A tissue microarray block containing 119 surgically resected lung adenocarcinoma specimens and paired non-cancerous surrounding tissues was constructed using a tissue microarrayer (Shanghai Outdo Biotech Co. Ltd., Shanghai, China).

A total of 39 cancer samples were randomly retrieved from lung adenocarcinoma patients who underwent curative resection at the Provincial Hospital Affiliated to Shandong University (Jinan, China) from September 2010 to December 2012. The procedure of human sample collection was approved by the Ethics Committee of the Provincial Hospital Affiliated to Shandong University. Written informed consent was obtained from the patients.

Total RNA was extracted using RNA Simple Total RNA kit (Tiangen Biotech, Co., Ltd., Beijing, China), according to the manufacturer's protocol. qPCR was performed using a SYBR Green PCR kit (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in an Applied Biosystems™ 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific, Inc.). The following thermal cycling conditions were used: 2 min at 50°C, 1 min at 94°C, 1 min at 55°C and 1 min at 72°C for 40 cycles, and 5 min at 72°C. The messenger RNA level of specific genes was normalized against 18S using the 2-∆∆Cq method. The sequences of the primers used in the present study are listed in Table I.

Total protein was extracted using Minute TM Total Protein Extraction Kit for Animal Cultured Cells/Tissues (Invent Biotechnologies, Inc., Plymouth, MN, USA). The lung cancer tissue extract was separated on 12% polyacrylamide-sodium dodecyl sulfate gels, and transferred to polyvinylidene difluoride membranes. The membranes were blocked with Tris-buffered saline (TBS) with 5% milk for 2 h. Subsequently, the membranes were washed with TBS with Tween 20 (TBST) and the membranes were incubated with a rabbit polyclonal anti-CHKA and anti-GAPDH (catalog nos., ab88053 and ab9485; 1 µg/ml; Abcam Trading Co., Ltd., Shanghai, China) at 4°C overnight. Then the membranes were washed with TBST three times and incubated with Dylight 680, goat anti-rabbit IgG (catalog no., A23720; 1:1,000; Abbkine, Inc., Redlands, CA, USA) for 2 h at room temperature. The protein band, specifically bound to the primary antibody, was detected with a LI-COR imaging system (LI-COR Biotechnology, Lincoln, NE, USA).

IHC analysis of tumor tissue microarray was performed using an anti-CHKA antibody (catalog no., AP8179b; Abgent Biotech Co., Ltd., Suzhou, China). The tissue array was incubated with this primary anti-CHKA antibody at 4°C overnight, followed by incubation with a horseradish peroxidase-conjugated secondary antibody at 37°C for 30 min. Next, the tissue array was incubated with 3,3′-diaminobenzidine and counterstained with hematoxylin for detection. Assessment of tissue microarray staining was based on the percentage of positively stained cells, and staining intensity was measured by Aperio ImageScope software (Leica Microsystems, Inc., Buffalo Grove, IL, USA).

Overall survival was defined as the interval between the dates of surgery and mortality. All statistical analyses were performed with SPSS version 18.0 software (SPSS, Inc., Chicago, IL, USA). Survival curves were calculated using the Kaplan-Meier method and compared with the log-rank test. The Cox proportional hazards model was used to determine the independent factors affecting survival based on variables selected by univariate analysis. P<0.05 was considered to indicate a statistically significant difference.

To examine the clinical relevance of expression deregulation of CHKA in lung adenocacinoma patients, two lung cancer tissue microarrays containing 119 lung adenocarcinoma samples were used for IHC staining. It was noticed that the expression of CHKA was significantly elevated in lung adenocacinoma samples compared with peri-tumor tissues (Fig. 1A and B). These results were further confirmed by RT-qPCR and western blot analyses (Fig. 1C and D). This finding prompted us to speculate whether CHKA is important in lung adenocarcinoma.

The clinical implication of CHKA expression in lung adenocarcinoma was analyzed. High levels of CHKA were observed to be associated with poor survival in lung adenocarcinoma patients (Fig. 2A). The association between CHKA expression in tumor tissues and the clinicopathological characteristics of the 119 patients was examined (Table II). Correlation regression analysis indicated that prognosis was correlated with several individual parameters, including CHKA expression, tumor diameter, tumor stage and differentiation (Table III). These individual parameters were further analyzed with a multivariate Cox proportional hazards model. The results indicated that CHKA expression, tumor diameter and differentiation were significant and independent factors that affected the survival of lung adenocarcinoma patients (Table III and Fig. 2B). Among these factors, the significant hazard ratio (HR) value for the cumulative survival of CHKA expression levels was 2.644 [95% confidence interval (CI), 1.217–5.746; P=0.05]. Tumor diameter and differentiation also exhibited a significant HR value for cumulative survival. For tumor diameter, the HR value was determined to be 2.462 (95% CI, 1.249–4.853; P=0.01). Poor differentiation displayed the greatest HR value (HR, 2.644; 95% CI, 1.217–5.746; P=0.01). Since poor differentiated lung cancer exhibited the greatest HR value for cumulative survival, the expression level of CHKA in poor- vs. well-differentiated cancer was further analyzed. Patients with poor differentiation tended to have high CHKA expression levels (Fig. 2C). As known, lung cancer patients with poor differentiation are more inclined to bad prognosis compared with high differentiation group (9); therefore, this result suggested that CHKA may act as an oncogene and mediate the poor prognosis and poor differentiation of lung cancer.