The specimens were obtained from endometrial cancer tissues, and corresponding adjacent normal tissues were obtained from 156 female patients who were diagnosed with primary endometrial cancer by pathological examination at Linyi Central Hospital (Linyi, China) between January 2012 and January 2014. The patients were aged between 31 and 72 years old, with an mean age of 51.4±7.8 years. The patients were diagnosed with endometrial adenocarcinoma through pathological diagnosis (17). Based on the clinical pathological stage of endometrial cancer, the patients were classified as follows: 87 were stage I; 35 were stage II; 23 were stage III; and 11 were stage IV. Among the 156 patients, 126 cases were free of distant metastasis, and 30 cases had distant metastasis. In addition, 45, 70 and 41 cases exhibited poorly differentiated, moderately differentiated and well-differentiated endometrial cancer, respectively. The inclusion criteria were as follows: i) patients treated at Linyi Central Hospital for the first time without hormone therapy, chemotherapy or radiotherapy prior to surgery; ii) patients who were confirmed to suffer from endometrial cancer by histopathology following surgery; and iii) patients whose follow-up data were complete and reliable. The exclusion criteria were as follows: i) patients who did not undergo surgery; ii) patients who had been treated with surgery at other hospitals following hysterectomy; and iii) patients with other types of cancer that may affect their prognosis. All patients were followed up by telephone or outpatient service until January 2018, following which they were discharged. The follow-up period ranged between 6 and 72 months, during which the progression-free survival and overall survival rates were recorded. The present study was approved by the Ethics Committee of Linyi Central Hospital, and informed consent was obtained from all patients prior to study commencement.

IHC was performed using the streptavidin-peroxidase method. The primary Ezrin antibody EP886Y (cat. no. ab40398, Abcam, Cambridge, MA, USA) was diluted 1:2,000, and the secondary polyoxadiazole-conjugated goat anti-rabbit antibody (cat. no. A5795, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was diluted 1:5,000. The IHC kits were purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. (Beijing, China). All cases were examined and confirmed by pathological examination. Following fixation in 10% formalin, the tissues were embedded in paraffin and were cut into 4-μm sections. Subsequently, the tissues sections were immediately placed into centrifuge tubes containing 1 ml TRIzol reagent and were stored in a refrigerator at −80°C. The tissue paraffin blocks obtained from the patients with endometrial cancer were cut (4-μm thickness) and were then placed in an oven at 60°C for 1 h. The sections were dewaxed with xylene (5 min, three times), hydrated and treated with 3% H₂O₂ in a microwave for 5 min (low heat) to block endogenous peroxidase activity. Sodium citrate was used to repair the antigen, and the sections were rinsed with deionized water, treated with phosphate-buffered saline (PBS) containing Tween-20 for 15 min and treated with PBS for 3 min. The sections were incubated with primary antibody for 2 h at room temperature, washed with PBS (5 min, three times), and dried. The sections were then incubated with secondary antibody at 37°C for 2 h, washed with PBS (5 min, three times), and stained with diaminobenzidine (DAB) (cat. no. C1501, Xi Tang Biotechnology Co., Ltd., Shanghai, China) for 2–3 min. The reaction was terminated with water. The sections were then re-stained with hematoxylin, washed with tap water, dehydrated, and sealed with neutral gum. Semi-quantitative analysis was performed based on the proportion of positive cells in each section and on the staining intensity, as follows: i) proportion of positively stained cells, with 0 points for 0%, 1 point for <10%, 2 points for 11–50%, 3 points for 51–75%, and 4 points for >75%; and ii) staining intensity, with 0 points for colorless, 1 point for pale yellow, 2 points for brownish yellow, and 3 points for dark brown. The final scores were calculated by the product of the two scores, as follows: <3 points was considered negative; and >3 points was considered positive. The scores were independently evaluated by two physicians who were familiar with the scoring criteria. The averages were considered as the final score.

