The NSCLC cell lines LTEP-a-2, A549 and NCI-H1650 were obtained from Anhui Provincial Key Laboratory of Clinical Basic Research on Respiratory Disease, The First Affiliated Hospital of Bengbu Medical College. Human bronchial epithelial cells (16HBE) were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. The cell lines were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 0.03% antibiotic-antimycotic (all from Gibco, Grand Island, NY, USA) at 37°C in a 5% CO₂ humidified chamber.

Cancer cells (A549 and NCI-H1650 cells) were seeded in a 96-well plate at a density of 3×10⁴ cells/well. The Testin overexpression vector and the empty vector (Cyagen Biosciences Inc., Guangzhou, China) were used to transfect cells using lentivirus transfection technique according to the manufacturers protocol to establish Testin overexpression cell lines and control cell lines. The total virus titer is 1×10⁸ TU/ml. The virus titer was diluted with HBSS or RPMI-1640 according to the cell number and the multiplicity of infection. The appropriate multiplicity of infection was 40. Furthermore, Polybrene (Sigma-Aldrich, St. Louis, MO, USA) was used to significantly increase the viral transfection efficiency, which plays an important role in the lentivirus transfection.

Total RNA was extracted with TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturers instructions. Total RNA was reverse transcribed to cDNA in a 20 µl volume using a reverse transcription kit (Invitrogen Life Technologies). Isoform 2 was used to design the PCR primers. Primers designed and utilized for Testin were: forward, 5-ACTGTGGCAGACATTACTGTGACA-3 and reverse, 5-GATAGCTATGGCTCGATACTTCTGGGTGC-3. The length of the Testin primer was 440 bp. β-actin was used as an endogenous control for quantitative DNA-PCR. Primers designed and utilized for β-actin were listed as follows: forward, 5-TCACCAACTGGGACGACAT-3 and reverse, 5-GCACAGCCTGGATAGCAAC-3. The length of the β-actin primer was 192 bp. Annealing was performed at 72°C for Testin. All PCR product electrophoreses were performed on a 2% agarose gel with ethidium bromide and visualized using the Gel Imager system (Asia Xingtai Mechanical and Electrical Equipment Co., Beijing, China). The relative expression value of Testin mRNA is expressed as the ratio between the target mRNA gray scale value and the β-actin gray scale value. The experiments were repeated in triplicate to confirm the findings.

Proteins were extracted using RIPA lysis buffer (Beyotime, China) containing 0.1% phosphatase inhibitor cocktail and protease inhibitor. The protein concentrations were determined using the BCA protein assay kit (Beyotime, China). Equal amounts of protein were separated by SDS-PAGE (Amresco, LLC, Solon, OH, USA), electrotransferred to PVDF membranes (Biosharp, China) and blocked in 5% non-fat dry milk.

The membranes were incubated overnight at 4°C with the following primary antibodies: polyclonal goat anti-Testin (sc-34737; 1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and monoclonal mouse anti-β-actin (ab8226; 1:2,000; Abcam, Cambridge, MA, USA) as control. Then, the membranes were washed with Tris-buffered saline and Tween-20 (TBST) for three times, and incubated with a 1:2,000 dilution of HRP-conjugated secondary antibodies: rabbit anti-goat IgG (ab6741; Abcam) and goat anti-mouse IgG (ab97023; Abcam) at room temperature for 1 h. The immunoblots were visualized using a chemiluminescence detection kit (Pierce Chemical Co., Rockford, IL, USA).

Cells were seeded in a 6-well plate (1×10⁴ cells/well) and incubated in 3 ml RPMI-1640 medium supplemented with 10% FBS in 5% CO₂ at 37°C. As the cells grew to the logarithmic phase of growth, the cells in the 6-well plates were collected by digestion with 1 ml 0.25% trypsin. After being washed with pre-cooled phosphate-buffered saline (PBS) twice, the cells were re-suspended in 300 µl binding buffer and mixed with 5 µl Annexin V-PE/7-AAD and 5 µl propidium iodide successively, followed by incubation at room temperature in the dark for 15 min. The apoptosis rate of the cells was detected on a flow cytometer within 1 h according to the manufacturer's instructions.

The MTT assay was used to assess cellular proliferation. Cells were seeded in a 96-well plate at a density of 2×10⁴ cells/well. Then the cells were incubated in 5% CO₂ at 37°C for 72 h. As the cells grew to 80% confluence, the freshly prepared MTT solution (5 mg/ml) was added to each well (20 µl/well), and then incubated for an additional 2 h. Subsequently, the supernatant was removed from the well and 150 µl DMSO was added. After shaking, the asorbance of each well at 490 nm was measured using a microplate reader.

The survival and proliferation potential of the cells were assessed using clonogenic assays. The cells were trypsinized, counted, and seeded in a 6-cm plate at 500 cells/well. After incubation for 2 weeks, the colonies were fixed with paraformaldehyde and stained with Giemsa staining solution, and the number of colonies containing more than 50 cells was scored.

Six hundred microliters of balanced mixture of the conditional medium from Matrigel fibroblasts and the complete medium was added to the lower compartment as the chemotactic factor. Serum-free RPMI-1640 with 1×10⁵ cells was added to the upper compartment of the chamber. At the indicated time, the non-invasive cells in the upper compartment were removed with a cotton swab. The cells in the lower compartment of the chamber were counted under a light microscope for a minimum of 10 random visual fields.

