Human NSCLC cell line A549 was obtained from the American Type Culture Collection and maintained in RPMI 1640 supplemented with 10% fetal bovine serum, penicillin (100 units/mL), and streptomycin (100 Ag/mL; Invitrogen, Corp.) at 37°C in a humidified atmosphere with 5% CO₂. Previous studies showed that α5-nAChR is expressed in several NSCLC cell lines (Lam et al., 2007). Total RNA was isolated from cells using the RNeasy kit from Qiagen according to the manufacturer’s instructions. Lung adenocarcinoma and normal lung tissue samples were homogenized using Omni plastic disposable probes and an Omni (TH-115) homogenizer (Omni International) for 1 min on dry ice after which total RNA was isolated using Trizol reagent according to the manufacturer’s instructions. Total RNA was quantified using a Nanodrop 1000 spectrophotometer.

RNA was isolated from lung cancer cells transfected with scrambled siRNA or α5-nAChR -specific siRNA (three replicates each). Double stranded siRNA oligonucleotides targeting CHRNA5 and a pair of negative control siRNAs were synthesized by GenePharma (China) as previously described (Ma et al., 2014). RNA samples were analyzed by microarray expression profiling using PrimeView Human Gene Expression Array platform (Affymetrix) according to the manufacturer’s instructions (Liu et al., 2014). A total of 2.5 mg of fragmented and labeled cDNA was generated using the Affymetrix GeneChip WT Terminal Labeling and Controls Kit and hybridized onto PrimeView Human Gene Expression Array according to the manufacturer’s instructions (Affymetrix). Arrays were washed, stained, and processed using Affymetrix GeneChip Fluidics Station 450 systems after which they were imaged using Affymetrix GeneChip Scanner 3000 7G for the subsequent generation of raw data. Genes differentially expressed between A549 lung cancer cells transfected with α5-nAChR-specific siRNA compared with cells transfected with scrambled siRNA were selected on the basis of a P < 0.001. Gene and functional analysis was conducted using the commercially available software GO & Pathways Analysis according to the manufacturer’s instructions.

A549 cells (2 × 10⁶) were plated in 100-mm plates with 15 ml of media, with or without nicotine or α5-nAChR-specific siRNA. Cells were harvested and fixed with 70% cold ethanol at 4°C overnight. After being washed in PBS, the cells were incubated in 1 mL of staining solution (20 mg/mL propidium iodide; 10 U/mL RNaseA) at room temperature for 30 min. Then, samples were measured using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, United States), and the percentage of cells in each phase of the cell cycle was obtained using Modfit software.

For the detection of apoptotic cells, A549 cells were trypsinized (0.25% Trypsin, 2.2 mM EDTA) from plates, washed with PBS, and stained with annexin-V-allophycocyanine (APC) according to the manufacturer’s instructions (BD Biosciences Pharmingen). Cells were analyzed with a FACSCalibur flow cytometer (BD Biosciences).

Cell monolayers were washed twice with PBS, harvested, and lysed with ice-cold assay buffer (Sigma-Aldrich) after which protein lysates (20 μg) were subjected to SDS-PAGE and Western blotting. The antibodies used for immunoblotting included those raised against cyclin D1, cyclin E2, cyclin D3 (1:500; Cell Signaling Technology), GAPDH (1:1000; Abcam) and horseradish peroxidase-conjugated anti-mouse/rabbit IgG antibody (1:10000; Santa Cruz Biotechnology). After a final wash, signals were detected with use of an enhanced chemiluminescence kit (Amersham Pharmacia, Buckinghamshire, United Kingdom). GAPDH levels were used as an internal standard.

All experimental protocols used were evaluated and approved by the Animal Care and Use Committee of Jinan Central Hospital affiliated to Shandong University. Groups of 4- to 6-week-old Balb/c athymic nude mice were purchased from Beijing HFK Bioscience (Beijing, China). To induce ectopic tumor formation, 2 × 10⁶ cells suspended in 100 μl medium were subcutaneously injected in right flank of the mice (n = 6). Tumor dimensions were measured every 3 days, and tumor volumes were calculated using the standard formula: length × width²/2. Whole body luciferase bioluminescent images were taken using a Xenogen IVIS-100, after the intraperitoneal injection of D-luciferin substrate (Caliper Life Sciences, 160 μl/mice). At the end of the experiment, mice were sacrificed by cervical vertebra dislocation, and tumors weights were measured after dissection. All experiments were approved by the Experimental Animal Ethics Committee of Shandong University (Jinan, P. R. China).

Tissue microarrays (HLug-Ade150Sur-02) were from Xinchao Biotechnology, Co., Ltd (Shanghai, China). HLug-Ade150Sur-02 contains 75 lung adenocarcinoma and para-carcinoma specimens collected from 2007 to 2012 with survival time data.

Immunohistochemical (IHC) analysis was conducted on histologic sections using purified rabbit polyclonal primary antibodies raised against α5-nAChR (1:100). α5-nAChR expression was determined by the percentage of stained tumor cells and staining intensity as previously described (Ma et al., 2014). We examined at least three different high-power (×200) fields of tumor infiltration. The percentage of positive tumor cells was rated as follows: negative expression, no positive staining; weak expression, ≤10% of cells stained positive; and moderate and strong expression, >10% of cells stained positive. Weak expression was rated as negative, and moderate and strong expression was rated as positive for statistical analysis. Expression was analyzed by two independent investigators who used a multi-headed microscope and were blinded to clinical data with consensus.