The total RNA was extracted using TRIzol based on the manufacturer's protocol (cat. no. 12183555, Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and qualitative analysis was performed using agarose gel electrophoresis and an ultraviolet spectrophotometer. The total RNA was reverse transcribed cDNA with reverse transcriptase, which was amplified using the SYBR Prime Script RT-PCR kit (cat. no. RR086A, Takara Bio, Inc., Otsu, Japan). The reaction condition was 20 μl, including 2X One Step TB Green RT-PCR Buffer 4 (10 μl), PrimeScript 1 Step Enzyme Mix (20.8 μl), Forward Primer (0.8 μl), Reverse Primer (0.8 μl), Total RNA (2 μl) and RNase Free dH₂O (5.6 μl). The amplification conditions were as follows: 40 cycles of 95°C for 5 sec, 62°C for 20 sec, and 72°C for 30 sec. U6 was used as an internal reference of miR-183-5p. The expression levels of the target genes were quantified using the 2^−ΔΔCq method (18). The experiments were repeated three times to obtain an average for the statistical analysis. The RT-qPCR primer sequences are listed in Table I.

Protein extraction reagent was added to the sample tissues at a ratio of 1:10 (g/l). The extracted protein was homogenized and centrifuged at 4°C at 13,975 x g for 15 min, and the supernatant was collected. RIPA lysate was added into the cultured cells (Shanghai Beyotime Biotechnology Company, Shanghai, China). The protein concentration was detected using a bicinchoninic acid quantitative kit (Thermo Fisher Scientific, Inc.). The protein extracts were heated at 100°C for 5 min, and equal quantities of samples (20 μl) were loaded onto 12% polyacrylamide gels for electrophoresis. Following transfer, the nitrocellulose membrane was incubated with Tris-buffered saline and Tween-20 (TBST) containing 5% bovine serum albumin (BSA) (Sigma-Aldrich; Merck KGaA) in a decolorization shaker at room temperature for 1 h. The blocking solution was removed, and the membrane was placed into a plastic container and incubated with 5% BSA containing the following antibodies: Ezrin antibody EP886Y (1:2,000, cat. no. ab40839, Abcam), E-cadherin antibody MB2 (1:1,000, cat. no. ab8993, Abcam), N-cadherin antibody 12F7 (1:2,000, cat. no. ab196628, Abcam), vimentin antibody RV202 (1:1,000, cat. no. ab8978, Abcam), β-catenin antibody 5D5 (1:2,000, cat. no. ab98952, Abcam) and β-actin antibody (1:1,000, cat. no. ab195055, Abcam). The transfer surface was placed upward, and the membrane was incubated overnight at 4°C with shaking.

The membrane was then washed with TBST (10 min, three times) and incubated with diluted secondary antibody (1:2,000, cat. no. ab6789, Abcam) at 4°C for 4–6 h, and the membrane was then washed with TBST (15 min, three times). Chemiluminescence reagents A and B (Yanhui Biological Co., Ltd., Shanghai, China) were mixed at a ratio of 1:1, and the mixture was added evenly to the nitrocellulose membrane. Following development, relative expression analysis was performed on all the western blots. β-actin was used as the internal reference. The experiment was performed three times to obtain a mean value. ImageJ 1.33u software (National Institutes of Health, Bethesda, MD, USA) was used to analyze the relative light density of the blot bands.

Target gene analysis for miR-183-5p was performed using the TargetScan biological prediction website (http://www.targetscan.org/vert_71/) to verify whether Ezrin is a direct target gene. The full length of the 3′ untranslated region (3′UTR) region of the Ezrin gene was cloned and amplified, and the PCR product was cloned into the polyclonal loci downstream of the pmirGLO luciferase gene (Promega, Madison, WI, USA). The bioin-formatics website was used to predict the binding site of miR-183-5p and its target gene, and site-directed mutation was then performed. The pRL-TK vector expressing Renilla luciferase (Takara Biotechnology Co., Ltd., Dalian, China) was used as the internal reference for transfection efficiency to adjust for the number of cells. miR-183-5p mimics and negative control (NC) were co-transfected with luciferase reporter vectors into 293T cells (CRL-1415; Shanghai Xinyu Biotechnology Pharmacuetical Co., Ltd., Shanghai, China), and the luciferase activity was detected according to the methods provided by Promega. At 48 h post-transfection, the culture medium was discarded, and the cells were washed twice with PBS. Passive lysis buffer (100 μl) was then added to each well, and the cells were gently shaken at room temperature for 15 min. The lysates were then collected. The program pre-reading value was set at 2 sec with a 10 sec reading value. LARIIStop & Glo reagent (Promega) was added to the cells (100 μl), and the prepared LARIIStop & Glo reagent was added to a luminescent tube or plate containing the cell lysate (20 μl per sample) for detection by a chemiluminescence detector (Modulus™; Turner BioSystems; Promega) at a wavelength of 560 nm. The ratio of firefly luciferase activity to Renilla luciferase activity was used as the relative luciferase activity. The experiment was independently repeated three times.

