NSCLC cell line H1299 was obtained from GeneChem Co., Ltd. (Shanghai, China). We maintained H1299 in Roswell Park Memorial Institute (RPMI)-1640 culture medium (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone, Logan, UT, USA) at 37°C with 5% CO₂ in a humidified atmosphere. All experiments were conducted in accordance with the Ethical Standards of the Declaration of Helsinki and according to national and international guidelines, and have been approved by the Ethics Committee of the Affiliated Hospital of Nantong University.

MTSS1-overexpressing lentivirus (LV-MTSS1) and the negative control lentivirus (LV-NC) were purchased from GeneChem Co., Ltd. The highest transfection efficiency was evident when the virus titer was 1×10⁹ Tu. A total of 2×10⁵ H1299 cells were planted and maintained on a 6-well plate (Corning Inc., Corning, NY, USA) to achieve 80–90% confluency and then transfection was carried out according to the manufacturer's instructions (GeneChem Co., Ltd.). After transfection, cells were starved for green fluorescent protein (GFP) and fluorescence detection was performed using an EVOS FL Auto (Invitrogen Life Technologies).

Cells in the different groups were cultured in 6-well plates with expansion medium (2 ml/well) at 80–90% confluency, and total RNA was isolated using a UNIQ-10 Spin Column RNA Purified kit (Sangon, Shanghai, China). The first strand of complementary DNA (cDNA) was synthesized using a RevertAid™ First Strand cDNA Synthesis kit (Fermentas, Burlington, Canada). and was subsequently subjected to processing by a Corbett RG-6000 polymerase chain reaction system (Qiagen, Dusseldorf, German) using FastStart Universal SYBR-Green Master Mix (Roche, Basel, Switzerland). The reactions were optimized by varying the annealing temperatures at 52–55°C, and the sense and antisense primers were synthesized as follows: glyceraldehyde 3-phosphate dehydrogenase, 5′-GCAAGTTCAACGGCACAG-3′ and 5′-GCCAGTAGACTCCACGACAT-3′; MTSS1, 5′-GAGGAGATGGAGGCTTGTGA-3′ and 5′-TGGTTGTCTGGGTGCTGTAG-3′. Fold-changes in mRNA expression were determined using the 2^−ΔΔCt method (17).

H1299 cells were lysed by RIPA lysis buffer on ice and total protein was extracted using a protein extraction kit (both from Beyotime, Jiangsu, China) according to the manufacturer's instructions. A bicinchoninic kit (Thermo Fisher Scientific, Pittsburgh, PA, USA) was used to detect the concentration of extracted protein. A total of 50 µg of protein was vertically electrophoresed through sodium dodecylsulfate-polyacrylamide gels and the separated proteins were electronically transferred to polyvinylidene difluoride membranes. Next, the non-specific interactions were blocked by 5% defatted milk-Tris-buffered saline and Tween-20 solution and incubated at 37°C for 1 h. After being washed, specific antibodies against MTSS1 (1:50; Abcam, San Francisco, CA, USA) and β-actin (1:2,000; Beyotime) were applied to incubate the membranes at 4°C for 12 h to detect corresponding proteins. The membranes were then washed and incubated with horseradish peroxidase-conjugated secondary antibodies (Bioss, Woburn, MA, USA) for 1 h at 37°C. Immunoblots were then detected by enhanced chemiluminescence reagents (Invitrogen Life Technologies) and finally exposed to X-ray films. Software Quantity One (Bio-Rad, Hercules, CA, USA) was used to analyze the relative protein expression levels, while β-actin was introduced as the internal reference.

Cells in the different groups were seeded onto 96-well plates in 100 µl of RPMI-1640 with 10% FBS at a density of 2,000 cells/well. Cell viability was determined after 24, 48 and 72 h using a Cell Counting Kit-8 (CCK-8) (Beyotime) according to the manufacturer's instructions. The optical density (OD) at 450 nm was detected using a Synergy 2 enzyme mark instrument (BioTek, Winooski, VT, USA).

After transfection, confluent monolayers of cells were scratched with a 1,000 µl pipette tip to induce a wound. The wound edges were imaged using a Leica DM IRB inverted microscope (Leica, Solms, Germany). Images were collected at 0, 24 and 48 h after wounding. Images were quantified by measuring the number of cells across the initial wounded edge (black line) at each time-point.

The migration and invasion abilities of the H1299 cells were assessed using Transwell plates with 8-µm pore membranes (Corning Inc.) according to protocols previously described (18). Briefly, the H1299 cells were cultured in RPMI-1640 medium for 24 h and the medium was collected as conditioning medium. For the invasion assay, Matrigel (0.1 mg/ml; GeneChem Co., Ltd.) was coated on the top surface of the Transwell chambers. The treated cells were seeded into the chambers and incubated in a humidified environment with 5% CO₂ at 37°C for 48 h. The cells that passed through the polyethylene membrane (migrated cells) or through the Matrigel (invaded cells) were fixed with methanol. Crystal violet staining was then used to indicate migration or invasion under the EVOS FL Auto.

