Anti-Hakai antibody (21179-1-AP) was purchased from ProteinTech Group, Inc. (Chicago, IL, USA). Anti-pAKT (Ser⁴⁷³) antibody (sc-7985-R), anti-AKT antibody (sc-8312), and the goat anti-rabbit (sc-2004) and anti-mouse (sc-2005) horseradish peroxidase-conjugated IgG secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-N-cadherin (AF0243) and anti-E-cadherin (AF0138) antibodies were obtained from Beyotime Institute of Biotechnology (Shanghai, China). Anti-actin (A5316), cisplatin (P4394) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Vetec V900888) were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Lipofectamine^® 2000 (Invitrogen 11668-019) was purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA).

The NSCLC cell lines A549 and NCI-H460 were obtained from the American Type Culture Collection (Manassas, VA, USA). The human normal bronchial epithelial cell line 16HBE was purchased from the Cell Resource Center of the Chinese Academy of Medical Sciences (Beijing, China) and cultured according to standard protocols. The human normal bronchial epithelial cells Beas-2B were provided by Professor Guangbiao Zhou at the Institute of Zoology, Chinese Academy of Sciences. Cells were cultured in Dulbecco modified Eagle medium (DMEM; HyClone; GE Healthcare Life Sciences, Logan, UT, USA), supplemented with 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences), 100 U/ml penicillin and 0.1 mg/ml streptomycin (Beyotime Institute of Biotechnology) under 5% CO₂ and 37°C in an incubator.

NSCLC cells were plated in 6-well plates with growth medium without antibiotics at densities ranging between 1×10⁵ and 2×10⁵ cells/well. Following incubation for 24 h, cells were transfected with synthesized Hakai siRNA or negative control (NC) siRNA (GenePharma Co., Ltd., Shanghai, China) at a concentration of 100 nM using Lipofectamine^® 2000 for siRNA delivery, according to the manufacturer's protocol. The specific transfection procedure was as follows: First, siRNA oligomer was diluted in 250 µl Opti-MEM I Reduced Serum Medium (Gibco; Thermo Fisher Scientific, Inc.) without serum. Next, 4 µl Lipofectamine^® 2000 was diluted in 250 µl Opti-MEM I Reduced Serum Medium, mixed gently and incubated for 5 min at room temperature. After 5 min of incubation, the diluted oligomer and Lipofectamine^® 2000 solutions were combined, mixed gently and incubated for 20 min at room temperature. Oligomer-Lipofectamine^® 2000 complexes were added to each well containing the cells, along with 500 µl growth medium without antibiotics, and cells were incubated at 37°C in a CO₂ incubator. After 6 h of transfection, the medium was replaced with fresh growth medium, and the cells were harvest for further analysis after 48-72 h of cultivation. The sequences for Hakai RNAi were as follows: Hakai RNAi-1, 5′-CUCGAUCGGUCAGUCAGGAAA-3′; and Hakai RNAi-2, 5′-CACCGCGAACUCAAAGAACUA-3′. The efficiency of siRNA transfection in downregulating the targeted gene was detected by a western blot assay, with an >50% reduction observed in the targeted protein expression.

Isolation of total RNA from cell lines was conducted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and the phenol-chloroform extraction method, according to the manufacturer's protocols. The 28S and 18S bands were detected by agarose gel electrophoresis for measuring the quality of the extracted RNA. cDNA was subsequently generated from total RNA using a PrimeScript II 1st Strand cDNA Synthesis kit (Takara Biotechnology Co., Ltd., Dalian, China). The RT conditions were as follows: 42°C for 1 h and 95°C for 5 min, followed by storage at −20°C. Next, the resultant cDNAs were used as templates for PCR amplification in 25 µl reaction mixture, including 10X ExTaq PCR buffer, deoxynucleoside triphosphate mixture (2.5 mM each), 0.2 µM of each primer and 1 U ExTaq polymerase (RR001A; Takara Biotechnology Co., Ltd.). The primers for Hakai were synthesized by Jinsirui Biotechnology Co., Ltd. (Nanjing, China), and the sequences were as follows: Hakai: 5′-GGACACCTTTTTTGGGACTT-3′ (forward) and 5′-CACCCTTGAACAATGCTACAC-3′ (reverse); Actin: 5′-ATCGTCCACCGCAAATGCTTCTA-3′ (forward) and 5′-AGCCATGCCAATCTCATCTTGTT-3′ (reverse). The PCR amplification conditions for Hakai and Actin were 94°C for 10 sec, 55°C for 15 sec and 72°C for 30 sec, for a total of 35 and 22 cycles, respectively. Actin was used as an internal control. PCR products were separated by electrophoresis on 1.5% (w/v) agarose gels and visualized under UV light via Goldview staining (Beijing Solarbio Science & Technology Co. Ltd., Beijing, China).

