Mouse anti-human AURKA polyclonal antibody was purchased from ProteinTech Group (Wuhan, Hubei, China); mouse anti-human AURKA monoclonal antibody was purchased from Abcam (Abcam, Cambridge, UK); Taq DNA polymerase was obtained from Fermentas, Inc. (Waltham, MA, USA); Lipofectamine 2000, Opti-MEM and the SuperScript III reverse transcriptase (RT) kit were purchased from Invitrogen (Carlsbad, CA, USA); Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and bovine serum albumin were purchased from Sigma Chemical Co. (St. Louis, MO, USA); VCR were purchased from Shenzhen Wan Le Pharmaceutical Co., Ltd. (Shenzhen, Guangdong, China); cell culture media, fetal bovine serum (FBS) and other supplementary materials were purchased from Gibco Co. (Grand Island, NY, USA).

Human lung adenocarcinoma H1299 cells and A549 cells were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) in 2013 and were identified by STR (short tandem repeat) method in 2014. All cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium) (Gibco Co.) supplemented with 10% fetal calf serum (FCS) and grown in a humidified incubator at 37°C and 5% CO₂.

From January 2005 to January 2010, 101 patients with lung adenocarcinoma underwent resection in the Kunshan First People’s Hospital affiliated to Jiangsu University. All cases of lung adenocarcinoma were clinically and pathologically proven. This study was approved by the medical ethics committee of the Kunshan First People’s Hospital with the reference number: 10. All participants have provided verbal informed consent to participate in this study. We recorded participant consents through the telephone communication. This consent procedure was approved by the ethics committees.

Immunohistochemistry (IHC) studies were performed according to the manufacturer’s instructions. Briefly, 3 μm-thick sections was deparaffinized and rehydrated, then incubated in 3% hydrogen peroxide for 15 min to block endogenous peroxidase activity. These tissue slides were boiled in EDTA buffer (pH 9.0) for 10 min for antigen retrieval. At room temperature, 10% normal rabbit serum was introduced for blocking non-specific binding. At 4°C refrigerator, the slides was incubated with polyclonal antibody against AURKA at a dilution of 1:100 in PBS for 1 h, rinsed five times with PBS, they were incubated with goat anti-mouse IgG conjugated with horseradish peroxidase for 30 min at room temperature. The histologic sections were developed with DAB (3,3′-diaminobenzidine-tetrahydrochloride-dihydrate) and lightly counterstained with haematoxylin. The histologic sections were read using light microscopy.

Each case was scored according to the percentage of positive cells to total cancer cells and the staining intensity of the positive cells. Regarding cell counting under microscope, at least 10 high-power fields were randomly selected. The area of staining was divided into four levels as follows: no staining of cells in any microscopic fields was scored 0; <30% of tissue stained positive was scored 1; between 30 and 60% stained positive was scored 2; >60% stained positive was scored 3. In each slice, no staining, weak staining, moderate staining and strong staining were scored as 0, 1, 2, and 3, respectively.

A total of 443 samples from 4 institutions [Moffitt Cancer Center (HLM), University of Michigan Cancer Center (UM), the Dana-Farber Cancer Institute (DFCI) and Memorial Sloan-Kettering Cancer Center (MSK)] were used to investigate the association of the AURKA expression and prognosis in lung adenocarcinoma, as previously published (12). Patients were separated into high or low AURKA expression groups based on the first quantile (25%) of the AURKA expression values of total samples. Kaplan-Meier survival analysis was used to estimate survival curves and difference between curves was evaluated by log-rank test. Multivariate Cox proportional hazards regression with covariate age, gender, and stage was carried out to measure the independent prognostic factors. All tests were two-tailed and p<0.05 were considered significant.

The TRIzol reagent (Invitrogen) following the protocol of the manufacturer (Invitrogen). RNA (1 μg) was subjected to reverse transcription. The PCR primers used were as follows: for AURKA: forward 5′-GCCCTGTCTTACTGTCATTCG-3′ and reverse 5′-AGGTCTCTTGGTATGTGTTTGC-3′; for GAPDH: forward 5-TGACTTCAACAGCGACACCCA-3′ and reverse 5′-CACCCTGTTGCTGTAGCCAAA-3′; PCR products were separated by electrophoresis in 1% agarose gel, visualized by staining with ethidium bromide and photographed under ultraviolet light.