The five endometrial cancer cell lines (Ishikawa, KLE, JEC, HEC-1-A, and HHUA cells) were purchased from Shanghai Fu Xiang Biotechnology Co., Ltd. (Shanghai, China) The cell lines were all cultured in Dulbecco's modified Eagle's medium (DMEM)-F12 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin in a 5% CO₂ incubator at 37°C. The cells were passaged every 3–4 days, and the fourth generation cells were used for the experiments. RT-qPCR analysis was performed to determine expression of miR-183-5p in the five endometrial cell lines to identify the cell line with the highest expression for the subsequent experiments.

The cells were assigned into the blank group (no transfection), the negative control of miR-183-5p (NC) group, the miR-183-5p mimic group (transfected with miR-183-5p mimics), the miR-183-5p inhibitor group (transfected with miR-371-5p inhibitors; GenePharma Biological Co., Ltd. Shanghai, China), the small interfering RNA (si)Ezrin group (transfected with siEzrin from GenePharma Biological Co., Ltd.) and the miR-183-5p inhibitor + siEzrin group (transfected with miR-183-5p inhibitors and siEzrin). The cells were seeded into a 50 ml culture flask and were cultured in complete medium to 70–80% density. Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) and DNA were prepared in a sterile Eppendorf tube, and 5 μl of Lipofectamine 2000 and 100 μl of serum-free medium were incubated at room temperature for 5 min. siRNA (50 nmol) and 100 μl of serum-free medium were incubated at room temperature for 20 min. The cells in the culture flask were washed. Serum-free medium (without antibiotics) was added to the complex, which was then mixed, and the mixture was added into the 50 ml culture flask for transfection. The flask was placed in an incubator containing 5% CO₂ at 37°C for 6–8 h, and the reagent was then replaced with complete culture medium. Finally, the cells were transfected for 48 h for further experiments.

When the Ishikawa cells of each group reached a density of ~80%, the cells were washed twice with PBS. The cells were detached with 0.25% trypsin and were then made into a single cell suspension. Following counting, the cells were seeded into 96-well plates (3–6×10³ cells/well) with 200 μl medium per well, and the above process was repeated six times. After 48 h, 20 μl of 5 mg/ml MTT solution (Sigma-Aldrich; Merck KGaA) was added to each well. Following incubation for 4 h at 37°C, the culture medium was removed. Dimethyl sulfoxide (150 μl) was then added, and the plate was gently shaken for 10 min. After 12, 24 and 48 h, the optical density (OD) of each well was determined at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (DNM9606, PuLang, Beijing, China). The cell viability curve was generated with time as the abscissa and OD as the ordinate. The experiment was repeated three times.

Ishikawa cells transfected for 48 h were collected from each group via detaching with 0.25% trypsin and washing with PBS. The number of cells was adjusted to 1×10⁶/ml, and the cells were seeded into a 6-well plate. A cell suspension was obtained by centrifugation at 252 x g for 10 min at room temperature and by removal of the supernatant, and 70% ethanol was then added. The cells were centrifuged at 252 x g for 10 min at room temperature, and the supernatant was removed. The cells were washed twice with PBS, and PBS was added to obtain 100 μl of cell suspension (number of cells ≥10⁶/ml). Subsequently, 25 μl of RNA enzyme (1 mg/ml) and 50 μl of propidium iodide (PI) (1 mg/ml) was added, and the cells were incubated in the dark for 30 min. The cells were filtered using a 100-mesh nylon filter, and flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) was used to record red fluorescence at 488 nm to detect the cell cycle. Annexin V-FITC/PI double staining was used to detect cell apoptosis. The cells were prepared as mentioned above and were mixed with binding buffer containing 10 μl of Annexin V-FITC (cat. no. ab14085, Abcam) and 5 μl of PI, and the cells were incubated in the dark for 15 min at room temperature. Following this, 200 μl of binding buffer was added to the cells, and flow cytometry was used to record the fluorescence at 488 nm to measure cell apoptosis. The experiment was repeated three times to obtain an average.