Cell viability was evaluated using the trypan blue dye exclusion assay (cat. no. C0011; Beyotime Institute of Biotechnology). Cells after 48 and 72 h of transfection were respectively detached with a trypsin-EDTA solution (HyClone; GE Healthcare Life Sciences). The mixture of detached cells was centrifuged at 1,000 × g for 1 min at room temperature and resuspended with fresh DMEM supplement with 10% FBS and penicillin/streptomycin. Subsequently, 100 µl of cell suspension was combined with 100 µl trypan blue solution (2X) and mixed gently. After 3 min staining at room temperature, viable cells that were not stained were counted manually using an inverted light microscope (Leica Microsystems GmbH, Wetzlar, Germany) using a x10 objective in 6-8 random fields of view in each group.

A549 or NCI-H460 cells were transfected with NC or Hakai-specific siRNA using the aforementioned method. After 48 h, the transfected cells were digested with trypsin and seeded onto 35 mm plates in triplicate (1,000 cells per plate). The medium were refreshed every 5 days. After 10 days of incubation, the cells were fixed with methanol for 10 min and stained with 0.005% crystal violet solution for ~20 min. Stained clones containing >50 cells were counted under a microscope.

A549 (2×10⁵/well) and NCI-H460 (4×10⁵/well) cells were seeded into 6-well plates and transfected the next day with Hakai RNAi or NC RNAi, while there was also a control group without siRNA transfection. After 48 h, when the cells approximated full confluence, a sterile 200-µl micropipette tip was used to create wounds in the cell monolayer of the three groups, and the culture medium was replaced with fresh serum-free medium. At 0 and 24 h, images of the scratched areas were captured with an inverted microscope (Leica Microsystems GmbH, Wetzlar, Germany) using a x4 objective. The wound widths were measured, and the relative wound widths were calculated.

A 24-well transwell unit (pore size, 8 µm; Costar; Corning Life Science, Woburn, MA, USA) was used to evaluate the ability of cell invasion. A total of 2×10⁵ transfected cells were seeded into the upper chamber coated with Matrigel (cat. no. 354248; BD Biosciences, Franklin Lakes, NJ, USA), and the bottom chamber was filled with DMEM with 20% FBS. After 24 h of incubation at 37°C in a 5% CO₂ atmosphere, invasive cells on the bottom sides of the membrane were fixed with ethanol for 10 min and stained with 0.2% crystal violet for 20 min. Invasive cells were manually counted under a microscope (Leica Microsystems GmbH) at a ×20 objective in 4-5 random fields-of-view in each group.

At 48 h post-transfection, A549 and NCI-H460 cells from the two different groups (Hakai RNAi and NC RNAi) were seeded into 96-well plates at a density of 5,000-10,000 cells/well, with three replicate wells used per group. When the cells in each well reached 70-80% confluence, different concentrations of cisplatin (0, 5, 10 and 15 µM) were added followed by 48 h incubation. Next, 10 µl MTT (5 mg/ml) was added to each well for an additional 2-4 h. The medium was discarded and 150 µl DMSO was added to dissolve the formazan crystals for measurement. Subsequently, the optical density was measured at an absorbance wavelength of 490 nm using a microplate reader (Multiskan FC; Thermo Fisher Scientific, Inc.). The cell survival rate was calculated as follows: Cell viability (%) = (average A490 of the experimental group − average A490 of the blank group)/(average A490 of the control group − average A490 of the blank group) × 100.

Proteins from different cell lines in the different transfection groups were extracted using radioimmunoprecipitation assay buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM Na₃VO₄, 1 mM NaF and 1 mM PMSF (19). The protein levels were determined using Bradford Protein Assay kit (Beyotime Institute of Biotechnology). Proteins (25-50 µg) were then subjected to SDS-polyacrylamide gel electrophoresis (8 and 10%), and transferred to a nitrocellulose membrane (Pall Corporation, East Hills, NY, USA). Subsequent to blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated overnight at 4°C with the indicated primary antibodies, then washed with Tris-buffered salin containing 1% Tween-20 for 20 min, followed by incubation with the corresponding secondary antibodies for 2 h at room temperature. Detection was performed using an enhanced chemiluminescence substrate (CW0049M; Cwbiotech, Beijing, China). Bands intensities were quantified using ImageJ software (version 1.48; National Institutes of Health, Bethesda, MD, USA).

