Fresh tumor tissues and matched normal tissues from 37 NSCLC patients (diagnosed in 2011 and 2012) were immediately transferred to liquid nitrogen and stored at −80°C for subsequent real-time PCR analysis, and 94 formalin-fixed paraffin-embedded samples of NSCLC tissues (patients diagnosed in 2007 and 2008) were obtained for immunohistochemical analysis. All patients were diagnosed in the First Affiliated Hospital of Liaoning Medical University (Jinzhou, China) and samples from the patients were stored in the pathology archive. Permission from patients was obtained prior to specimen collection. None of the patients had received chemotherapy, radiotherapy or immunotherapy prior to surgery. The study was approved by the Ethics Committee of the First Affiliated Hospital of Liaoning Medical University. Histopathological evaluation was conducted independently by two pathologists. All cases were classified according to the World Health Organization revised proposal for histological types of lung and pleural tumors (14). TNM patient evaluation was performed according to the criteria indicated in the staging procedures of the International Association for the Study of Lung Cancer (15). The patients enrolled in the IHC analysis were followed-up for five years to determine survival time, which was defined as the time period between the date of surgery on the primary tumor and when the patient succumbed to disease or the date of the final follow-up. Patient survival times were individually provided by family members by telephone. The clinicopathological data are summarized in Table I.

Total RNA was isolated from the frozen NSCLC and matched normal tissues using the TRIzol RNA kit (Invitrogen, Carlsbad, CA, USA) and quantified using an Eppendorf Biophotometer (Eppendorf, Hamburg, Germany). Reverse transcription was conducted using a PrimeScript™ RT reagent kit with gDNA Eraser (Takara Bio, Inc., Shiga, Japan). Under the thermal conditions recommended by the manufacturer, 2 μg total RNA was transcribed to cDNA in a 20 μl reaction using random hexamers. The following primers were used for quantification of MIIP mRNA expression levels: Forward, 5′-GGT CCA TCC TGG CTC AAC AGA-3′ and reverse, 5′-GCA ATC CAG TCA TAG CCC AGG TA-3′. The length of the PCR product was 118 bp. Real-time PCR was performed using Mastercycler^® ep realplex (Eppendorf, Hamburg, Germany) and the SYBR^® Premix Ex Taq^™ kit (Takara Bio., Inc.). GAPDH served as a control with the following primers used: Forward, 5′-GCA CCG TCA AGG CTG AGA AC-3′ and reverse, 5′-TGG TGA AGA CGC CAG TGG A-3′, with the length of PCR product at 138 bp. PCR was run for 40 cycles with 5 sec per 95°C denaturation, 30 sec/55°C annealing and 30 sec/72°C elongation. To verify the accuracy of the amplification, a melting curve analysis was conducted subsequent to amplification. In addition, the PCR products were verified by electrophoretic analysis on a 3% agarose gel. To determine the relative expression levels of MIIP, the comparative Ct method was used. The Ct value of the target gene was normalized to that of the endogenous reference [ΔCT = CT(target) − CT(GAPDH)] and compared with a calibrator [ΔΔCT = ΔCT(target) − ΔCT (calibrator)]. The relative expression levels of the target gene were calculated via the 2^−ΔΔCT method.

MIIP protein expression on the formalin-fixed paraffin sections was determined by IHC. Briefly, 5-μm tissue sections were dewaxed in xylene, rehydrated and incubated in 0.3% (v/v) hydrogen peroxide in 0.01 M phosphate-buffered saline (PBS; pH 7.6) for 20 min to inactivate endogenous peroxidase. Antigen retrieval was performed by heating the sections with 10 mm citrate buffer (pH 6.0) for 15 min in a microwave. The sections were incubated with rabbit anti-human polyclonal primary anti-MIIP antibody (1:300; Sigma-Aldrich, St. Louis, MO, USA) at 4°C overnight, washed in PBS three times for 15 min and incubated with horseradish peroxidase/Fab Polymer-conjugated mouse anti-rabbit monoclonal secondary antibody (Zhongshan Biotechnology Inc., China) for 30 min at room temperature. Thereafter, the antibody was revealed by incubation with diaminobenzidine at room temperature for 1 min. The sections were counterstained with hematoxylin, dehydrated and mounted. The MIIP immunoreactivity level was classified using the proportion of positive cells: 0, <5% positive cells; 1+, 5–30% positive cells; 2+, 31–50% positive cells; and 3+, >50% positive cells. The intensity of MIIP expression was scored as follows: 0, negative to weak; 1, moderate; and 2, strong. The final staining score was the sum of the intensity and the percentage of positive cells scores. A score of ≤1 was applied as a cut-off point for loss of MIIP expression.