The cells (10⁶ cells) were seeded in a 6-well culture plate previously coated with matrix adhesive. In each group, three parallel samples were set up, and the cells were cultured to form cell monolayers. At the bottom of the culture plate, a 200-μl pipette tip was used to form an I-shape scratch. The cells were washed gently with PBS (0.1 mol/l), and serum-free medium was added. The cells were cultured in a 5% CO₂ incubator at 37°C for 24 h. Cell growth at the scratch was observed using a fluorescence microscope (Olympus CKX-41, Olympus Corporation, Tokyo, Japan). The rate of the cells migrating to the injured area was calculated as follows: Migrating cells =(1–24 h scratch width/initial scratch width) ×100%. The experiment was repeated three times to obtain an average value.

Matrigel (frozen at -20°C) was thawed overnight at 4°C and was then diluted to 1 mg/ml using pre-cooled serum-free medium on ice at 4°C. The upper surface of a polycarbonate membrane was coated with 40 μl diluted Matrigel, incubated at 37°C for 3–5 h and coagulated for future use. Following transfection for 24 h, 4×10⁵ Ishikawa cells were suspended in 400 μl of serum-free medium, and the mixed solution was added onto the upper layer of the Matrigel. Medium containing 20% fetal bovine serum was added to the lower chamber and was used as a chemotactic factor. Following culture at 37°C and 5% CO₂ for 24 h, the chamber was removed, and the medium in the chamber was aspirated. The cells on the upper layer were gently wiped off. The chamber was washed twice with PBS, fixed with methanol at room temperature for 10 min, stained with 0.5% crystal violet solution for 5 min, washed with water three times and inverted to be dried. The film was uncovered with a blade, and the slices were sealed with neutral balata. Using a microscope, five visual fields from each group were randomly selected for image capture, and the cells in each field were counted. The experiment was repeated three times to obtain an average value.

The data analysis was performed using SPSS 21.0 statistical analysis software (IBM SPSS. Armonk, NY, USA). The measurement data were expressed as the mean ± standard deviation. One-way analysis of variance was applied to perform comparisons among multiple groups, and Tukey's post hoc test was performed to compare data of multiple groups with normal distribution. An unpaired t-test was used for the comparison between two groups, and a paired t-test was used to compare data between adjacent normal tissues and endometrial cancer tissues. The enumeration data are expressed as the number of cases and the percentage. The association between the protein expression of Ezrin and the clinicopathological characteristics of endometrial cancer was measured using the χ² test. The progression-free survival and overall survival rates of patients were analyzed by Kaplan-Meier curves and were compared by the Log-Rank test. P<0.05 was considered to indicate a statistically significant difference.

Initially, RT-qPCR analysis was performed to determine the expression of miR-183-5p and Ezrin in endometrial cancer tissues and adjacent normal tissues. In comparison with the adjacent normal tissues, the expression of miR-183 in the endometrial cancer tissues was significantly decreased, whereas the mRNA expression of Ezrin in endometrial cancer tissues was significantly increased (both P<0.05). The mRNA expression levels of miR-183-5p and Ezrin in endometrial cancer tissues and adjacent normal tissues are shown in Fig. 1. The above results demonstrated that miR-183-5p was downregulated and that Ezrin was upregulated in endometrial cancer tissues.

