Human endometrial carcinoma cell line RL-952 cells were purchased from the Shanghai Academy of Life Sciences Cell Bank (Shanghai, China).

Lipofectamine 2000 transfection reagent was purchased from Invitrogen (Carlsbad, CA, USA); small interfering RNA (si-RNA) was synthesized by Shanghai Gemma (Pudong New Area, Shanghai, China); rabbit anti-human EZH2 monoclonal antibody and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody were purchased from Abcam (Cambridge, UK); immunohistochemical SP kit, DAB color kit and hematoxylin were purchased from Zhongjin Jinqiao (Beijing, China); horseradish peroxidase conjugated secondary antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA); Transwell Chamber was purchased from Corning Inc. (Corning, NY, USA); and CCK-8 kit was purchased from Nanjing Kaiji Biological Co., Ltd. (Nanjing, China).

A total of 104 patients admitted to Daqing Longnan Hospital from May, 2014 to June, 2016 with the pathological diagnosis of EC were selected as subjects. All patients underwent total uterine, double attachment resection and pelvic lymphadenectomy. Patients had not received chemotherapy, radiotherapy or biological targeted therapy before surgery. The patients ages ranged from 31 to 67 years (average age, 47.56±15.49 years). According to the degree of pathological differentiation, 38 cases were well differentiated, 34 cases were moderately differentiated and 32 cases were poorly differentiated. According to the revised staging for carcinoma adopted by the 2009 International Union of Obstetrics and Gynecology (FIGO), 26 cases were in stage I, 31 cases were in stage II, 29 cases were in stage III, and 18 cases were in stage IV. Pathological types: endometrial adenocarcinoma was present in 59 cases, clear cell carcinoma in 23 cases and serous papillary carcinoma in 22 cases. A total of 104 corresponding adjacent tissue samples were obtained from the patients. This study was approved by the Medical Ethics Committee of Daqing Longnan Hospital (Heilongjiang, China), and all patients and their families signed the informed consent.

The paraffin embedded tissue blocks of patients with EC were collected and 4 µm consecutive paraffin sections were cut and heated at 65°C for 2 h. After dewaxing and hydration, an immunohistochemical SP method was used in accordance with the protocol. Sections were incubated with anti-EZH2 primary antibody (1:500) overnight at 4°C followed by 3 washes in phosphate-buffered saline (PBS). The sections were then incubated with enzyme-conjugated secondary antibody at 37°C for 1 h; after washing, the sections were incubated with diaminobenzidine (DAB) as the chromogen and counterstained with hematoxylin. Assessment of results was as follows: EZH2 positive cells showed brown or yellow granules in the nuclei. Six different fields of view were randomly selected under microscope (Olympus, Tokyo, Japan) at ×200 magnification and the result was evaluated according to the percentage of positive cells and the severity of the pigment. First, scoring according to the severity of the pigment was: negative as 0 points; light yellow as 1 point; brown as 2 points; strong brown as 3 points. Second, scoring according to the percentage of positive cells in the total cells was: 0–30% as 1 point, 30–70% as 2 points and 70–100% as 3 points. Two scores were multiplied for each section: 3 points or more represented positive expression; otherwise, the scores represented negative expression.

The endometrial cancer RL-952 cells were removed from the −80°C refrigerator and placed in a 37°C water bath until they were completely thawed. The cells were then incubated in a 37°C incubator and the medium was replaced periodically. Lipofectamine 2000 transfection reagent was used to transfect siRNA-EZH2 three times and the control group siRNAs once into endometrial cancer RL-952 cells, respectively, for 4 samples total. The protocol was performed according to the user manual. Three primers, EZH2-Homo-1741 (si-EZH2-1), EZH2-Homo-803 (si-EZH2-2) and EZH2-Homo-2167 (si-EZH2-3), were designed to ensure the efficiency of the transfection. The sequences are shown in Table I.