The real-time PCR values are presented as mean ± standard error of the mean, and MIIP protein expression was dichotomized as either ‘negative’ or ‘positive’, according to the criteria described above. A two-sample t-test for independent samples was used for continuous variables. The correlation between MIIP expression and clinicopathological characteristics was then analyzed using the χ²-test. One-way analysis of variance was used to compare the means of two or more independent groups. The survival curves for patient with MIIP-positive and -negative tumors were plotted using the Kaplan-Meier method and the log-rank test was used to assess the statistical difference between the groups. Univariate and multivariate analyses were conducted using the Cox proportional-hazards regression model, and the results are expressed as hazard ratios with 95% confidence intervals. Two-tailed P<0.05 was considered to indicate a statistically significant difference. All analyses were performed using SPSS for Windows, version 13.0 (SPSS, Inc., Chicago, IL, USA).

The MIIP mRNA expression levels in cancer tissues and matched normal tissues from 37 NSCLC patients were examined using the ΔΔCT method. Melting curve analysis confirmed the specific amplification of the target and reference genes. Furthermore, gel electrophoresis analysis of the amplification products revealed single bands of the expected sizes for MIIP (118 bp) and GAPDH (138 bp) (Fig. 1). The results demonstrated that MIIP expression was downregulated in the cancer tissues. The average MIIP mRNA expression level in the 37 cancer tissue samples was 0.1867±0.0217.

The association between MIIP mRNA expression levels in the cancer tissues and various clinicopathological parameters was further analyzed. The results revealed that the MIIP mRNA expression levels in adenocarcinoma were significantly higher than those in squamous cell carcinoma (P=0.002). A statistically significant correlation was also observed between MIIP mRNA expression and tumor stage (P=0.014), with reduced MIIP mRNA expression levels in advanced tumor stage samples, as compared with specimens from tumors at the lower stages (Fig. 2A). However, no significant correlations were observed between MIIP expression levels and gender, age or differentiation status (all P>0.05), as shown in Table II.

Immunohistochemical staining of MIIP in the cancer tissue sections was conducted. Immunoreactivity for the MIIP antibody was predominantly identified in the cytoplasm of cancer cells with marginal immunoreactivity in the nucleus (Fig. 3). Among the 94 tissue samples, MIIP protein expression was positive in 36 (38.3%) and negative in 58 (61.7%) cases. The results revealed that MIIP protein expression was downregulated in cancer tissues, a finding in concordance with the mRNA expression result. The association between MIIP expression in NSCLC tissues and various clinicopathological parameters was also analyzed. Analysis of MIIP expression revealed that expression was significantly associated with pathology and tumor staging (P=0.014 and P=0.002, respectively), but not with gender, age or NSCLC differentiation status (all P>0.05), as shown in Table III. To determine whether the downregulation of MIIP protein expression was correlated with disease progression, the staining degrees within tumor staging groups were compared. The results demonstrated that the positive percentage of MIIP protein expression was significantly reduced in advanced tumor stage samples, as compared with specimens from less advanced tumors (Fig. 2B; Table III).

The total follow-up time period for the patients who were alive at the time of analysis was five years. A total of 66 (70.2%) of the 94 patients succumbed to disease during the follow-up period. The five-year survival rate within the patient population was 29.8%. With regard to MIIP expression status, the five-year survival rate for patients who were MIIP-positive was 41.7% as compared with 22.4% for patients who were MIIP-negative. According to the Kaplan-Meier survival analysis, the overall survival rate curve demonstrated statistically significant differences between NSCLC patients with and without MIIP expression (P=0.028; Fig. 4). Cox proportional-hazards regression model was employed to perform univariate and multivariate analysis of survival. The results revealed that positive MIIP expression is an independent and significant factor associated with improved five-year survival. The detailed results are shown in Table IV.