The present study was conducted with the approval of the Ethics Committee of China Medical University. Primary tumor specimens were obtained from the First Affiliated Hospital of China Medical University during the period of 2008 to 2013. Through the surgery consent form, patients were informed that the resected specimens were kept by our hospital and may be used for scientific research, and that their privacy would be maintained. Follow-up information was obtained by reviewing patient medical records. None of the patients received radiotherapy or chemotherapy before surgical resection, and all patients were treated with routine chemotherapy after the operation. The histological diagnosis and grade of differentiation of the tumors were defined by evaluation of the hematoxylin and eosin-stained tissue sections, according to the World Health Organization guidelines of classification. All 150 specimens were re-evaluated with respect to their histological subtypes, differentiation status and tumor stages. For NSCLC samples, squamous-cell carcinoma (SCC) and adenocarcinoma were identified in 69 and 81 of the 150 cases, respectively. Lymph node metastases were observed in 60 of the 150 patients. The p-TNM staging system of the International Union Against Cancer (7th edition) was used to classify specimens.

A549 and H1299 were obtained from the American Type Culture Collection (Manassas, VA, USA), and H157 was purchased from the Cell Bank, Chinese Academy of Sciences (Shanghai, China). The BE1 were gifts from Dr Jie Zheng (College of Medicine, Beijing University, China). The cells were cultured in RPMI-1640 containing 10% fetal calf serum (both from Invitrogen), 100 IU/ml penicillin and 100 μg/ml streptomycin (both from Sigma). The cells were seeded in 6-well plates 24 h prior to the experiment. Transfection with siRNA for Mig-6 or non-targeting siRNA (GenePharma, Shanghai, China), was conducted using DharmaFECT 1 (5 μl/well; Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer’s recommendations. The sequences of siRNAs were: sense, 5′-GGAUAUCCAACUGUUGUAUTT-3′ and antisense, 5′-AUACAACAGUUGGAUAUCCTT-3′ (6). For control scrambled siRNA: sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-ACGUGACACGUUCGGAGAATT-3′. Cells were transfected with control vector (pcDNA3) or Mig-6 overexpression vector (pcDNA3-Mig-6) (both from Takara Biotechnology, Dalian, China) using Attractene transfection reagent (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. The mRNA and protein levels were assessed 48 h following transfection.

Surgically excised tumor specimens were fixed with 10% neutral formalin, embedded in paraffin and 4-μm thick sections were prepared. Immunostaining was performed using the avidin-biotin-peroxidase complex method (Ultra Sensitive TM, Maixin, Fuzhou, China). The sections were deparaffinized in xylene, rehydrated in graded alcohol series and boiled in 0.01 M citrate buffer (pH 6.0) for 2 min in an autoclave. Endogenous peroxidase activity was blocked using hydrogen peroxide (0.3%), which was followed by incubation with normal goat serum to reduce non-specific binding. Tissue sections were incubated with Mig-6 rabbit polyclonal antibody (1:100 dilution; Sigma). Rabbit immunoglobulin (at the same concentration as the antigen specific antibody; Maixin) was used as a negative control. Staining for all primary antibodies was performed at room temperature for 2 h. HRP-polymer conjugated anti-mouse/rabbit IgG (ready-to-use; Maixin) was used as the secondary antibody. After washing, the sections were incubated with horseradish peroxidase-conjugated streptavidin-biotin, followed by 3,3′-diaminobenzidine tetrahydrochloride to develop the peroxidase reaction. Counterstaining of the sections was carried out with hematoxylin, and they were then dehydrated in ethanol before mounting. Two independent investigators examined all tumor slides randomly. Five views were examined per slide, and 100 cells were observed per view at ×400 magnification. Immunostaining of Mig-6 was scored following a semi-quantitative scale by evaluating in representative tumor areas. The intensity of Mig-6 staining was scored as 0 (no staining); 1 (weak) and 2 (marked). Percentage scores were assigned as 1, 1–25%; 2, 26–50%; and 3, 51–100%. The scores of each tumor sample were multiplied to give a final score of 0–6 and the total expression of Mig-6 was determined as either negative or low expression (−), score <3 or positive expression or high expression (+), score ≥3.

Total protein from cell lines were extracted in lysis buffer (Thermo Fisher Scientific) and quantified using the Bradford method. Sixty micrograms of protein were separated by SDS-PAGE (12%). After transferring, the polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) were incubated overnight at 4°C with the following antibodies: Mig-6 (1:2,000; Sigma), Bcl-2 and β-actin (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-ERK (1:2,000; Cell Signaling Technology, Danvers, MA, USA). After incubation with peroxidase-coupled anti-mouse or rabbit IgG (Santa Cruz Biotechnology) at 37°C for 2 h, bound proteins were visualized using ECL (Thermo Fisher Scientific) and detected using BioImaging Systems (UVP Inc., Upland, CA, USA). The relative protein levels were calculated based on β-actin as the loading control.

