The A549 human lung cancer cell line was purchased from the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). The A549 cells were cultured in RPMI-1640 medium with 10% heat inactivated fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a humidified incubator with 5% CO₂ at 37°C.

The Livin (Gene ID: 79444) and Survivin (Gene ID: 332) mRNA sequences were obtained from the NCBI database (http://www.ncbi.nlm.nih.gov/pubmed) according to a previous study (14). The pSILENCER-3.0H1 expression vector (Ambion; Thermo Fisher Scientific, Inc.) contained a human H1 polymerase-III promoter for shRNA expression. According to the methods described previously (15,16), the following 19 nucleotide siRNA target sequences were designed: Livin, 5′-GGAAGAGACTTTGTCCACA-3′; Survivin, 5′-GGACCACCGCATCTCTACA-3′. Each shRNA insert was designed as a synthetic duplex with overhanging ends identical to those created by BamHI restriction enzyme digestion at the 5′ end and HindIII restriction enzyme digestion at the 3′ end. There was a nine-nucleotide ring sequence (TTC AAG AGA) between the strand and antisense strands. Following digestion by the BamHI/HindIII restriction endonucleases, the two sequences were inserted into the pSILENCER-3.0H1 plasmid by ligating the same restriction enzyme-digested shRNA fragment to the vector.

Transfection was performed using Lipofectamine^® LTX reagent according to the manufacturer's protocol. At 24 h prior to transfection, the A549 cells were harvested and 2.5×10⁵ cells were plated in each well of a 6-well plate in DMEM (Gibco; Thermo Fisher Scientific, Inc.) with 10% FBS at 5% CO₂, 37°C, 95% humidity. When the cells reached 50–80% confluence, the culture medium was removed and replaced with 2 ml of complete culture medium. For the transfection of cells in each well, 2.5 µg of plasmid diluted in 100 µl of Opti-MEM^® I reduced serum medium (0% serum), following which Lipofectamine LTX^® reagent (3.75–8.75 µl) was added into the above diluted solution. The DNA-Lipofectamine LTX^® reagent complexes formed following gentle mixing of the solution and incubation at room temperature for 30 min. The DNA-Lipofectamine LTX^® reagent complexes (500 µl) were added directly to each well containing 2.5×10⁵ cells and mixed gently by rocking the plate back and forth for 30 min. The complexes were not removed following transfection. Prior to transgene expression assaying, the transfected cells were incubated at 37°C, 5% CO₂ for 24 h following transfection. The cells were divided into four groups: pSilencer-Livin transfection group; pSilencer-Survivin transfection group; pSilencer-Survivin and pSilencer-Livin double-transfection group; and no transfection group (control group).

Fluorescent reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis was performed to determine the expression levels of Survivin and Livin according to a previous study (17). Following transfection for 72 h, the cells were harvested and total RNA extraction was performed using TRIzol reagent (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany). PrimeScript RT Master Mix (Takara Bio, Inc., Otsu, Japan) was used to perform reverse transcription according to the manufacturer's protocol. Briefly, 2 µl 5X Primer Script mix, 3 µl RNase-free water and 5 µl total RNA were added to the reaction system. The mixture was incubated for 15 min at 37°C, and then at 85°C for 5 sec, following which the mixture was maintained at 4°C. The primer sequences were as follows: Survivin, forward 5′-ATTCGCAGACTGGCCCTTTA-3′ and reverse 5′-AGAGGAAACCACAGTTGGCAG-3′; Livin, forward 5′-AGGGCGTGGTGGGTTCTTG-3′ and reverse 5′-CGGCACAAAGACGATGGACA-3′. The ABI PRISM 7000 sequence detection system and SYBR-Green PCR Master mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) were used to perform RT-qPCR. The PCR sample volume of 25 µl consisted of 12.5 µl SYBR-Green PCR Master mix, 300 nM forward primers and reverse primers, and 2.0 µl template cDNA (diluted 1:20). The PCR cycle consisted of a total of 40 cycles, the parameters were as follows: Pre-degeneration for 30 sec at 95°C; followed by denaturation at 95°C for 5 sec and annealing at 60°C for 20 sec, and then melting curve analysis for 15 sec at 65°C. The 2^−ΔΔCq method (18), with a reference gene (β-actin, forward 5′-GAACGGTGAAGGTGACAG-3′ and reverse 5′-TAGAGAGAAGTGGGGTGG-3′) as an internal standard, was used to determine the relative gene expression.

