A total of 86 patients with NSCLC (46 males and 40 females) were enrolled in the present study. All patients underwent treatment at the People's Hospital of Dongyang City (Dongyang, China) between August 2007 and October 2010. The stage of cancer of patients was classified according to the tumor-node-metastasis (TNM) staging system. The tumor tissues and matched adjacent normal tissues were frozen in liquid nitrogen following surgical resection and preserved at −80°C prior to use. Clinicopathological data were collected, including age, gender, tumor size, tobacco smoking status, alcohol drinking status, TNM stage and differentiation. Patients characteristics are summarized in Table I. The present study was approved by the ethics committee of The People's Hospital of Dongyang City. Written informed consent was obtained from all patients prior to enrollment in the present study.

The A549 and NCI-H460 human NSCLC cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). For cell culture, Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 U/ml streptomycin were used, and a 100% humidified atmosphere was maintained (37°C, 5% CO₂).

The sequences of Tbx3-specific short hairpin RNA (shRNA) and control shRNA were described in a previous study (13) and obtained from Invitrogen (Thermo Fisher, Scientific, Inc.). A549 and NCI-H460 cell lines stably expressing Tbx3 shRNA were synthesized by retroviral transduction of the pSUPER.Retro.Puro (Addgene, Inc., Cambridge, MA, USA) vector encoding shRNA against Tbx3, followed by selection in 1 µg/ml puromycin.

The MTT assay was performed to evaluate the level of cell proliferation. Following transfection, cells with Tbx3-specific shRNA or control shRNA were seeded in 96-well plates at a density of 4,000 cells/well and incubated under the aforementioned conditions (37°C, 5% CO₂). Following 12, 24, 48 and 72 h culture, 20 µl of 5 mg/ml MTT (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added to each well. The plates were incubated for a further 4 h under the same conditions (37°C, 5% CO₂), and subsequently 150 µl of dimethylsulfoxide was added until the purple formazan was completely dissolved. The absorbance of each well was determined using the Multiskan^® Spectrum (Thermo Fisher Scientific, Inc.) at a wavelength of 490 nm. Each independent experiment was performed three times.

Total RNA was extracted from the transfected cells, tumor tissues and matched normal tissues using an RNeasy Mini kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's protocol, and first-strand complementary DNA was synthesized from 5 ng total RNA using the QuantiTect Reverse Transcription kit (Qiagen GmbH).

The expression level of Tbx3 in transfected cells, tumor tissues and normal tissues was quantified using SYBR^®-Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). RT-qPCR was performed using an ABI-7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and analyzed using ABI SDS software (version 2.0.6; Applied Biosystems; Thermo Fisher Scientific, Inc.). The primers used for RT-qPCR were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.) and listed as follows: Human Tbx3 sense 5′-CCCGAAGAAGACGTAGAAGATGAC-3′ and antisense 5′-CCCGAAGAAGAGGTGGAGGACGAC-3′; human β-actin (ACTB) sense 5′-CCTCCATCGTCCACCGCAAATG-3′ and antisense 5′-TGCTGTCACCTTCACCGTTCCA-3′. The following optimized cycling conditions were used: 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 55°C for 15 sec. Each sample was analyzed in triplicate and the mean value was determined. The expression level of Tbx3 was normalized to the endogenous control, ACTB. The relative expression level of Tbx3 was evaluated using the 2^−ΔΔCq method (14).