Quantitative real-time PCR was performed using SYBR-Green PCR master mix in a total volume of 20 μl on 7900HT Fast Real-Time PCR System (both from Applied Biosystems) as follows: 95°C for 30 sec, 40 cycles of 95°C for 5 sec, 60°C for 30 sec. A dissociation step was performed to generate a melting curve to confirm the specificity of the amplification. β-actin was used as the reference gene. The relative levels of gene expression were represented as ΔCt = Ct gene - Ct reference, and the fold-change of gene expression was calculated by the 2^−ΔΔCt method.

Apoptosis was detected using an Annexin V-FITC/PI double staining kit (KeyGen, Nanjing, China). Cells were harvested and washed twice with cold phosphate-buffered saline (PBS) by gentle shaking. Cells were then resuspended and added to binding buffer (1X); cell density was adjusted to 2–5×10⁵/ml. In the dark, 5 μl Annexin V-FITC was added to the cell suspension volume of 195 μl and incubated for 10 min at room temperature before the addition of 190 μl binding buffer (1X) and 10 μl PI. Ten thousand events per sample were acquired using a FACScan flow cytometer and the percentage of cell apoptosis was analyzed using CellQuest analysis software (both from Becton-Dickinson, San Jose, CA, USA).

Cell proliferation assay was performed using Cell Counting Kit-8^® solution (Dojindo, Gaithersburg, MD, USA) according to the manufacturer’s protocol. Briefly, cells were seeded at a concentration of 5×10³ cells/100 μl/well in 96-well culture plates and treated with 10 μl/well of Cell Counting Kit-8^® solution during the last 4 h of the culture. Optical density of the wells was measured at 450 nm using a microplate reader.

For colony formation assays, cells were grown on 6 cm dishes at a density of 5,000 cells/dish, and transfected with PC or Mig-6. Colonies were washed twice with PBS, fixed, and stained with formaldehyde-crystal violet 13–15 days after transfection. All experiments were independently repeated a minimum of three times under identical conditions.

Cell invasion assay was performed using a 24-well Transwell chamber with a pore size of 8 μm (Costar, Cambridge, MA, USA). The inserts were coated with 20 μl Matrigel (1:3 dilution; BD Biosciences, San Jose, CA, USA). Forty-eight hours after the transfection, cells were trypsinized and 3×10⁵ cells in 100 μl of serum-free medium were transferred to the upper Matrigel chamber and incubated for 16 h. After incubation, the non-invaded cells on the upper membrane surface were removed with a cotton tip, and the cells that passed through the filter were fixed with 4% paraformaldehyde and stained with hematoxylin. The number of invaded cells was counted in 10 randomly selected high power fields under the microscope. This experiment was performed in triplicate.

SPSS version 19.0 for Windows was used for all analyses. The χ² test was used to examine possible correlations between Mig-6 expression and clinicopathological factors. The Kaplan-Meier method was used to estimate the probability of patient survival, and differences in the survival of subgroup of patients were compared by using log-rank test. The Cox regression mode laws were used for multivariate analysis. Student’s t-test was used to compare differences between groups. p-values were based on the two-sided statistical analysis, and p<0.05 was considered to indicate a statistically significant difference.

We first stratified these 150 lung cancer patients as Mig-6-positive and -negative groups according to the Mig-6 staining of tumor sections (Fig. 1A–D). As shown in Table I, statistical differences were found between Mig-6 expression and the characteristics of tumor size (T1+T2 vs. T3+T4, p=0.002), differentiation (high vs. poor-moderate, p=0.002), TNM stage (I+II vs. III+IV, p=0.014), as well as histological type (adenocarcinoma vs. squamous cell carcinoma, p=0.005). We followed up on 60 patients and divided them into two groups according to Mig-6 expression. Patients with high expression of Mig-6 had a statistically significantly longer survival (median survival, 45±11.132 months; 95% CI, 23.182–66.818 months) than those with low expression of Mig-6 (median survival, 13±1.458 months; 95% CI, 10.143–15.857 months). Furthermore, a multivariate analysis using a Cox regression model indicated that pTNM stage and loss of Mig-6 expression were independent, unfavorable prognostic factors [TNM stage: Exp (B), 3.715, 95.0% CI, 2.227–6.198, p=0.000; Mig-6 expression: Exp (B), 0.329, 95.0% CI, 0.152–0.713, p=0.005) (Table II)].

