LPP-Z3392-Lv105-400 (lentiviral particles for GSTA1), LPP-e green fluorescent protein (GFP)-Lv105-100 (lentiviral particles for eGFP), LP-HSH008475-5-LvRH1GP-200 [lentiviral particles for small interfering RNA, (si-RNA)] and LP-CSHCTR001-LVRH1GP-050 (lentiviral particles for scramble control) were prepared by GeneCopoeia, Inc. (Rockville, MD, USA). The human lung adenocarcinoma cell line A549 was purchased from Shanghai Institute of Cellular Biology of Chinese Academy of Sciences (Shanghai, China). Cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum and 0.1% penicillin/streptomycin (all from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a humidified atmosphere with 5% CO₂ at 37°C.

A total of 24 healthy BALB/c nude mice (half male and female; aged ~3 weeks; weight 17~20 g) were purchased from Guangdong Medical Experimental Center (Guangzhou, China). Mice were maintained under specific pathogen-free conditions at a temperature of 20–25°C with humidity of 40–70%. They were fed in groups, and had free access to water and food. The mice were randomly divided into four groups: GSTA1, vector (lentiviral particles for eGFP), si-control (scramble control) and si-GSTA1 groups with six mice/group. To establish the above four groups of animal A549 tumor models, each nude mouse was injected with 4×10⁵ A549 cells (transfected with GSTA1, vector, si-control and si-GSTA1 respectively) into the right flank. Tumor size was measured using a caliper four times/week. Tumor volumes were calculated by the following formula: 0.5× largest diameter (mm) × smallest diameter² (mm). Mice were sacrificed and xenograft tumors were removed en bloc following 3 weeks. All xenograft experiments were performed under a protocol approved by the Animal Care and Use Committee. All animal experiments were approved by the Animal Care Committee of Guangdong Pharmaceutical University (Guangzhou, China).

Total protein was extracted from the tumor tissues of the four groups or cells cultured in six-well plates. The tumor tissues were ground and lysed in radioimmunoprecipitation assay lysis buffer (#P0013B, Beyotime Institute of Biotechnology, Haimen, China) containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 1 µg/ml leupeptin and 0.1% SDS on ice, and then centrifuged at 12,000 × g, 4°C for 20 min to obtain the total protein supernatants. The protein concentration of each extract was measured using a BCA Protein assay kit (Thermo Fisher Scientific, Inc.). A total of 40 µg protein per lane was loaded and separated via 12% SDS-PAGE, and then transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% (w/v) non-fat milk powder in Tris buffered saline with Tween-20 (TBS-T) pH 7.6 solution at room temperature for 1 h. Membranes were then incubated with the primary antibodies goat anti-GSTA1 (#ab53940; 1:3,000 dilution; Abcam, Cambridge, UK) or mouse anti-β-actin (#ab8226; 1:3,000 dilution; Abcam) overnight at 4°C. The membrane was washed with TBS-T buffer three times and incubated with horseradish peroxidase-conjugated polyclonal donkey anti-goat IgG (#ab97110; 1:5,000 dilution; Abcam) or goat anti-mouse IgG (#ab136815; 1:5,000 dilution; Abcam) at room temperature for 1 h. After washing with TBS-T buffer three times, the membranes were visualized after incubation SuperSignal^™ West Pico PLUS Chemiluminescent Substrate (#34580, Thermo Fisher Scientific, Inc.). β-actin was used as loading control. The gray value was analyzed by using ImageJ software (version 1.46; National Institutes of Health, Bethesda, MD, USA).

Total RNA from the tumor lysates or cells was extracted with TRIzol solution (Life Technologies; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. cDNA was synthesized using the PrimeScript^™ II First Strand cDNA Synthesis kit according to the manufacturer's protocol using 1 µg of total RNA with the Bio-Rad Reverse Transcription system. The temperature protocol was as follows: 42°C for 2 min, then at 37°C for 15 min and 85°C for 5 sec. The RT-qPCR assay was run in a 25 µl reaction system containing 12.5 µl X2 PCR Master mix, 1 µl forward and reverse primer each, 2 µl cDNA and 8.5 µl nuclease-free water on a CFX96^™ Real-Time PCR Detection system with SYBR^® Premix Ex Taq^™ II (Takara Bio, Inc., Otsu, Japan). The primers were synthesized according to the designed sequence by Shanghai Shenggong Biology Engineering Technology Service, Ltd. (Shanghai, China) and were used for qPCR: GSTA1 forward, 5′-GCCTCCATGACTGCGTTATT-3′ and reverse, 5′-CCTGCCCACAGTGAAGAAGT-3′; GAPDH forward, 5′-AACGGATTTGGTCGTATTGGG-3′ and reverse 5′-CCTGGAAGATGGTGATGGGAT-3′. qPCR was performed under the following conditions: 95°C for 30 sec; 40 cycles of 95°C for 5 sec, 60°C for 30 sec; 95°C for 5 sec, 60°C for 1 min; 50°C for 30 sec. GAPDH served as a reference gene. The relative quantity of mRNA expression was calculated by using the 2^−ΔΔCt method (18). All experiments were performed in triplicate.

A549 cells were seeded at a density of 5×10³ cells/well in 96-well flat bottom plates. A total of 4 µl of 1 ×10⁸ TU/ml lentivirus particles (GSTA1, vector, si-control and si-GSTA1) were serially diluted to 4×10⁶ TU/ml by serum free OptiMEM medium (Gibco; Thermo Fisher Scientific, Inc.) containing 5 µg/ml Polybrene (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). When the cells reached 30–50% confluence, they were transfected with the aforementioned lentivirus particles using 2 µg/ml puromycin (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for screening stable cell lines. The cells were cultured for 5 days. During this time, 20 µl MTT solution (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) in PBS (5 mg/ml) were added to each well at 1-day interval for 4 h at 37°C, then, 200 µl DMSO was added after removing the supernatants. The absorbance of each well was measured with a microplate reader at 570 nm. All experiments were performed in triplicate.