Subsequently, a correlation analysis was performed between the protein expression of Ezrin and the clinicopathological characteristics of endometrial cancer (Table II). Among the 156 cases of endometrial cancer, there were 125 cases with a positive expression of Ezrin protein (Ezrin-positive percentage: 80.12%). There were 31 cases of adjacent normal tissues with positive expression of Ezrin protein (Ezrin-positive percentage: 19.88%). Compared with the adjacent normal tissues, the protein expression of Ezrin was higher in the endometrial cancer tissues (P<0.05). In addition, compared with the endometrial cancer tissues, which had a myometrial invasion depth ≤1/2, the protein expression of Ezrin was significantly increased in endometrial cancer tissues, which had a myometrial invasion depth ≥1/2 (P<0.05).

The correlations between the expression intensity of Ezrin protein and the progression-free and overall survival rates of patients with endometrial cancer were also analyzed. Patients with a positive expression of Ezrin protein exhibited significantly lower progression-free survival and overall survival rates, compared with those with a negative expression of Ezrin protein (Fig. 2A and B). Together, these data suggested that the protein expression of Ezrin was positively associated with the severity and of endometrial cancer and poor prognosis.

RT-qPCR and western blot analyses were performed to determine the expression of EMT-related genes in endometrial cancer tissues and in adjacent normal tissues. Compared with the adjacent normal tissues, the mRNA and protein expression levels of E-cadherin, an epithelial marker, and the relative expression of β-catenin, were markedly decreased in the endometrial cancer tissues (P<0.05), and the mRNA and protein expression levels of vimentin and N-cadherin, two mesenchymal markers, and the mRNA expression level of β-catenin were significantly increased (P<0.05) (Fig. 3A-C). Therefore, these data demonstrated that the expression level of E-cadherin was decreased and that the expression levels of vimentin and N-cadherin were increased in endometrial cancer tissues.

A target prediction program and luciferase activity were utilized to elucidate whether miR-183-5p targets Ezrin. Based on the online analysis software, there was a specific binding region between the Ezrin gene 3′UTR and the miR-183-5p sequence, suggesting that Ezrin is the target gene of miR-183-5p, which was verified using a luciferase reporter gene. The miR-183-5p mimic reduced the luciferase activity in the Ezrin-3′-UTR-wild-type group (P<0.05), indicating the inhibition of miR-183-5p binding to the Ezrin-3′-UTR (Fig. 4A and B). Therefore, these findings suggested that Ezrin is a target gene of miR-183-5p.

RT-qPCR analysis was used to determine the expression of miR-183-5p in human endometrial cancer cell lines, including Ishikawa, KLE, JEC, HEC-1-A and HHUA, as shown in Fig. 5. The expression of miR-183-5p in the Ishikawa cell line was the highest among the five cell lines (P<0.05). Therefore, Ishikawa cells were selected for subsequent cell experiments.

Additional RT-qPCR and western blot analyses were performed to investigate the effects of miR-183-5p and Ezrin on the expression of EMT-related genes. Following transfection for 48 h, the expression level of miR-183-5p was significantly increased in the miR-183-5p mimic group and was significantly decreased in the miR-183-5p inhibitor group and miR-183-5p inhibitor + siEzrin group compared with that in the blank group (all P<0.05; Fig. 6A-C). No significant differences in the expression level of miR-183-5p were detected among the blank group, the NC group and the siEzrin group (all P>0.05). Compared with the blank group, the mRNA and protein expression levels of Ezrin, vimentin and N-cadherin in the miR-183-5p mimic group and the siEzrin group were significantly decreased, whereas the mRNA and protein expression levels of E-cadherin and β-catenin were significantly increased in the miR-183-5p mimic group and the siEzrin group (all P<0.05). The miR-183-5p inhibitor group showed the opposite tendency to that of the miR-183-5p mimic group and the siEzrin group in terms of Ezrin and EMT-related gene expression. Compared with the siEzrin group, the mRNA and protein expression levels of Ezrin, vimentin and N-cadherin in the miR-183-5p inhibitor + siEzrin group were significantly increased, whereas the mRNA and protein expression levels of E-cadherin and β-catenin were significantly decreased in the miR-183-5p inhibitor + siEzrin group (all P<0.05). The above results suggested that the upregulation of miR-183-5p and downregulation of Ezrin inhibited the EMT of Ishikawa cells.

