All samples were obtained from 74 patients with diagnosed gastric cancer who underwent surgery (surgical resection) at The First Affiliated Hospital of Anhui Medical University in 2014, as previously described (7). The median age of the study population was 63.4 years (range, 33–84 years) and the sex distribution was 58 males and 16 females. The clinical characteristics are summarized in Table I. None of the patients had received any other therapies, including radiotherapy or chemotherapy prior to surgery. The hepatic, renal and bone marrow functions of all patients were normal, and the Eastern Cooperative Oncology Group Performance Status scores were between 0–2 (8). Patients with abnormal function rest results, and those who were pregnant or breast-feeding were excluded.

All patients provided written informed consent. The study protocol was approved by the Clinical Research Ethics Committee of Anhui Medical University (Hefei, China). All methods were performed in accordance with the human ethics guidelines in the clinical research project (9).

The human gastric cancer SGC-7,901 [American Type Culture Collection (ATCC), Manassas, VA, USA] and BGC-823 (ATCC) cell lines and human normal gastric epithelial GES-1 (ATCC) cell line were employed to detect REV-ERBα expression. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing high glucose, 1 mmol/l L-glutamine, pyridoxine hydrochloride, 110 mg/l sodium pyruvate and bicarbonate. Additionally, 10% heat inactivated foetal calf serum (Clark Bioscience, Richmond, VA, USA), 100 U/ml penicillin and 100 µg/ml streptomycin were added to the DMEM. Cells were cultured at 37°C in a humidified atmosphere of 5% CO₂.

REV-ERBα expression in human gastric cancer and normal tissues (5 cm from the tumor site) was measured by immunohistochemistry as described previously (7). Immunohistochemistry was performed on 4-µm-thick sections from 10% formalin-fixed paraffin-embedded tissue specimens. Sections were deparaffinized with 100% xylene at 25°C for 10 min and removed xylene through a graded series (100, 85 and 70% ethanol) and were subjected to microwaving with 10 mm citrate buffer (pH=6.0) at 100°C for 5 min. Briefly, the sections were blocked with 3% hydrogen peroxide and 10% normal goat serum (Clark Bioscience) each for 10 min at room temperature, and then incubated with REV-ERBα rabbit antibody (cat no. AB174309; 1:100 dilution; Abcam, Cambridge, MA, USA) overnight at 4°C. Following incubation with a biotin-conjugated secondary antibody (cat. no. PV6000; 1:100 dilution; ZSGB-BIO; OriGene Technologies, Inc., Beijing, China), the tissue slides were incubated with a streptavidin-biotin horseradish peroxidase complex for 30 min at room temperature, followed by incubation with 3,3′-diaminobenzidine (ZSGB-BIO; OriGene Technologies, Inc.) for 5 min at room temperature. The counterstaining with 20% hematoxylin was then performed for 60 sec at room temperature, and images of stained samples were captured in a single-blinded manner under a fluorescent microscope at a magnification of ×200. The relative protein expression of all images was calculated using mean optical density units, and the sequences were analyzed using IPWIN Application software version 6.0.0260 (Media Cybernetics, Inc., Rockville, MD, USA). The staining intensity was scored according to the number of cells: 0, no staining; 1 (≤25%), weakly stained; 2 (25–50%), moderately stained; or 3 (≥50%), markedly stained. A low REV-ERBα expression was defined as score ‘0’, ‘1’ or ‘2’, and a high REV-ERBα expression was defined as score ‘3’. The patients were divided into two groups: The low expression group (n=43) and high expression group (n=31), as summarized in Table I.

Gastric tissues and cells were lysed in lysis buffer (25 mM HEPES, 2 mM MgCl2, 2 mM DTT, 1 mM EDTA, 1 mM PSMF, 5 µg/ml leupeptin, pH 7.4). Subsequent to freeze-thawing the suspension liquid containing the extracted protein 3 times, the lysates were centrifuged at 32,869.2 × g at 4°C for 30 min. The concentration of all the extracted protein was determined by the BCA assay. The protein extracts (10 µl) were separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Following non-specific blocking with 5% skimmed milk at room temperature for 2 h, PVDF membranes were washed 2 times with TBST (TBS contained 0.1% Tween-20) for 10 min each time. The membranes were then incubated with primary antibodies against: REV-ERBα (rabbit; cat no. AB174309; 1:500 dilution; Abcam); cleaved caspase-3 (mouse; cat no. SC70497; 1:500 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); B-cell lymphoma 2 (Bcl-2; mouse; cat no. SC23960; 1:500 dilution; Santa Cruz Biotechnology, Inc.); Bcl-2-associated X protein (Bax; mouse; cat no. SC23959; 1:500 dilution; Santa Cruz Biotechnology, Inc.); and β-actin (mouse; cat no. AB8226; 1:1,000 dilution; Abcam) overnight at 4°C, and then washed 3 times with TBST for 10 min each time. The membranes were incubated with the corresponding Goat anti-mouse horseradish peroxidase-conjugated secondary antibody (mouse; cat no. AP124P; 1:1,000 dilution; EMD Millipore, Billerica, MA, USA) or rabbit anti-Goat horseradish peroxidase-conjugated secondary antibody (rabbit; cat no. AP106P; 1:1,000 dilution; EMD Millipore) for 2 h at 20°C, and then washed 3 times with TBST again for 10 min each time. Finally, the detection of the molecules of interest was performed using enhanced chemiluminescence (Beyotime Institute of Biotechnology, Haimen, China). The bands were quantified to calculate relative protein expression levels using Quantity one software version 4.99.5.2.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Total RNA was extracted from human tissues and cells using TRIzol^® (Life Technologies; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA was reverse transcribed by cDNA synthesis using a PrimeScript RT Reagent kit with gDNA Eraser (Perfect Real Time; Takara Bio, Inc., Otsu, Japan) at 37°C for 30 min and 85°C for 5 sec. qPCR was performed using a 7,900 Thermal Cycler (ABI, Applied Biosystems; Thermo Fisher Scientific, Inc.) with GoTaq^® Green Master Mix (Promega Corporation, Madison, WI USA) at an initial denaturation at 95°C for 30 sec, followed by 40 cycles of denaturation for 5 sec at 95°C, annealing for 30 sec at 60°C and extension for 15 sec at 72°C. The qPCR primers for REV-ERBα were 5′-ACAGAATCGAACTCTGCACTTCT-3′ (forward) and 5′-GGGGAGGGAGGCAGGTATT-3′ (reverse) (10). The primers for β-actin were 5′-CATGTACGTTGCTATCCAGGC-3′ (forward) and 5′-CTCCTTAATGTCACGCACGAT-3 (reverse). The cycle threshold (Cq) values were obtained in each sample. Relative levels of mRNA were determined using the 2^−ΔΔCq method (10). β-actin was used as an internal gene for normalization.