Four-week old female BALB/c athymic nude mice (Comparative Medicine Centre of Yangzhou University, China) were housed in an environmentally controlled room (22±2°C, 40–60% humidity and a 12-h light cycle). Cancer cells (1×10⁶) were subcutaneously inoculated into the fossa axillaris of mice at 5 weeks of age. The injection was made through the subcutaneous layer of the cervicodorsal part of the animals. The growth of primary tumors was monitored by measuring the tumor diameters for 5 weeks. Tumor length (L) and width (W) were measured twice a week using a caliper, and tumor volume (V) was calculated by the equation: V = (W²xL)/2. After 5 weeks, the mice were scarificed under anesthesia, the tumor masses were removed, weighed and fixed in 10% neutral buffered formaldehyde solution and paraffin-embedded for histological analysis or preserved at −80°C.

Tumor sections (3-µm) were cut from formalin-fixed paraffin-embedded blocks and mounted on positive-charged slides. The primary antibody was goat anti-Testin polyclonal antibody (Santa Cruz Biotechnology). The paraffin sections were placed in a xylene bath for 10 min to remove paraffin, and repeated again and then placed in an ethanol gradient for rehydration. Antigen retrieval was performed with EDTA (pH 8.0) repair solution in a microwave, cooled to room temperature, treated with 3% H₂O₂ for 10 min for inactivation of endogenous peroxidase, rinsed with 1X PBST (0.1% Tween), incubated with 5% rabbit serum at room temperature for 15 min, and then incubated with primary antibody (1:100) at 4°C overnight. The sections were then rinsed and incubated with biotin-labeled secondary antibody (SP KIT; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) at 37°C for 15 min, rinsed in 1X PBST (0.1% Tween) and then incubated with horseradish peroxidase (SP KIT; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) at 37°C for 15 min. The sections were then treated with DAB for 10 min and the reaction was terminated. H&E staining was performed. The sections were fixed with hydrochloride ethanol and then mounted for analysis. All sections were observed in at least five areas at a magnification of ×400 by at least two investigators in a blinded manner. Cytoplasm and nuclei were counterstained with hematoxylin solution. The total number of cells and positive cells were counted and the staining was scored as the percentages of positive cells: 0 (no staining) for specimens with positive cells ≤5%; 1 (weak staining) for specimens with positive cells >5% and ≤25%; 2 (moderate staining) for specimens with positive cells >25% and ≤50%; 3 (strong staining) for specimens with positive cells >50%. Specimens with scores of ≤1 were regarded as negative; specimens with scores of >1 were regarded as positive.

Data are expressed as the mean and standard deviation (SD), and statistical analysis was performed using software SPSS version 18.0. The differences among groups were analyzed by one-way ANOVA followed by Bonferronis multiple comparison test. Differences were considered significant at P<0.05.

In order to clarify the relationship between Testin and NSCLC, we first compared the expression level of Testin in NSCLC cell lines LTEP-a-2, A549 and NCI-H1650 and 16HBE cells by semi-quantitative PCR and western blot analysis. The Testin mRNA (Fig. 1A) and protein levels (Fig. 1B) were significantly reduced in the LTEP-a-2, A549 and NCI-H1650 cells compared with these levels in the 16HBE cells (P<0.01), suggesting an association between decreased expression of Testin mRNA and protein levels and the carcinogenesis of NSCLC.

In order to explore additional functions of Testin in NSCLC, we used the NSCLC cell lines A549 and NCI-H1650 to establish stable cells that constitutively overexpressed the Testin mRNA and protein (Fig. 2). The transfection efficiency was confirmed using semi-quantitative PCR (Fig. 3A)and western blot analysis (Fig. 3B). A549 and NCI-H1650 cells transfected with the Testin overexpression vector showed significantly increased Testin mRNA levels and protein expression compared with the control cells.

We next investigated the effect of Testin overexpression on cell proliferation. The MTT assay showed that overexpression of Testin significantly inhibited proliferation of the A549 and NCI-H1650 cells compared with control cells (P<0.05) (Fig. 4A). Clonogenic assay showed that overexpression of Testin in the A549 and NCI-H1650 cells markedly reduced colony formation efficiency compared with the control cells (P<0.05) (Fig. 4B). Invasion assay showed that overexpression of Testin significantly inhibited invasion of the A549 and NCI-H1650 cells compared with the control cells (P<0.05) (Fig. 4C). Flow cytometric analysis showed that overexpression of the Testin gene in the A549 and NCI-H1650 cells significantly induced cancer cell apoptosis compared with the control cells (P<0.05) (Fig. 4D). These results suggest that Testin plays a significant role in inhibiting the proliferation, invasion and colony formation of NSCLC cells.

In order to investigate the tumor-suppressing function of Testin in vivo, the Testin-overexpressing A549 cells (A549-Testin-OVER), A549-Control cells and untreated A549 cells (1×10⁶) were subcutaneously inoculated into the fossa axillaris of 5-week-old female BALB/c athymic nude mice. During the process, we found that the mice injected with the A549-Testin-OVER cells formed tumors later than those in the control groups. Whereas large tumors were formed in the mice injected with the control cells within 5 weeks, tumor growth was greatly reduced in the mice injected with the A549-Testin-OVER cells (Fig. 5A).

After sacrifice at 5 weeks, the tumor masses were removed, weighed and fixed in 10% neutral buffered formaldehyde solution. Tumors were stained for Testin and representative images are shown in Fig. 5B. The A549-Testin-OVER group (98.32±1.76%) had higher Testin expression while the A549-control (22.92±21.46%) and untreated A549 group (20.14±22.5%) had lower Testin expression (P<0.05). These data showed that Testin plays a critical role in the inhibition of NSCLC cell xenograft formation and growth in vivo.