The human AURKA-specific small interfering RNA (siRNA) sequence is 5′-GAAAGCTCCACATCAATAA-3′, designed with an online software of Invitrogen using AURKA sequence (GeneBank code: NM_003600) as a reference. The non-silencing (NS) sequence (5′-TTCTCCGAACGTGTCACGT-3′) was used as scrambled control that has been widely used (13). The short hairpin RNA (shRNA) cassette against AURKA is 5′-CCGGCAGAAAGCTCCACATCAATAATTCAAGAGA TTATTGATGTGGAGCTTTCTGTTTTTG-3′, with two cohesive ends for ligation into the pGCSIL-GFP vector. The double stranded shRNA oligonucleotide were ligated into pGCSIL-GFP vector linearized by restriction enzyme EcoRI and AgeI.

Next, lentiviral vector that expressed the AURKA-specific siRNA or negative control siRNA, together with pHelper 1.0 and pHelper 2.0 plasmids were co-transfected into HEK293T cells with Lipofectamine 2000 for lentivirus generation, according to the manufacturer’s instructions (Invitrogen). After 48 h of transfection, the lentiviral particles were harvested and purified with ultracentrifugation. Due to the produced lentiviruses carrying green fluorescence protein (GFP), the viral titer was determined by counting green cells with serial dilutions under fluorescence microscopy at 5 days after infection. For lentivirus infection, H1299 and A549 cells were grown in 6-well plates at 70–80% confluence and infected with AURKA-specific siRNA lentivirus or control lentivirus at MOI of 20. Five days after infection, cells expressing GFP protein were observed using fluorescence microscopy to determine the infection efficiency. There were two experimental groups for each cell line: LV-AURKA infected cells (AURKA-siRNA) and LV-NS infected cells (scr-siRNA).

Total RNA was initially extracted from cultured lung adenocarcinoma cells using TRIzol (Invitrogen) and treated with RNase-free DNase I. Standard reverse transcription reaction was performed using a Promega M-MLV cDNA synthesis kit following the manufacturer’s instructions. The real-time reverse transcription polymerase chain reaction was performed using the SYBR Green One-Step qRT-PCR kit (Invitrogen) according to the kit’s procedure manual. GAPDH was used as an internal control. The PCR primers used were: AURKA: forward 5′-GCCCTGTCTTACT GTCATTCG-3′, AURKA: reverse 5′-AGGTCTCTTGGTAT GTGTTTGC-3′; GAPDH: forward 5′-TGACTTCAACAGC GACACCCA-3′, and GAPDH: reverse 5′-CACCCTGTTGCT GTAGCCAAA-3. The relative gene expression levels were calculated using the 2^−ΔΔCT algorithm.

The monolayer culture growth rate was determined by using a Cellomics Arrayscan (Thermo Fisher Scientific Inc., Waltham, MA, USA). Briefly, after the lung adenocarcinoma H1299 cells were infected with virus for 3 days, cells were seeded into 96-well plates and cultured in a humidified atmosphere of 5% CO₂ at 37°C. The Cell viability was measured at 0, 1, 2, 3, 4 and 5 days with Cellomics Arrayscan to observe the cell growth with GFP signal. Consequently, the statistical analysis of the data collected was performed to create a growth curve for the six days. Each experiment was performed in triplicate.

The effects of AURKA silence on proliferation of A549 cells were analyzed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma Chemical Co.). The A549 cells were plated at a final concentration of 5×10³ cells/well in 96-well culture plates for different culture times. MTT (10 μl) (5 mg/ml in PBS) was added to each well and incubated for an additional 3 h at 37°C. The formazan crystals were dissolved in 100 μl of DMSO, and the absorbance was read at 490 nm by an ELISA reader (ELx808, Bio-Tek Instruments, Winooski, VT, USA).

H1299 cells in all experimental groups were trypsinized and resuspended in complete medium. Two groups (scr-siRNA, AURKA-siRNA) of H1299 cells were plated in 96-well plates at the rate of 500 cells per perforation. Three compound perforations were set in each experimental group. The medium was changed and cells were monitored every 3 days. After 2 weeks of culture, the cells were washed with PBS. These perforations were scanned and photographed with Cellomics ArrayScan, the number and size of clones within the perforations were analyzed.

The infected cells were synchronized by exposure to serum-free medium for 24 h to induce starvation. Then adherent cells were harvested by trypsinization, washed twice with ice-cold PBS, fixed in 70% ethanol and incubated for 30 min at 4°C. After the ethanol was discarded by centrifugation, the fixed cells suspended in PI/RNase/PBS (100 μg/ml propidium iodide and 10 μg/ml RNase A) for 45 min at room temperature in the dark. After the suspension was filtered through a 50-μm nylon mesh, the DNA content of the stained nuclei was analyzed by a flow cytometer to determine the percentage of cells for each phase of the cell cycle. Each experiment was performed in triplicate.