Data were collected and analyzed using GraphPad Prism 6.0 software (GraphPad Software, Inc., La Jolla, CA, USA), and are expressed as the mean ± standard deviation. Student's t-test was used to compare data between two groups. One-way or two-way analysis of variance, followed by Sidak post hoc test, was used to analyze data of multiple groups. P<0.05 was considered to be an indicator of a statistically significant difference.

Hakai expression was assessed by RT-PCR and western blot analysis in the NSCLC cell lines A549 and NCI-H460, as well as in human normal bronchial epithelial cells 16HBE and Beas-2B. The results demonstrated that Hakai mRNA was highly expressed in A549 and NCI-H460 cell lines compared with its expression in 16HBE and Beas-2B cells (Fig. 1A). Consistent with mRNA expression, western blot analysis revealed significantly higher protein expression levels of Hakai in A549 and NCI-H460 cell lines, with weaker expression observed in 16HBE and Beas-2B cells (P<0.0001; Fig. 1B and C). These results indicated that Hakai is overexpressed in NSCLC cells.

In order to investigate the potential function of Hakai in NSCLC cell lines, A549 and NCI-H460 cells were transfected with siRNA against Hakai, and the results revealed that the two siRNAs were able to decrease the expression of Hakai (Fig. 2A). Thereafter, Hakai RNAi-1 was used for further experiments, owing to its higher effect. Hakai silencing was found to markedly inhibit the proliferation of A549 and NCI-H460 cells, and an enhanced inhibitory effect was observed when the incubation time increased, as demonstrated by trypan blue dye exclusion assay (P<0.05; Fig. 2B and C). Similarly, a flat colony formation assay indicated that Hakai knockdown in A549 and NCI-H460 cells led to a significant decrease in the colony numbers (P<0.01 and P<0.0001, respectively; Fig. 2D). These data demonstrated that Hakai serves a critical role in NSCLC cell proliferation.

Hakai is implicated in the regulation of cell substratum adhesions and in epithelial cell invasion (14). Thus, the present study next investigated whether Hakai was involved in the cell migration progress in NSCLC cells. When comparing with the cells of the NC and non-transfected control groups, the results of wound healing assay demonstrated that Hakai depletion led to a significant decrease of the migration ability of A549 and NCI-H460 cells transfected with Hakai RNAi (P<0.001 and P<0.01, respectively; Fig. 3A). A transwell invasion assay revealed a low level of invasion in Hakai RNAi cells compared with the NC and non-transfected control cells (P<0.01 and P<0.001; Fig. 3B).

Hakai was firstly described as the E3 ubiquitin ligase for the E-cadherin and mediated its degradation (13). Thus, the current study explored whether downregulation of Hakai was able to regulate the expression levels of E-cadherin and N-cadherin in NSCLC cells. The results of western blot analysis demonstrated that Hakai depletion upregulated E-cadherin and downregulated N-cadherin in both A549 and NCI-H460 cells (Fig. 3C), which may contribute to the inhibitory effect of Hakai decrease on cell migration and invasion. These results indicate that Hakai influences cell migration and invasion in NSCLC cells.

A growing body of evidence suggests that AKT perturbations serve an important role in human malignancy (20). Consistently, high activation of AKT has been reported in various types of human cancer, including NSCLC (21,22). Thus, whether Hakai inhibition regulated the activity of the AKT signaling pathway was examined in NSCLC cells. As shown in Fig. 4A and B, the western blot results revealed that downregulation of Hakai decreased AKT phosphorylation at the Ser-473 site in both A549 and NCI-H460 cells, with no apparent change in total AKT level.

Cisplatin resistance was associated with AKT overexpression and gene amplification in human lung cancer cells that acquired drug resistance (23). Next, the current study tested whether Hakai was involved in the sensitivity of NSCLC cells to cisplatin. The results demonstrated that silencing of Hakai potentiated the susceptibility to cisplatin in A549 and NCI-H460 cells (P<0.05; Fig. 5A and B). This finding indicates that the level of Hakai expression influences the sensitivity to cisplatin in NSCLC cells.