NSCLC tissues were collected from surgical resection specimens of 223 patients who had not undergone radiotherapy or chemotherapy in the Affiliated Hospital of Nantong University between September 2006 and December 2010. Tumor tissues including 136 SCC and 87 ADCA were used to construct a tissue microarray. Briefly, the tumor of each patient was represented by 2.0-mm cores. Histotypes were confirmed using hematoxylin and eosin staining. The Tumor-Node-Metastasis (TNM) staging was according to the 7th edition of the TNM Staging System for Lung Cancer (19). Written consent was obtained from all patients before sample analysis, and all investigations were conducted in accordance with the Ethical Standards according to the Declaration of Helsinki and according to national and international guidelines and were approved by the Ethics Committee of the Affiliated Hospital of Nantong University.

The immunohistochemical analysis was performed as previously described (20). Rabbit anti-MTSS1 polyclonal (1:50; Abcam) antibody was used for detection and Leica microscopy was used to capture the images. The immunostaining of these sections was evaluated by two independent experienced pathologists who were unaware of the clinicopathological data and patient outcomes. The MTSS1 labeling score was defined by multiplying the percentage of positive cells and staining intensity (21). The percentages of MTSS1-positive cells were scored and placed into four categories according to staining proportion: <10%=1; 10–50%=2; >50–75%=3; and >75%=4. The MTSS1 staining intensities were divided into four grades: no staining, 0; weak staining 1; moderate staining 2; and strong staining 3. MTSS1 positivity was determined using the following formula: Overall scores = percentage score × intensity score. Overall scores of <3 and ≥3 were defined as negative and positive, respectively. All samples were evaluated at a magnification of ×200.

The data were subjected to Student's t-test or one-way analysis of variance test. Associations between clinicopathological variables and MTSS1 were examined by χ² tests. Survival curves were calculated by the Kaplan-Meier method and analyzed by the log-rank test. The multivariate analysis was performed using a Cox regression model. P-values <0.05 were considered statistically significant. Data were analyzed using SPSS 22.0 for Windows (SPSS, Inc., Chicago, IL, USA).

Transfection efficacy was detected using an immunofluorescence technique. H1299 cells stably expressing MTSS1-GFP (LV-MTSS1 group) or GFP alone (LV-NC group) were obtained after lentivirus transfection (Fig. 1A). The transfection efficiency was ~95% (Fig. 1B). Next, real-time polymerase chain reaction and western blotting were utilized to detect the relative expression of MTSS1 in each group. The results indicated that MTSS1 expression in H1299 cells (control group) was low and the mRNA and protein levels of MTSS1 were significantly increased in the LV-MTSS1 group compared to the LV-NC and the control groups (Fig. 1C–E). However, there was no difference between the latter two groups.

We examined cell proliferation following MTSS1 overexpression in the cells using the CCK-8 assay. The OD value (450 nm) did not significantly differ among the three groups at 24, 48 or 72 h (Fig. 2). MTSS1 had no effect on H1299 proliferation.

The Transwell results displayed that the number of cells that penetrated the membrane in the LV-MTSS1 group was significantly lower than those of the other two groups in both the cell migration (Fig. 3A and C) and cell invasion assays (Fig. 3B and D). The same result was obtained with the wound healing assay at 24 and 48 h in the LV-MTSS1 group (Fig. 3E and F). This finding implied that MTSS1 suppressed cell migration and invasion.

Representative tissue microarray immunohistochemical staining in NSCLC observed for MTSS1 are shown in Fig. 4. The MTSS1 protein was mainly located in the cytoplasm and strong staining was detected in well-differentiated tissues, moderate staining was seen in medium-differentiated tissues, and weak staining was seen in poorly differentiated tissues (both SCC and ADCA). The overall MTSS1 expression level scores changed from high to low as the differentiation degree changed from high to low.

An overview of MTSS1 expression and clinicopathological parameters is shown in Table I. MTSS1 expression was significantly associated with tumor tissue differentiation degree (P<0.05), TNM stage (P<0.05) and lymph node metastases (P<0.001), respectively. There was no significant correlation between MTSS1 and variables such as age, gender, tumor histology, tumor size or smoking history (all P>0.05).

Univariate survival analysis of these 223 patients revealed that only 27 of the 105 (26%) patients in the MTSS1-negative group were alive vs. 48 of 118 (38%) in the MTSS1-positive group (Table II). The Kaplan-Meier survival curves showed that decreased or absent MTSS1 expression was significantly correlated with poor survival (Fig. 5). When all variables were compared separately with survival status, only differentiation stage (P<0.001), TNM stage (P<0.001), lymph node metastases (P<0.001), MTSS1 (P<0.05) tumor size (P<0.01) significantly affected postoperative outcome (Table II). The Cox proportional hazards regression model proved that MTSS1 (P<0.05), differentiation stage (P<0.05), TNM stage (P<0.001), lymph node metastases (P<0.01) and tumor size (P<0.05) were independent prognostic factors in patients with NSCLC (Table III).