A total of 1.0×10⁶ cells were collected and washed with ice-cold PBS for twice. Cells were resuspended in 100 μl of Annexin V binding buffer, incubated with APC labeled Annexin V at room temperature for 15 min. Apoptotic cells were detected by a flow cytometer. Each experiment was performed in triplicate.

The cell pellets were lysed in lysis buffer that was supplemented with protease and phosphatase inhibitor cocktails. Total protein (50 μg) was separated by SDS-PAGE and electroblotted onto nitrocellulose membranes after protein quantitation using Coomassie brilliant blue assay. Membranes were blocked by 5% non-fat dry milk and incubated for 1 h with mouse monoclonal antibodies against Aurora-A, WEE1, CDK4, EGFR, PAK4, RAF-1, CCND2, CCND3. After incubation with the secondary antibodies (peroxidase-conjugated anti-mouse IgG) for 1 h, protein bands were visualized by enhanced chemiluminescence. GAPDH was used as an internal positive control.

The A549 cells infected with control lentivirus or AURKA-siRNA lentivirus were cultured for 72 h at 37°C in a humidified incubator with 5% CO₂. Then cells were trypsinized, resuspended, spread onto 96-well plates and VCR was added after 12 h. The treated cells were cultured for 24 and 96 h, respectively, and incubated with BrdU for 4 h. Subsequently, the cells were fixed, washed and incubated with mouse anti-BrdU antibody for 1 h and horseradish peroxidase-conjugated secondary antibodies for 30 min following the manufacturer’s protocol (Chemicon International Inc., Temecula, CA, USA). The immune complexes were detected by the subsequent 3,3′,5,5′-Tetramethyl-benzidine (TMB) substrate reaction, and the levels of BrdU incorporated into cells were quantified by measuring the absorbance at 490 nm using a microplate reader (Bio-Rad 680, Bio-Rad, Hercules, CA, USA).

To investigate whether the transfection with lentivirus encoding a AURKA siRNA increases the chemosensitivity of lung adenocarcinoma cells, A549 cells were treated with VCR at 50, 100 nM for 48 h after transfection with AURKA-siRNA and scr-siRNA, respectively.

Then A549 cells in each group were harvested and cells stained with the Annexin V apoptosis kit (Invitrogen) according to the manufacturer’s instructions. Analysis of apoptosis were performed using a FACScan flow cytometer (Becton-Dickinson).

All quantitative data are represented as mean ± SD in this study. Statistical analysis was performed by Student’s t-test and one-way ANOVA using GraphPad Prism 5.0 software. p<0.05 was considered to be statistically significant.

Clinical characteristics of the 101 lung adenocarcinoma patients including age, gender, tumor differentiation, lymph node status, are summarized in Table I. There were 56 males and 45 females, aged from 30 to 80 years, with a median age of 62 years. Of the patients, 46 demonstrated no lymph node metastasis (N0), whereas 55 were identified with lymph node involvement (N+). The grades of differentiation were 24 with grade I (well differentiated) and 77 with grade II or III (moderately to poorly differentiated). No significant association was found between AURKA overexpression and clinicopathological features such as age, gender, tumor differentiation, lymph node status in our study.

Finally, the AURKA expression and prognosis in lung adenocarcinoma was tested. AURKA was highly expressed in lung adenocarcinoma tissue (Fig. 1A). Patients with high AURKA expression had shorter overall survival than low expression group (p=0.0016, log-rank test). The adjusted hazard ratio is 1.55 (95% CI=1.11–2.17, p=0.011) of AURKA expression. It indicated that AURKA expression was an independent prognostic factor after adjusted the effects of age, gender, and stage (Fig. 1B).

To determine the expression of AURKA in lung carcinoma cells, RT-PCR assay was performed. As showed in Fig. 1C, AURKA was highly expressed in lung adenocarcinoma cell lines H-125, H1299, LTEP-A-2, SPC-A-1 and human small cell lung cancer NCL-H446 cells. These results suggests a correlation between the overexpression of AURKA and the occurrence of NSCLC.

To further illuminate the role of AURKA in lung adenocarcinoma, we constructed the lentivirus-delivered AURKA-specific siRNA vector (AURKA-siRNA) and scramble-siRNA vector (scr-siRNA). Fluorescent microscope was used to investigate the lentiviral infection efficiency. The results showed that >90% of the cells exhibited the green fluorescence indicative of infection after the transfection (Fig. 2A). To determine the silencing efficiency, the expression levels of AURKA mRNA and protein were detected by real-time PCR and western blotting. The results indicated that the levels of RNA (Fig. 2B) and protein (Fig. 2C) expression of AURKA were dramatically decreased in both H1299 and A549 cells compared to scr-siRNA treatment groups. Thus, these results confirmed that the AURKA-siRNA could downregulate the AURKA expression effectively.