Student’s t-test or a one-way ANOVA test was used for univariate analyses. A two-way ANOVA test was used to determine the differences in tumor xenograft volume between groups. Pearson’s correlation test was used to consider correlations between α5-nAChR and cyclin D1. Associations of α5-nAChR IHC protein expression with patient outcome (survival time) was estimated using the Kaplan–Meier method and compared among groups by log-rank statistical tests. Statistical analyses were performed using the Statistical Package of Social Science (SPSS) software, version 18.0. All tests were two-sided. A P-value < 0.05 was considered statistically significant.

Our previous studies have showed that α5-nAChR mediates nicotine-induced cell proliferation, migration, and invasion in lung NSCLC in vitro (Ma et al., 2014; Sun and Ma, 2015). To gain insights into the mechanisms of α5-nAChR function, we further compared the transcriptome of cells transfected with scrambled siRNA and CHRNA5-specific siRNA. Gene expression profiling, using PrimeView Human Gene Expression Array, identified 1,972 transcripts that were significantly differentially expressed (P < 0.01), in CHRNA5 knockdown A549 lung cancer cells compared with control (Figure 1A) (Datasets submitted to GEO datasets, with GEO accession: GSE101979). Moreover, pathway analysis of these genes using KEGG¹ and Biocarta² revealed that CHRNA5 knockdown significantly modulates key pathways, including KEGG_Cell_Cycle, KEGG_DNA_replication, KEGG_Pathway_in_cancer, Carta_MCM _pathway (CDK Regulation of DNA Replication) (Aya-Bonilla et al., 2014), KEGG_P53_signaling_pathway sorting by P-value (P < 0.01; Figure 1B). Notably, pathway analysis revealed that cell cycle signaling was the top modulated canonical pathway following CHRNA5 knockdown (P < 0.01; Figure 1B). In this regard, we observed significant modulation of the cell cycle genes cyclin D1, cyclin E2 and cyclin D3, which was confirmed by Western blotting (Figure 1C). This global transcriptome analysis suggests a functional role for α5-nAChR in human lung cancer cell cycle progression.

To investigate how α5-nAChR affects the NSCLC cell cycle, fluorescence-activated cell sorting (FACS) was used to analyze the cell cycle state of control, nicotine, CHRNA5-siRNA and CHRNA5-siRNA+nicotine groups. As shown in Figure 2A, FACS revealed that 52.56 and 51.79% of A549-shCHRNA5 and A549-shCHRNA5+nicotine cells were in G1 phase, and 44.16 and 43.73% were in S phase, respectively. In contrast, 63.50 and 67.99% of the A549-shControl and A549-shControl+nicotine cells, were in G1 phase, while 33.90 and 28.89% were in S phase, respectively (P < 0.01). These results suggest that silencing of α5-nAChR expression induces S phase cell cycle arrest.

Furthermore, A549 apoptotic frequencies were 5.67% in the negative control (NC), 5.17% in the NC plus nicotine group (NC+Nic), 7.71% in the CHRNA5 Knockdown group (KD), and 10.83% in the KD plus nicotine group (KD+Nic) (Figure 2B). These results indicate that α5-nAChR regulates A549 cell apoptosis.

Based on our observation of a functional role for CHRNA5 in regulating the cell cycle, proliferation and apoptosis in A549 cells, we next sought to investigate the tumorigenic potential of α5-nAChR expression in vivo. A549 cells stably suppressing CHRNA5 or control cells were injected subcutaneously into nude mice. Tumors derived from CHRNA5-suppressing A549 cell clones were measurably smaller than those produced from control cells (Figure 3A), and ex vivo imaging of organs at the termination of the experiment confirmed these results (Figure 3B). Moreover, mice implanted with shcontrol-luc cells that received nicotine had significantly larger tumors compared to those receiving vehicle (Figure 3C). Taken together, these results confirm that α5-nAChR is indispensable for optimal lung tumor growth, especially in the context of nicotine exposure.

Our findings demonstrating α5-nAChR’s involvement in lung tumor growth in nude mice prompted us to examine α5-nAChR protein expression as it relates to lung cancer patient outcome. The HLug-Ade150Sur-02 microarray contains 75 specimens of adenocarcinoma and para-carcinoma tissues with survival data (from 2007 to 2012). As measured by immunohistochemistry, α5-nAChR is highly expressed in lung cancer (Figure 4A, 62.7%, 47/75) but not in para-carcinoma tissue (Figure 4A, 24.0%, 18/75). The association between α5-nAChR expression and clinicopathological variables is shown in Table 1. While there is no statistically significant association between α5-nAChR protein expression and age or sex, α5-nAChR expression is correlated with the clinical T and N stages (Table 1). Moreover, there is a trend for α5-nAChR expression to correlate with the clinical TMN stage (P = 0.083; Table 1).

GraphPad Prism software was used to analyze the correlation between overall survival and α5-nAChR expression in adenocarcinoma (ADC) patients. This analysis revealed that high α5-nAChR expression patients is predictive of shorter survival time (Figure 4B), and, high α5-nAChR expression in Stage II ACD predicts shorter survival time compared with low α5-nAChR expression patients (P = 0.007; Figure 4C). These results are consistent with data from the Kaplan-Meier plotter web-based tool (KMplot.com) showing that high levels of α5-nAChR correlate with decreased survival probability across all histological subtypes and variants of NSCLC (Schaal and Chellappan, 2016). The association of high α5-nAChR expression with shorter survival time in adenocarcinoma patients suggests a potential tumor-promoting function for α5-nAChR in human lung cancer.