Western blotting was performed to assess the expression level of Tbx3 in transfected cells, tumor tissues and normal tissues. Protein concentration was determined using the standard Bradford assay kit (cat. no. P0006; Beyotime Institute of Biotechnology, Haimen, China). Equal amount of total protein was loaded onto a 10% SDS-PAGE gel and blotted onto a nitrocellulose membrane in a humid environment. The membrane was blocked with 5% milk in TBS containing 0.05% Tween 20 (TBST) and incubated with anti-Tbx3 antibody (dilution, 1:2,000; cat no. ab89220; Abcam, Cambridge, UK) at room temperature for 1 h. The anti-β-actin antibody (dilution, 1:2,000; cat. no. ab6276; Abcam) was used as the internal control and incubated at room temperature for 1 h. Subsequently, the membrane was washed three times with TBST and incubated with a secondary anti-rabbit immunoglobulin G antibody conjugated to horseradish peroxidase (dilution, 1:5,000; cat. no. SC-2054; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature for 1 h. Blots were developed using the enhanced chemiluminescence method, according to the manufacturer's protocol (Pierce; Thermo Fisher Scientific, Inc.). Autoradiography signals were analyzed using an ImageQuant RT ECL gel imaging system (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). The gray-scale ratio of Tbx3 to β-actin in each sample was considered as the relative protein expression level. Patients who exhibited Tbx3 expression levels in tumor tissues that was higher compared with adjacent noncancerous tissues were classified into the high Tbx3 expression group. Patients with a lower Tbx3 expression level in tumor tissues compared with the adjacent noncancerous tissues were classified into low Tbx3 expression group.

Following transfection of Tbx3-specific shRNA or control shRNA, the A549 and NCI-H460 cells were collected by trypsinization (Sigma-Aldrich; Merck KGaA), washed with PBS and fixed with 70% cold ethanol overnight at −20°C. The ethanol-suspended cells were then collected, washed and stained with propidium iodide (PI) staining buffer (5 µg/ml PI and 0.25 µg/ml RNase) for 30 min in the dark at room temperature. Finally, cell cycle analysis was performed by evaluating the DNA content of control shRNA and Tbx3-specific shRNA-transfected NSCLC cell lines using a FACSCalibur™ flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer's protocol. The results were analyzed using the ModFit LT software (version 3.2; Verity Software House, Inc., Topsham, ME, USA).

Cell migration in culture was determined using a two-dimensional in vitro scratch motility assay, as previously described (15). The A549 and NCI-H460 cell lines transfected with control shRNA or Tbx3-specific shRNA were seeded at 2×10⁶ cells/well in 6-well plates. A linear wound was made by scratching cells with the tip of a p1000 pipette (Axygen; Corning Life Sciences, Tewksbury, MA, USA). To prevent cell proliferation, mitomycin C (Sigma-Aldrich; Merck KGaA) was added at a final concentration of 10 µg/ml. Cells were incubated at 37°C in 5% CO₂. The wound widths were measured at the time of the scratching (0 h) and at various time points (2, 4 and 6 h). Images were captured using a BX53 microscope (Olympus Corporation, Tokyo, Japan), and migration distances were measured using AxioVision software version 4.8 (Zeiss GmbH, Jena, Germany). The difference in width represents the distance migrated in µm.

All the data collected in the present study were analyzed using SPSS software (version 20.0; IBM SPSS, Armonk, NY, USA). Data are expressed as the mean ± standard deviation. Differences between groups were determined by the χ² test. The Kaplan-Meier log-rank test was used for survival analysis, whereas prognostic factors were examined by univariate and multivariate analyses (Cox's regression test). P<0.05 was considered to indicate a statistically significant difference.

In order to evaluate the expression level of Tbx3 in NCLC tissues, RT-qPCR and western blot analysis were performed to quantify the Tbx3 expression levels. As presented in Fig. 1A and B, the mRNA expression level of Tbx3 in normal or tumor tissues was determined. The present study demonstrated that 61/86 patients with NSCLC (70.9%) displayed a significantly higher Tbx3 gene expression level in tumor tissues compared with that in adjacent normal tissues (overexpressed group); thus, in the majority of cases, Tbx3 was overexpressed in patients with NSCLC. The remaining 25 cases displayed lower Tbx3 gene expression level in tumor tissues compared with those in normal tissues (underexpressed group). Furthermore, the results of the western blot analysis verified the conclusions drawn from RT-qPCR. The protein level of Tbx3 in the overexpressed group was significantly higher in tumor tissues compared with that in normal tissues (P<0.001, Fig. 1C and E). Additionally, the expression level of Tbx3 was lower in tumor tissues compared with that in the normal tissues of the underexpressed group (P<0.05; Fig. 1D and F).

