from Cell Signaling Technology, Inc. Incubation with primary antibodies was performed overnight at 4°C. Following washes with TBS with Tween 20, membranes were incubated with the secondary antibody for 1 h at room temperature and detected using an enhanced chemiluminescence detection system (GE Healthcare, Chicago, IL, USA).

Values are presented as the mean ± standard deviation of three independent experiments. Analyses were performed using SPSS 17.0 software (IBM Corp., Armonk, NY, USA). Comparisons between two groups were analyzed using Student's t-test (two-tailed). Comparisons among groups were analyzed using one-way analysis of variance followed by the Student-Newman-Keuls post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

Stable EZH2-overexpressing 769-P/EZH2 cells and empty vector control 769-P/EV cells were established following blasticidin screening. Western blot analysis demonstrated that 769-P/EZH2 cell clones had elevated expression of EZH2 (Fig. 1A). To knock down EZH2 in 786-O cells, EZH2-specific siRNA was transfected into 786-O cells, and western blotting was performed to assess the expression of EZH2 following transfection. As exhibited in Fig. 1A, 786-O/siEZH2 cells demonstrated decreased expression of EZH2 following transfection.

In a previous study, the authors demonstrated that downregulation of EZH2 led to apoptosis in RCC cells. In the present study, the effect of EZH2 alteration on proliferation capability in 769-P and 786-O cells was determined. As exhibited in Fig. 1B, following BrdU incorporation, 769-P/EZH2 cells demonstrated a significantly higher absorbance value compared with parental 769-P cells or control 769-P/EV cells, which indicated that EZH2 overexpression was able to accelerate 769-P cell proliferation (P<0.05). By contrast, knockdown of EZH2 significantly repressed the proliferation of 786-O cells (P<0.05; Fig. 1C).

To investigate the effect of EZH2 expression on adhesion ability in RCC cells, Petri dishes were coated with Matrigel to mimic the extracellular matrix (ECM). The ability of the cells to adhere to Matrigel was determined. The value of absorbance at 590 nm of 769-P/EZH2 cells significantly decreased by 1.7-fold compared with parental 769-P cells (P<0.05; Fig. 2A). By contrast, following 6 h of attachment, the value of A590 in EZH2-knockdown 786-O/siEZH2 cells significantly increased by 0.7-fold relative to parental 786-O cells (P<0.05; Fig. 2B). These results indicated that EZH2 expression was negatively associated with the ability of RCC cells to adhere.

The effect of EZH2 expression on invasive ability of tumor cells was subsequently investigated. The 769-P/EZH2 cells exhibited significantly enhanced invasive potential compared with parental 769-P cells, whereas 786-O cells with EZH2 knockdown exhibited a significantly decreased invasive ability compared with parental 786-O and 786-O/control cells (P<0.01; Fig. 2C-E).

Previous reports have demonstrated that STAT3 serves an important role in metastatic progression in RCC (15,16). For this reason, STAT3 activity following altered EZH2 expression was investigated. As exhibited in Fig. 3, EZH2 overexpression increased the phosphorylation of STAT3 (Tyr⁷⁰⁵) in 769-P/EZH2 cells, while 786-O/siEZH2 cells demonstrated reduced phosphorylation compared with the parental 786-O cells in response to EZH2 knockdown (Fig. 3). MMP-2, which associated with the degradation of various components of the ECM, is involved in tumor invasion and metastasis. Therefore, the expression of MMP-2 in cells with altered EZH2 expression was examined. EZH2 upregulated the expression of MMP-2 in 769-P/EZH2 cells, whereas 786-O/siEZH2 cells revealed a reduced MMP-2 level (Fig. 3).

It has been reported that STAT3 may elevate a number of downstream targets, including MMP-2, to facilitate metastasis in cancer cells (17). For this reason, the effect of STAT3 on invasive capability in 769-P/EZH2 and 786-O cells was analyzed. Following treatment with siRNA targeting STAT3 or Stattic, an inhibitor of STAT3, 769-P/EZH2 and 786-O cells exhibited a significantly reduced invasive capability compared with the mock-transfected or vehicle control (P<0.05; Fig. 4A-C). Western blot analysis demonstrated reduced expression of MMP-2 in response to knockdown or deactivation of STAT3 in the two cell lines (Fig. 4D). Based on the above, it was hypothesized that EZH2 enhanced the invasive potential of RCC cells via STAT3 phosphorylation and upregulation of MMP-2.