The rabbit anti-human BTG1 monoclonal (14879-1-AP and 14102-1-AP) antibodies were purchased from Proteintech Group (Chicago, IL, USA). Rabbit anti-human cyclin D1 polyclonal (ab7958), mouse anti-human Bcl-2 monoclonal (ab117115), and rabbit anti-human MMP-9 polyclonal (ab7299) antibodies were purchased from Abcam (Cambridge, UK). Goat anti-rabbit fluorescent secondary antibody (IRDye800) was obtained from LI-COR Biosciences, Inc. (Lincoln, NE, USA). The β-actin primary antibody (A1978), polyoxymethylene and crystal violet were purchased from Sigma (St. Louis, MO, USA). The pLenti6/V5-DEST vector, Lentiviral Packaging Mix, Opti-MEM, Lipofectamine 2000 and TRIzol were obtained from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). An immunohistochemistry and Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit were purchased from 4A Biotech Co., Ltd. (Beijing, China). Fetal bovine serum (FBS), cell-culture media and supplementary materials were obtained from Gibco (Thermo Fisher Scientific). Reverse transcriptase reagents and SYBR Premix ExTaq (Perfect real-time) were purchased from Takara (Shiga, Japan). Invasion chambers and Matrigel for invasion and migration assays were purchased from BD Biosciences (Franklin Lakes, NJ, USA).

The human kidney cancer cell line ACHN was maintained in RPMI-1640 medium supplemented with 10% FBS, which was changed every 2–3 days. Upon reaching confluence, cells were subcultured with 0.25% trypsin and 1% ethylenediaminetetraacetic acid. BTG1 cDNA sequences were cloned into the BamHI and AscI sites of the pLenti6/V5-DEST vector, and cells were transfected with the pLenti6-BTG1 or pLenti6/V5-DEST vector using Lipofectamine 2000. Transfected cells were maintained in blastidicin (5 μg/ml)-containing RPMI-1640 medium for selection of stable vector-containing sublines.

Immunohistochemistry was performed as previously described (14). Briefly, 4-μm sections were prepared from paraffin-embedded biopsy samples and dehydrated. Sections were incubated in 3% hydrogen peroxide for 10 min to block endogenous peroxidase, followed by 20 min in 0.05% trypsin. Sections were incubated for 20 min at room temperature in a blocking solution containing 10% goat serum followed by the BTG1 antibody (1:100) at 4°C overnight. For a negative control, the primary antibody was replaced with phosphate-buffered saline (PBS). Sections were subsequently incubated for 20 min each in secondary and tertiary antibodies at room temperature, visualized by 3,3′-diaminobenzidine staining and countered with a hematoxylin stain. Two pathologists blind to the patient condition examined and quantified the sections. Five randomly-selected fields from three slides for each specimen were examined under a microscope and counted. BTG1 expression was determined based on the percentage of positive cells (0 points, ≤5%; 1 point, 5–25%; 2 points, 25–50%; and 3 points, >50% positive cells) and the staining intensity [0 points, no staining; 1 point, weak staining (light yellow); 2 points, moderate staining (yellowish-brown); and 3 points, strong staining (brown)]. The final score of BTG1 expression was the product of the BTG1 expression rate (percentage score) and intensity: - for 0 points, ⁺ for 1–3 points, ⁺⁺ for 4–6 points and ⁺⁺⁺ for 7–9 points.

Total RNA was extracted from ACHN cells using the TRIzol reagent according to the manufacturer's instructions. Total RNA (500 ng) was reverse transcribed using reverse transcriptase, and RT-qPCR was performed on an ABI Prism 7300 Real-Time PCR system (Applied Biosystems Inc., Life Technologies/Thermo Fisher Scientific) according to the standard manufacturer’s instructions for SYBR Premix ExTaq. Gene specific primers used include: BTG1, sense, 5′-GGAATTCATGCATCCCTTCTACACCCGG; and anti-sense, 5′-CGACGCGTTTAACCTGATACAGTCATCAT; and β-actin for normalization, sense, 5′-ATCGTCCACCGCAAATGCTTCTA and antisense, 5′-AGCCATGCCAATCTCATCTTGTT. Thermal cycling conditions were 95°C for 1 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The expression level relative to β-actin was calculated using the 2^−ΔΔCt method in SDS 1.3 software (Applied Biosystems, Inc.).

Western blotting was performed as previously described (15). Briefly, 50 μg of protein [determined using a bicinchoninic acid Protein Assay kit (Tiangen Biotech Co., Ltd., Beijing, China)] per samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Membranes were incubated for 2 h in 5% skimmed dry milk followed by an overnight incubation at 4°C in primary antibody (BTG1, 1:1,000; β-actin, 1:5,000). Subsequent to washing, the membranes were incubated with goat anti-rabbit fluorescent secondary antibody (IRDye800, 1:20,000 dilution) in the dark for 1 h at room temperature. The blots were scanned and analyzed using the Odyssey Infrared Imaging System (LI-COR Biosciences, Inc.). Western blot data were quantified by normalizing the BTG1 signal intensity of each sample to that of β-actin.