An MTT assay was performed to investigate the effect of miR-183-5p or Ezrin on the cell viability of Ishikawa cells (Fig. 7). Compared with the blank and NC groups, cell growth in the miR-183-5p mimic group and the siEzrin group was significantly inhibited (P<0.05). No significant difference in the OD value was found between the blank group and the NC group at any time point (all P>0.05). The OD values of the miR-183-5p inhibitor group at 12, 24 and 48 h were higher than those of the blank group and the NC group (all P<0.05). No statistical significance was found among the miR-183-5p inhibitor + siEzrin group, the blank group and the NC group (P>0.05). These findings demonstrated that the overexpression of miR-183-5p and the decreased expression of Ezrin inhibited the viability of the Ishikawa cells.

Flow cytometry was performed to investigate whether miR-183-5p or Ezrin affected the cell cycle and apoptosis of Ishikawa cells (Fig. 8A and B). Following trans-fection for 48 h, the Ishikawa cells in the miR-183-5p mimic group and the siEzrin group were arrested in the G₀/G1 phase, compared with cells in the blank and NC groups (all P<0.05). The percentage of cells in the S phase was higher in the miR-183-5p inhibitor group than in the blank and NC groups (all P<0.05).

Following transfection for 48 h, the apoptotic rates of the Ishikawa cells in the miR-183-5p mimic group and siEzrin group were 27.53±3.52 and 25.14±3.36%, respectively, which were higher than rates in the blank group (7.94±0.78%) and the NC group (8.13±0.81%) (all P>0.05; Fig. 8C and D). The apoptotic rate of the miR-183-5p inhibitor group was 2.13±0.29%, which was significantly lower than the rates in the blank and NC groups (P<0.05). The apoptotic rate of the miR-183-5p inhibitor + siEzrin group was 8.02±0.76%, which did not differ significantly from that of the blank or NC group (P>0.05). Taken together, these data suggested that the upregulation of miR-183-5p and downregulation of Ezrin repressed cell cycle progression and enhanced the apoptosis of Ishikawa cells.

A scratch test was performed to investigate whether miR-183-5p or Ezrin affect cell migration in Ishikawa cells (Fig. 9A). Culturing for 24 h post-scratch wound showed that the scratch distances of the miR-183-5p mimic group and the siEzrin group were more marked and wider than those of the blank and NC groups. The scratch distance of the miR-183-5p inhibitor group was significantly decreased (P<0.05). However, the scratch distance of the miR-183-5p inhibitor + siEzrin group did not differ significantly from that of the blank or NC group (P>0.05). Compared with the blank and NC groups, the numbers of migrated cells in the miR-183-5p mimic group and the siEzrin group were significantly decreased, and the number of migrated cells in the miR-183-5p inhibitor group was markedly increased (both P<0.05). However, the number of migrated cells in the miR-183-5p inhibitor + siEzrin group did not differ significantly (P>0.05). The cell migration rate of each group is shown in Fig. 9B.

A Transwell assay was performed to elucidate whether miR-183-5p or Ezrin affect cell migration in Ishikawa cells (Fig. 9C and D). The number of cells migrating to the back of the Transwell membrane through the Matrigel gel was 112.67±10.26 in the blank group, 101.67±11.55 in the NC group, 37.67±3.79 in the miR-183-5p mimic group, 197.00±18.03 in the miR-183-5p inhibitor group, 42.33±5.13 in the siEzrin group and 105.33±15.04 in the miR-183-5p inhibitor + siEzrin group. The numbers of invasive cells in the miR-183-5p mimic group and the siEzrin group were significantly lower than those of the NC and blank groups (all P<0.05). No difference in the number of invasive cells were found between the blank group and the NC group (P>0.05). The number of invasive cells in the miR-183-5p inhibitor group was significantly higher than that of the miR-183 mimic group and the siEzrin group (both P<0.05). The number of invasive cells on the miR-183-5p inhibitor + siEzrin group did not differ significantly from that in the blank or NC groups (P>0.05). Therefore, the above data demonstrated that the upregulation of miR-183-5p and downregulation of Ezrin inhibited the migration and invasion of Ishikawa cells.