To determine whether Mig-6 influences the apoptosis of NSCLC cells, we explored Mig-6 knockdown and overexpression. Interference efficiency was examined by mRNA and protein expression levels after 48 h of transfection treatment (Fig. 2A–C). Annexin V staining was performed using flow cytometry to determine the effect of Mig-6 on apoptosis in NSCLC cells. Mig-6 knockdown decreased apoptosis in H1299 and BE1 cells (H1299 control vs. Mig-6 siRNA: 11.54±0.65 vs. 6.10±1.29%, p<0.01; BE1 control vs. Mig-6 siRNA: 12.63±0.96 vs. 6.42±0.83%, p<0.01) (Fig. 3A). Mig-6 overexpression increased apoptosis in A549 and H157 cells (A549 empty vector vs. Mig-6 plasmid: 7.05±0.82 vs. 13.98±0.79%, p<0.01; H157 empty vector vs. Mig-6 plasmid: 12.46±0.87 vs. 17.35±1.44%, p<0.05) (Fig. 3B).

It has been established that apoptosis within cells is chiefly involved in the expression of Bcl-2, Bax, or p53 (15–17). To determine a possible mechanism by which Mig-6 promotes apoptosis of NSCLC cells, western blotting was performed. As shown in Fig. 3C the expression at both the protein and mRNA levels of anti-apoptotic Bcl-2 was upregulated in the Mig-6 depletion group but downregulated in the Mig-6 overexpression group. There were no significant differences in Bax and p53 expression between each group (data not shown). These results indicated that the function of Mig-6 as a promotor of apoptosis in NSCLC cells is predominantly implemented possibly by the pathways of Bcl-2 but not Bax and p53.

Our previous study confirmed that the suppression of Mig-6 by a specific siRNA led to a marked increase in EGFR-ERK signaling (14). Numerous reports have shown that the ERK pathway plays an important role in the apoptosis of NSCLC (18–20). We investigated whether ERK mediated Mig-6 induction of Bcl-2 downregulation. Cells were treated with PD98059, a selective inhibitor of MEK that disrupts the activation of downstream ERK, for 1 h. We found that basal Bcl-2 production and anti-apoptosis ability were inhibited by treatment of H1299 and BE1 cells with 50 mol/l PD98059. Furthermore, PD98059 significantly abolished Mig-6 siRNA-mediated Bcl-2 production and anti-apoptotic effects (Fig. 4A). Consequently, we overexpressed Mig-6 in A549 and H157 cells, we also blocked ERK activities using PD98059. As shown in Fig. 4B, Mig-6 and PD98059 can inhibit the expression of P-ERK and Bcl-2. Similar to Mig-6 overexpression, inhibition of ERK by PD98059 can also promote apoptosis of both cell lines. To further validate the role of ERK signaling in Mig-6-induced apoptosis, we used siRNAs to deplete ERK1/2 expression in H1299 and BE1 cells. Then, we transfected Mig-6 siRNA and examined Bcl-2 protein expression, as well as the apoptosis ability of cells. As shown in Fig. 4C, the effect of Mig-6 siRNA-mediated Bcl-2 production and anti-apoptotic effects were abolished in ERK1/2-depleted H1299 and BE1 cell lines.

A549 and H157 cells were used in the present study to investigate the effects of Mig-6 upregulation on proliferation and invasion. These cells have low endogenous Mig-6 protein levels. The Mig-6 expression levels were unchanged upon transient transfection with the empty vector, whereas pcDNA3-Mig-6 plasmid significantly upregulated both mRNA as well as protein expression levels in the A549 and H157 cell lines (Fig. 2A). Next, we examined the cell growth rate. With pcDNA3-Mig-6 plasmid transfection, A549 cells displayed reduced growth rates when compared to the control (Fig. 5A). Similar to A549 cells, we found that upregulation of Mig-6 also reduced the growth rate of H157 cells (Fig. 5A). We utilized an independent method, colony formation assay, to validate the antiproliferative effects of Mig-6 in lung cancer cells. pcDNA3-Mig-6 plasmid led to a clear reduction of the colony formation capacity of two tested lung cancer cell lines compared to empty vector control (empty vector vs. Mig-6 plasmid: A549, 210.33±19.66 vs. 84.33±10.97, p<0.01; H157, 163.33±13.01 vs. 119±17.35, p<0.01) (Fig. 5B). These overexpresssion studies demonstrated that Mig-6 inhibits tumor cell proliferation. To further examine whether Mig-6 contributes to the invasive capabilities of NSCLC cells, we conducted Matrigel invasion assays. The expression of Mig-6 was enhanced in transfected cells in comparison to the control cells. Our results demonstrated that the invasive capabilities of Mig-6 overexpressed A549 and H157 cancer cells were reduced compared to the control cells (empty vector vs. Mig-6 plasmid: A549, 58±8 vs. 34.33±4.04, p<0.05; H157, 53.67±5.13 vs. 16.33±3.21, p<0.01) (Fig. 5C). Collectively, these results demonstrate that increased expression of Mig-6 significantly inhibited invasion of A549 and H157 cells.