Cell viability was determined using the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, as previously described (16). Briefly, ACHN cells were plated into 96-well culture plates at an optimal density of 5×10³ cells/ml with 200 μl/well. After 24–96 h, 20 μl of 5 mg/ml MTT was added to each well and incubated at 37°C for 4 h. Subsequently, the medium was gently aspirated and 150 μl of dimethyl sulfoxide was added to each well to solubilize the formazan crystals. The optical density of each sample was immediately measured at 570 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

An Annexin V-FITC-flow cytometry assay was used as previously described (17) to detect the apoptosis rate. Cells were plated into 60-mm dishes for 48 h and grown to 70–75% confluency Cells were subsequently collected, washed with ice-cold PBS and resuspended at a density of 1×10⁶ cells/ml in a binding buffer and incubated for 15 min in the dark at 25°C with 5 μl Annexin V-FITC and 10 μl PI (20 μg/ml). A total of 10,000 cells were analyzed with a FACScan flow cytometer with CellQuest software (BD Biosciences) for the apoptosis rate determination. For cell cycle distribution, 1×10⁶ cells were fixed in 70% ethanol and resuspended in 1 ml of a solution containing 3.8 mM sodium citrate, 50 μg/ml PI, and 0.5 μg RNase A, and analyzed with the flow cytometer using the ModFit software program (Verity Software House, Topsham, ME, USA).

Invasion and migration assays were performed as previously described (18). Briefly, 8×10⁵ cells were plated into Invasion Chambers with Costar Transwell 8-μm inserts coated with 50 μg reduced serum Matrigel according to the manufacturer's instructions. Medium supplemented with 10% FBS was used in the lower chamber. Migration assays were performed in the same manner excluding the Matrigel. After 12 h, non-invading cells and media were removed with a cotton swab. Cells on the lower surface of the membrane were fixed with polyoxymethylene and stained with 0.1% crystal violet for 30 min. Stained cells were counted under a microscope in four randomly-selected fields and the average was used to indicate cell migration and invasion.

All the statistical analyses were performed using the SPSS 16.0 software (IBM, Armonk, NY, USA), according to published guidelines (19). Survival distributions were estimated with the Kaplan-Meier method and compared with the log-rank test. Student’s t-test, χ² and Fisher’s exact tests were used to analyze the differences between groups. Data are presented as the mean ± standard error, and P<0.05 was considered to indicate a statistically significant difference.

Immunohistochemistry for BTG1 revealed light yellow to brown staining in 77.5% (31/40) of normal kidney tissues, and negative or weak staining in 34.1% (29/85) kidney cancer tissues (P <0.05) (Table I, Fig. 1). Furthermore, BTG1 protein expression was significantly lower in cancer lesion samples compared to adjacent normal tissue, as determined by western blot analysis (0.481±0.051 vs. 0.857±0.081; P<0.05) (Fig. 2). BTG1 expression levels correlated with T stage, lymph node metastasis, clinical stage and pathological differentiation (P<0.05), regardless of age, gender, tumor size and pathological types (P>0.05) (Table II).

Follow-up examinations were performed on cancer patients for up to 60 months, with 37 patients remaining at the conclusion of the study. Overall survival (OS) rates were determined between patients positive for BTG1 expression and those negative for expression. Thirteen of the 56 individuals showing no BTG1 expression remained at the conclusion of the study, with an OS rate of 32.1%. Patients positive for BTG1 expression had a significantly higher OS rate of 65.5% (19/29) (P<0.05) (Fig. 3).

BTG1 overexpressing ACHN cells (known as LeBTG1) were obtained by a stable transfection of BTG1 cDNA, and compared to ACHN cells overexpressing an empty vector (named LeEmpty) as a control. Analysis of RT-qPCR data showed that LeBTG1 cells had a significantly higher expression of BTG1 mRNA compared to LeEmpty cells (0.911±0.095 vs. 0.508±0.055; P<0.05) (Fig. 4A). Furthermore, western blot analysis showed that LeBTG1 cells had a significantly higher expression of BTG1 protein compared to LeEmpty cells (0.871±0.089 vs. 0.429±0.045; P<0.05) (Fig. 4B).

LeBTG1 cells had a significantly lower viability at 24, 48, 72 and 96 h compared to LeEmpty cells as assessed by an MTT assay (P<0.05) (Fig. 5). Cell cycle analysis using flow cytometry showed that the proportion of LeBTG1 cells in the G₀/G₁ and S phases of the cell cycle were significantly different compared to the control LeEmpty cells (66.8±5.3 and 22.2±1.5 vs. 44.4±3.1 and 34.5±2.3%, respectively; P<0.05) (Fig. 6). In addition, there was a large increase in the early apoptosis rate in LeBTG1 cells compared to control LeEmpty cells (16.6±2.5 vs. 6.1±0.7%; P<0.05) (Fig. 7). Furthermore, LeBTG1 cells had a reduced capability for invasion and migration through Transwell inserts (74.0±9.0 and 53.0±7.0, respectively) compared to control LeEmpty cells (118.0±15.0 and 103.0±13.0, respectively; P<0.05) (Fig. 8).

To further identify the mechanisms by which BTG1 overexpression regulated these cellular changes in cancer cells, expression levels of proteins critical for the regulation of cell cycle, apoptosis and migration were examined. Western blot analysis revealed that LeBTG1 cells had significantly reduced levels of cyclin D1, Bcl-2 and MMP-9 (0.118±0.018, 0.169±0.015 and 0.207±0.027, respectively) compared to control LeEmpty cells (0.632±0.061, 0.651±0.063 and 0.443±0.042, respectively; P<0.05) (Fig. 9).