To evaluate the role of Tbx3 in the process of tumorigenesis, Tbx3-specific shRNA was introduced to the A549 and NCI-H460 NSCLC cell lines with the aim of downregulating the expression level of Tbx3. The gene expression level of Tbx3 in transfected cells was determined by RT-qPCR. As demonstrated in Fig. 2A, the mRNA expression level of Tbx3 in A549 and NCI-H460 cells transfected with Tbx3-specific shRNA was significantly decreased (P<0.05) compared with that in the control shRNA-transfected cells. Western blot analysis confirmed that the level of Tbx3 was significantly downregulated with the transfection of Tbx3-specific shRNA (P<0.05; Fig. 2B). These results indicated that the knockdown of Tbx3 significantly decreased the mRNA and protein expression levels of Tbx3, which suggested the highly regulated control of Tbx3-specific shRNA (P<0.05).

Furthermore, the role of Tbx3 in cell proliferation was assessed by MTT assay. The results demonstrated that the optical densities of A549 or NCI-H460 cells transfected with Tbx3-specific shRNA were significantly lower compared with those of the A549 or NCI-H460 cells with the control shRNA (P<0.05; Fig. 2C), which indicated that Tbx3 overexpression may promote cell proliferation.

It has been well established that Tbx3 participates in the regulation of the cell cycle (16). In order to investigate the role of Tbx3 in regulating the cell cycle, flow cytometry was performed to determine whether Tbx3 overexpression affects the cell cycle of A549 and NCI-H460 cells. The results (Fig. 2D and E) indicated that the Tbx3-specific shRNA-transfected cells displayed a slight increase in cell population in the G₁ growth phase, whereas the proliferation of cells in the S and G₂/M phases were decreased compared with those of the control cells. The aforementioned results suggested that the expression level of Tbx3 may affect the cell cycle distribution and lead to the change in cell proliferation.

Investigating the clinicopathological function of Tbx3 is crucial for understanding tumorigenesis. The effect of Tbx3 overexpression on cell migration required further study. Therefore, the present study analyzed the mobility of cells transfected with Tbx3-specific shRNA or control shRNA by performing a two-dimensional scratch assay. As presented in Fig. 2F, the migration distance of cells transfected with Tbx3-specific shRNA was significantly lower (P<0.05) than that of the control shRNA-transfected group at all the selected time points (2, 4 and 6 h). The results presented (Fig. 2F) confirmed that Tbx3 overexpression promoted cell migration in A549 and NCI-H460 cells.

The clinical significance of Tbx3 overexpression in colorectal cancer (10) and pancreatic carcinoma (7) has been revealed in previous studies. Therefore, the clinical significance of Tbx3 expression in NSCLC patients was analyzed in the present study by evaluating the correlation of Tbx3 expression with clinicopathological characteristics. It was demonstrated that Tbx3 expression was significantly associated with tumor size (P=0.035), tobacco smoking status (P=0.036), TNM stage (P=0.002) and differentiation (P=0.014) (Table I). In addition, the 5-year overall survival rates were 13.14 and 49.56% in the overexpressed and underexpressed groups, respectively. Notably, the survival rates of these two groups were significantly different (P=0.046); thus, the patients with high Tbx3 expression levels had decreased 5-year survival rates compared with those with low Tbx3 expression (Fig. 3).

The association between Tbx3 expression and prognosis in patients with NSCLC was evaluated by univariate and multivariate analyses. Multivariate analysis using Cox's proportional hazards model was performed to analyze whether one factor can be considered as an independent factor for the prognosis of NSCLC patients, focusing on the factors that significantly affect the survival rate of patients with NSCLC obtained from univariate analysis. From the univariate and multivariate analysis results, the same conclusions could be drawn: Tumor differentiation, tumor size, tobacco smoking status, tumor stage and Tbx3 expression level were identified as independent prognosis factors for patients with NSCLC (Table II).