The morphological changes associated with apoptosis were assayed using fluorescence microscopy following Hoechst33258 staining. The number of cells was counted in five random fields under a fluorescence microscope at a magnification of ×200. Briefly, SGC-7901 and BGC-823 cells (3,500 cells/well) were fixed for 30 min at room temperature in 70% ethanol, followed by Hoechst33258 (10 µg/ml) staining for 2 min at 37°C and then visualization using the UV fluorescence microscope. Apoptotic cells were considered as cells exhibiting nuclear and cytoplasmic shrinkage, chromatin condensation and apoptotic bodies. A minimum of 400 cells were counted, and the percentage of apoptotic cells, or the apoptotic index, was calculated as described previously (11).

Data are expressed as the mean ± standard deviation. Data analysis was performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). Comparison between different groups was performed using analysis of variance. The Student-Newman-Keuls test was the post-hoc test used following analysis of variance. The χ² test was utilized to analyze the associations between REV-ERBα expression levels and clinicopathological variables and survival time. A Kaplan-Meier test was utilized to describe survival curves and the log-rank test was used to analyze survival curves. P<0.05 was considered to indicate a statistically significant difference.

The REV-ERBα expression levels in normal gastric and mucous gastric cancer tissues, and gastric cancer tissues with different TNM stages, were determined by immunohistochemistry. As demonstrated in Fig. 1A and B, REV-ERBα protein levels were decreased in gastric cancer tissues, which was associated with increased TNM stage. Additionally, REV-ERBα expression in mucous gastric cancer tissues was also decreased compared with normal gastric tissues. Furthermore, the REV-ERBα expression in normal gastric and gastric cancer tissues was confirmed by western blot analysis (Fig. 1C and D). The mRNA levels of REV-ERBα were also measured by RT-qPCR (Fig. 1E). It was identified that the levels of REV-ERBα mRNA were also decreased in gastric cancer tissues, which was associated with incremental TNM stage. These results suggest that REV-ERBα levels are decreased significantly in gastric cancer tissues with higher TNM stages.

The association between the REV-ERBα expression and clinicopathological factors was analyzed using immunohistochemistry data (Table I). A low expression of REV-ERBα was significantly associated with poor differentiation (P=0.009), T stage (P=0.001), TMN stage (P=0.001) and lymph node metastasis (P=0.007). The results indicated that REV-ERBα level is associated with the progression of gastric cancer.

The different survival times of patients with low and high REV-ERBα expression levels are summarized in Table II. The 3- and 5-year survival times of patients were significantly associated with REV-ERBα expression (P=0.009 and P=0.002, respectively). The patients with low REV-ERBα expression exhibited poor prognosis (P<0.05) compared with patients with high REV-ERBα expression (Fig. 2).

To determine whether REV-ERBα was associated with the expression of cleaved caspase-3, Bcl-2 and Bax, western blot analysis was employed to detect the levels of these proteins. As indicated in Fig. 3A and B, the expression levels of cleaved caspase-3 were downregulated in gastric cancer tissues, which was associated with enhanced TNM stage. The Bcl-2 expression levels were upregulated in gastric cancer tissues, which were also associated with enhanced TNM stage. Additionally, the ratio of Bcl-2 to Bax, according to the densitometry of the western blot analysis bands (Fig. 3C), was increased in gastric cancer tissues compared with normal gastric tissues. Therefore, these results suggest that the level of REV-ERBα is decreased in human gastric cancer, which is associated with decreased levels of apoptosis.

To additionally investigate the REV-ERBα expression in gastric cancer, GES-1, SGC-7901 and BGC-823 cell lines were employed to determine the expression level of REV-ERBα (Fig. 4A and B). The REV-ERBα expression levels were significantly decreased in SGC-7901 (moderately differentiated) and BGC-823 (undifferentiated) compared with the GES-1 cell line. The mRNA levels of REV-ERBα were also decreased in SGC-7901 and BGC-823 cells compared with the GES-1 cell line (Fig. 4C). Furthermore, SGC-7901 and BGC-823 cells were treated with the REV-ERBα activator GSK4112 (20 and 40 µM; MedChemExpress, Monmouth Junction, NJ, USA), and it was identified that GSK4112 treatment for 48 h at 37°C caused an increase in apoptosis in a dose-dependent manner (Fig. 5). Therefore, these results suggest that REV-ERBα activation induces apoptosis in human gastric cancer cells.