We tested the effect of AURKA-siRNA on the cell viability of H1299 and A549 cells in vitro. Cellomics analysis showed that AURKA knockdown significantly inhibited cell growth of H1299 cells comparing with scr-siRNA treatment, and the difference was more pronounced with time-dependent manner (p<0.01) (Fig. 3A). The results of the colony formation assay show that the number of colonies in the AURKA-siRNA group (4.67±2.08) was significantly less than that in the scr-siRNA group (19.33±2.52) in H1299 cells (p<0.01) (Fig. 3B). These results demonstrate that the reduction in AURKA expression decreases the ability of H1299 cells to form colonies.

MTT assay was performed to study the effect of AURKA-siRNA on A549 cell growth. As shown in Fig. 3C, A549 cells show a significant (p<0.01) reduction in cell viability 5 days after infection. The results suggests that silencing of AURKA gene inhibits the proliferation of lung adenocarcinoma cells.

In order to study the mechanisms underlying RNAi-mediated proliferation inhibition, the changes in the cell cycle was detected by flow cytometric analysis of the DNA content. As shown in Fig. 4A and B, treatment with AURKA-siRNA results in an increase in the percentage of H1299 cells in the G2/M phase from 7.40±1.57% to 25.28±2.18% (p<0.01). In accordance with this increase in the percentage of cells in the G2/M phase, there was a significant decrease in the percentage of cells in the G0/G1 phase from 60.94±3.14% to 45.82±2.75% (p<0.01), but no significant change in the percentage of cells in the S phase from 31.66±4.69% to 28.90±3.04% (p>0.05). Treatment with AURKA-siRNA also results in an decrease in the percentage of A549 cells in the G2/M phase from 10.57±0.53% to 6.27±0.57% (p<0.01). Again, there was also a significant increase in the percentage of cells in the S phase from 31.77±0.61% to 37.74±1.01% (p<0.01), but no significant change in the percentage of cells in the G0/G1 phase from 58.63±1.30% to 55.99±0.67% (p>0.05).

These results suggest that depletion of AURKA inhibits the cellular proliferation of lung adenocarcinoma cells via G2/M and S phase arrest of the cell cycle in H1299 and A549 cells, respectively. The inconsistent results of cell cycling may derive from the endogenous differences in cell cycle in different cell types.

To determine whether knockdown of AURKA could induce cell apoptosis, flow cytometry was used to analyze the apoptosis of lung adenocarcinoma cells after infection with AURKA-siRNA for 72 h. As shown in Fig. 5A and B, the percentage of apoptotic H1299 cells was 4.14±0.46% in scr-siRNA group and the percentage of apoptotic cells increased to 18.04±2.69% in AURKA-siRNA group (p<0.01) (Fig. 5A). The percentage of apoptotic A549 cells was 0.98±0.02% in scr-siRNA group cells and increased to 8.27±0.61% in AURKA-siRNA group (p<0.01) (Fig. 5B). These data suggest that the depletion of AURKA specifically induced apoptosis of the lung adenocarcinoma cells.

To test the possible mechanisms underlying the lung adenocarcinoma cell proliferation inhibition and apoptosis after AURKA knockdown, we checked cell cycle-related gene expression. Our results reveals that the knockdown of AURKA downregulated RAF-1, CCND2, CCND3, CDK4, PAK4, EGFR and upregulated WEE1 expression in H1299 cells. These results indicate that the heightened apoptosis associated with AURKA downregulation may be partly mediated by cell cycle-related proteins in H1299 cells (Fig. 6A).

The cooperative effects of AURKA knockdown and VCR on repressing lung adenocarcinoma cell proliferation were investigated in this study. AURKA knockdown cooperated with VCR to inhibit A549 cell proliferation. As showed in Fig. 6B, at both low (50 nM) and high concentration (100 nM) of VCR, AURKA depletion could cooperatively inhibit A549 cell growth in vitro.

The number of apoptotic cells was determined by Annexin V staining. A549 cells were transfected with scr-siRNA or AURKA-siRNA, and then treated with VCR for 48 h. As shown in Fig. 6C, the percentage of apoptotic A549 cells was 9.79±0.18% in scr-siRNA + VCR (50 nM) group and the percentage of apoptotic cells increased to 38.81±0.88% in AURKA-siRNA + VCR (50 nM) group (p<0.01). The percentage of apoptotic A549 cells was 16.30±0.11% in scr-siRNA + VCR (100 nM) group cells and increased to 48.58±0.63% in AURKA-siRNA + VCR (100 nM) group (p<0.01). These data suggest that the depletion of AURKA cooperatively induces apoptosis of the lung adenocarcinoma cells.