Lentivirus-meditated GSTA1 overexpression and si-GSTA1 in A549 cell models. The aforementioned lentiviral particles (1×10⁸ TU/ml) were diluted and added (multiplicity of infection: 40, 20, 10, 5) to transfect A549 cells for 24 h. The supernatants containing lentivirus were replaced with OptiMEM medium, and the cells were cultured for an additional 48 h. Successful infection was detected by the presence of green fluorescence in cells due to GFP as observed under an inversed fluorescence microscope (×100).

The si-GSTA1 and si-control A549 cells were seeded in 12-well plates at a density of 1×10⁵ cells/well, and then cultured for up to 5 days. Following treatment, the cells were fixed with 4% formaldehyde for 30 min at room temperature, exposed to Hoechst 33258 (15 µM) for 30 min at room temperature, then observed and imaged using an inverted fluorescence microscope (×100).

The si-GSTA1 and si-control A549 cells were cultured in 6-well plates at a density of 5×10⁵ cells/well for 5 days. The cells were collected every day, washed twice with ice-cold PBS and resuspended in binding buffer (cat. no. KGA106; Nanjing KeyGen Biotech, Nanjing, China) containing PBS (pH 7.4), 1% fetal bovine serum and 0.1% NaN₃ at a concentration of 1×10⁶ cells/ml. Then, 5 µl of Annexin V-fluorescein isothiocyanate and 5 µl of propidium iodide were added. The cells were cultured for 15 min at room temperature in the dark, after which apoptotic cells were quantified using flow cytometry (FACSCalibur/Calibur; BD Biosciences, Franklin Lakes, NJ, USA). All experiments were performed in triplicate.

Data are presented as the mean + standard deviation. Statistical analysis was performed using SPSS software (version 17.0; SPSS, Inc., Chicago, IL, USA). One-way analysis of variance followed by Tukey's post-hoc test was used for multiple comparisons and the paired Student's t-test was applied when only two groups were compared. P<0.05 and P<0.01 were considered to indicate statistically significant difference.

GSTA1 protein and mRNA expression levels were investigated using western blot analysis, and RT-qPCR in cell samples from the GSTA1 and vector groups (Figs. 1 and 2). Si-GSTA1 protein expression (0.56-fold lower) was significantly suppressed compared with the si-control group (Fig. 1A; P<0.01). The data demonstrated that the relative mRNA levels in the si-GSTA1 group were 0.38-fold significantly lower compared with that of the si-control (P<0.01; Fig. 1B). In addition, the GSTA1 protein levels in the GSTA1 overexpression group were 1.83-fold higher compared with that of the vector group (P<0.05; Fig. 2A). Likewise, the GSTA1 mRNA expression in the GSTA1 overexpression group was 2.57-fold higher compared with that of the vector group (P<0.01; Fig. 2B). These results implied that the si-GSTA1 and GSTA1 overexpression cell models were successfully established.

The results of the MTT assay indicated that cell viability in the GSTA1 group after culturing for 3 days were significantly improved when compared with the vector group (P<0.05; Fig. 3A). In addition, si-GSTA1 transfection resulted in significantly decreased cell viability compared with the si-control following 3 days of culture (Fig. 3B; P<0.05). Hoechst 33258 staining was used to observe the morphological changes in the cell nuclei. As shown in Fig. 4, the cell florescence of A549 cells transfected with si-GSTA1 was relatively brighter compared with the si-control groups. When compared with si-control A549 cells, the cell amounts of si-GSTA1 were few. In order to further confirm that the cell death induced by si-GSTA1 was apoptosis, AnnexinV-FITC/PI staining was used to detect early apoptosis cells and middle-late apoptotic cells (19). A time-dependent increase in apoptotic cells was observed from 6.42 on day 1 to 10.06, 15.8, 21.3 and 77.61% in si-GSTA1 A549 cells cultured for 5 days compared with the control counterparts, which saw an increase from 1.38 on day 1 to 4.63, 5.30, 4.84 and 7.22% (Fig. 5). These results demonstrated that the downregulation of GSTA1 in A549 cell lines is able to suppress cell proliferation and induce cell apoptosis in vitro.

Based on the aforementioned results in vitro, the potential effect of GSTA1 in vivo in A549 cell xenografts was explored (Fig. 6A). As shown in Fig. 6B, the tumor volume in the GSTA1 group was 2,709.27 mm³, whereas that of the vector group was 1,395.43 mm³ after 21 days. The tumor volume in the si-GSTA1 group was 204.12 mm³ compared with that of the si-control group (1,066.07 mm³). Furthermore, as shown in Fig. 6C, the tumor weight in vector, GSTA1, si-control and si-GSTA1 groups were 0.73, 1.66, 0.64, and 0.12 g, respectively. These results indicated that GSTA1 significantly induced tumor growth compared with the vector control, while si-GSTA1 significantly inhibited tumor growth compared with the si-control (Fig. 6).

In order to further verify the role of GSTA1 in lung cancer cell growth in vivo, western blotting and RT-qPCR were used to analyze the expression of GSTA1. The gray values of GSTA1/β-actin in the vector, GSTA1 overexpression, si-control and si-GSTA1 groups were 0.50, 1.28, 0.52, and 0.38, respectively (Fig. 7A). As shown in Fig. 7B, the relative GSTA1 mRNA levels of the four tumor groups were 1.00, 3.30, 0.98 and 0.28, which demonstrated that tumor growth may have a significant association with the expression of GSTA1.