After transfection, the total protein in the cells was extracted according to the RAPI lysis and extraction buffer manual. Protein concentration was quantified by Coomassie brilliant blue method. A total of 60 µg of protein was separated by 10% SDS-PAGE and the separated protein was transferred to PVDF membrane after 1.5 h of electrophoresis. The membrane was incubated in 5% skim milk powder at room temperature for 1 h, and then rabbit anti-human EZH2 primary antibody (1:1,500) was added and the membrane was incubated overnight at 4°C. After washing the membrane with PBS, the secondary antibody IgG (1:2,000) was added and the membrane was incubated again at 37°C for 2 h. ECL was added on the membrane and blots were developed in the dark. Images were recorded with a gel imaging system (Bio-Rad Laboratories, Irvine, CA, USA) and the gray-scale values were calculated. GADPH was used as the internal reference and the ratio of EZH2 to GAPDH protein was interpreted as the relative expression level of EZH2.

The cells were collected after transfection (control group, cells transfected with control siRNAs; negative control group, normal untransfected cells; si-EZH2 group, cells transfected with si-EZH2-3) and cultured in an incubator. The number of cells was 5,000 in each well, with 5-wells for each group. After incubation at 37°C and 5% CO₂ for 24 h, 5 µl of 5 mg/ml MTT was added to each well. After 4 h, 200 µl of dimethyl sulfoxide (DMSO) was added to each well with shaking for 10 min. The absorbance value of each well at 490 nm was detected by a microplate reader (Bio-Rad 680). The absorbance values at days 1, 2, 3 and 4 were detected by MTT assay and the cell growth curve was plotted.

The cells were collected after transfection (control group, cells transfected with control siRNAs; negative control group, normal untransfected cells; si-EZH2 group, cells transfected with si-EZH2-3). Matrigel was diluted to a 1:1 ratio with precooled serum-free culture medium and 20 µl of diluted Matrigel was evenly added to the above culture wells made of 8 µm polycarbonate membrane at 37°C until fully solidified. A total of 200 µl cells diluted with serum-free medium (1×10⁵ cells/ml) were then added at the lower culture wells and the Transwell plate was incubated at 37°C and 5% CO₂ for 24 h. The Transwell plate was removed and fixed with methanol for 30 min; 0.1% crystal violet was added for 10 min for staining. After carefully wiping the bottom of the wells with a wet cotton swab to remove Matrigel and non-invasive cells, the wells were observed under an inverted microscope (Leica Microsystems, Wetzlar, Germany). Five fields of view were randomly selected and the cells were counted, with the average number of cells interpreted as the cells passing through the basement membrane.

Data were analyzed using SPSS 21.0 software (IBM, New York, NY, USA). Measurement data between the two groups were compared by t-test and among multiple groups it was analyzed by the analysis of variance. The enumeration of data among groups was compared by the χ² test. Differences with a P<0.05 were considered statistically significant.

Under microscope, EZH2 protein was observed in the nuclei showing brown or yellow particles. In this study, immunohistochemical results showed that, among 104 cases of EC specimens, 71 cases showed positive expression of EZH2, with an expression rate of 68.27%. In contrast, among 104 cases of adjacent tissue, positive expression of EZH2 was evident in 25 cases, with a 24.03% expression rate. The expression of EZH2 in endometrial carcinoma tissue was significantly higher than that in adjacent tissue (P<0.05) (Fig. 1 and Table II).

The expression of EZH2 in EC was not correlated with the menopausal status or age of patients (P>0.05), but was correlated with the histological grade, depth of tumor invasion, lymph node metastasis and TNM stage (P<0.05) (Table III).

As shown in Fig. 2, compared with the control group, the expression of EZH2 in cells was significantly inhibited after the three siRNA-EZH2 samples were transfected into endometrial carcinoma RL-952 cells, in which the inhibition of the third siRNA was the most markedly pronounced.

Compared with the control group, the proliferative ability of the cells was significantly decreased after being treated with siRNA-EZH2-3, and the difference was statistically significant (P<0.05) (Fig. 3).

The Transwell results showed that the number of cells passing through the basement membrane was significantly reduced after interference of the EZH2 gene with siRNA-EZH2-3 (Fig. 4A). The average number of cells in the si-EZH2 group was significantly higher than that in the control group and the negative control group (P<0.05) (Fig. 4B).