The rabbit anti-human BTG1 monoclonal, rabbit anti-human cyclin D1 polyclonal, rabbit anti-human Bcl-2 polyclonal and rabbit anti-human MMP-9 monoclonal antibodies were purchased from Abcam (Cambridge, UK). Goat anti-rabbit fluorescent secondary antibody (IRDye800) was obtained from LI-COR Bioscience, Inc. (Lincoln, NE, USA). The β-actin primary antibody, 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 kit and the Annexin V-FITC/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, Waltham, MA, USA). Reverse transcriptase reagents and SYBR Premix ExTaq (Perfect Real-Time) were purchased from Takara (Otsu, Shiga, Japan). Invasion chambers and Matrigel for invasion and migration assays were purchased from BD Biosciences (Franklin Lakes, NJ, USA).

All patients enrolled in this study provided informed consent in advance. There were 23 males and 60 females, aged from 26 to 73 years old, with a median age of 44 years. The studies contained 46 cases of papillary cancer, 22 cases of follicular cancer, and 11 cases of medullary cancer, and 4 cases of undifferentiated cancer. Of the 83 cases of thyroid cancer, 31 had lesions ≤4 cm and 52 had esions >4 cm. Fifty-five of the patients demonstrated lymph node metastasis. Twenty-seven patients had grade I (well differentiated) tumors, and 56 patients had grade II or III (moderately to poorly differentiated) tumors. Clinical staging showed 25 cases of stage I–II, and 58 cases of stage III–IV. Two endoscopic biopsy samples were collected from each patient and either immediately stored in liquid nitrogen for western blot analysis, or fixed in a 4% formaldehyde solution and paraffin-embedded for immunohistochemistry.

The human thyroid cancer cell line FTC-133 was maintained in DMEM 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 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 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 DAB staining and countered with a hematoxylin stain. Two pathologists blind to 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, + to +++ for positive (+ for 1–3 points, ++ for 4–6 points and +++ for 7–9 points).

Total RNA was extracted from FTC-133 cells using TRIzol reagent according to the manufacturer’s protocol. Total RNA (500 ng) was reverse transcribed using reverse transcriptase, and quantitative real-time RT-PCR (qRT-PCR) was performed on an ABI PRISM 7300 Real-Time PCR system (Applied Biosystems, Inc., by Life Technologies/Thermo Fisher Scientific, Waltham, MA, USA) according to the standard manufacturer’s protocol for SYBR Premix ExTaq. Gene specific primers used include: BTG1, sense 5′-GGAATTCATGCATCCCTTCTACACCCGG and antisense 5′-CGACGCGTTTAACCTGATACAGTCATCAT; and β-actin for normalization, sense 5′-ATCGTCCACC GCAAATGCTTCTA and antisense 5′-AGCCATGCCAA TCTCATCTTGTT. 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.

Western blotting was performed as previously described (15). Briefly, 50 μg of protein (determined using a BCA 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% non-fat dry milk followed by an overnight incubation at 4°C in primary antibody (BTG1, 1:1000; β-actin, 1:5000). After 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 then scanned and analyzed using the Odyssey Infrared Imaging System (LI-COR Bioscience, 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, FTC-133 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. The medium was then 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, 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 then 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 of Annexin V-FITC and 10 μl of propidium iodide (20 μg/ml). Ten thousand cells were analyzed with a FACScan flow cytometer with CellQuest software (BD Biosciences) for 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 propidium iodide, and 0.5 μg of 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, 10×10⁵ 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 statistical analyses were performed using 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 ± the standard error, and a P<0.05 was considered to be statistically significant.

Immunohistochemistry for BTG1 revealed light yellow to brown staining in 80.0% (28/35) of normal thyroid tissues, and negative or weak staining in 36.1% (30/83) thyroid 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 (0.251±0.021 vs. 0.651±0.065; P<0.05) (Fig. 2). BTG1 expression levels correlated with 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 120 months, with 36 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. Sixteen of the 53 individuals showing no BTG1 expression remained at the conclusion of the study, with an OS rate of 30.2%. Patients positive for BTG1 expression had a significantly higher OS rate of 66.7% (20/30) (P<0.05) (Fig. 3).

BTG1 overexpressing FTC-133 cells (named LeBTG1) were obtained by a stable transfection of BTG1 cDNA, and compared with FTC-133 cells overexpressing an empty vector (named LeEmpty) as a control. Analysis of qRT-PCR data showed LeBTG1 cells had a significantly higher expression of BTG1 mRNA compared to LeEmpty cells (0.912±0.097 vs. 0.423±0.042; 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 (0.873±0.086 vs. 0.395±0.042; 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 the MTT assay (P<0.05) (Fig. 5). Cell cycle analysis using flow cytometry showed that the proportion of LeBTG1 cells in G0/G1 and S phases of the cell cycle were significantly different compared to the control LeEmpty cells (81.8±6.3 and 10.2±1.0%, vs. 62.4±4.9 and 25.5±2.6%, 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 (11.6±2.1 vs. 2.1±0.4%; P<0.05) (Fig. 7). Furthermore, LeBTG1 cells had a reduced capability for invasion and migration through Transwell inserts (72.0±8.0 and 55.0±7.0, respectively) compared to control LeEmpty cells (113.0±16.0 and 89.0±9.0, 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 (0.234±0.018, 0.209±0.021 and 0.155±0.017, respectively) compared to control LeEmpty cells (0.551±0.065, 0.452±0.043 and 0.609±0.072, respectively; P<0.05) (Fig. 9).