The protein expression levels of Livin and Survivin were determined using western blot analysis (19). At 48 h post-transfection, cells were lysed with lysis buffer (Invitrogen; Thermo Fisher Scientific, Inc.) with protease inhibitor cocktail tablets (Roche Diagnostics GmbH, Mannheim, Germany) and phosphatase inhibitors (1 mM NaF and 1 mM Na3CV04). Protein concentration was measured using a protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with bovine serum albumin (Bio-Rad Laboratories, Inc.) as the standard. Proteins (15 µg/lane) were separated on 10% SDS-PAGE gels, and these were transferred onto nitrocellulose membranes. Subsequently, 5% w/v non-fat dry milk in Tris-buffered saline and Tween-20 (TBST) buffer, containing 20 mmol/l Tris-HCl (pH 7.4), 150 mmol/l NaCl and 0.05% Tween-20, was used to block the membranes at room temperature for 1 h. The membranes were then incubated with primary antibodies at room temperature for 1 h. Primary antibodies were as follows: Survivin rabbit mAb (cat. no. 2808; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) and livin rabbit mAb (cat. no. 5471; 1:1,000; Cell Signaling Technology, Inc.). Following three 15-min washes in TBST, the membranes were incubated with individual secondary antibodies [anti-rabbit IgG horseradish peroxidase (HRP)-linked antibody, cat. no. 7074 and anti-biotin HRP-linked antibody, cat. no. 7075; both 1:2,000; Cell Signaling Technology, Inc.] at room temperature for 1 h, followed by three 10-min washes with TBST. The subsequent signal generation was performed using a Perfect Protein Western Blot kit (Novagen, Inc., Madison, WI, USA). The membrane was visualized with ECL Western Blotting Detection reagents (GERPN2209; Sigma-Aldrich; Merck KGaA). Image J software (ImageJ2× 2.1.4.7; National Institutes of Health, Bethesda, MD, USA) was used for densitometric analysis and the relative protein levels were evaluated using GADPH (cat. no. 8884; 1:1,000; Cell Signaling Technology, Inc.) as the control.

The cells were seeded into each well of a 96-well plate (1×10³ cells/well) 24 h prior to transfection and were divided into the four groups described above. The transfection procedure was performed according to the protocol described above. Following transfection, the cells were cultured for 24, 48 or 72 h at 37°C in 5% CO₂, respectively. The optical density (OD) values were measured at an absorbance of 490 nm. The inhibition rate of cell proliferation was calculated using the following formula: (1-experimental group OD value/control group OD) × 100%.

The use of animals in the present study was approved by the Institutional Approval Board of The Second Affiliated Hospital of Fujian Medical University (Quanzhou, China). The human cancer cell xenograft models were constructed according to a previous study (20). In brief, the A549 cells were obtained and washed with PBS three times, re-suspended in RPMI-1640 medium with 10% FBS, and injected (2×10⁶ cells) into the flank regions of athymic nude (nu/nu) mice (male; age, 8 weeks; weight, 20 g). A total of 36 mice were supplied by the Laboratory Animal Center, Fujian Medical University and housed under standard animal housing conditions, with a temperature of 22±1°C, a 12-h light/dark cycle and free access to food and water. Following tumor development, the tumor fragments were transplanted subcutaneously into the flanks of both sides of nude mice, following which the mice were randomly divided into four groups (n=6/group): shRNA-Livin injection group; shRNA-Survivin injection group; shRNA-Survivin and shRNA-Livin double-injection group; and PBS group (control group). When the volume of the tumors reached 50–100 mm³, the mice were administered with shRNA plasmids in 500 µl PBS via injection into the tail vein three times each week for 6 weeks. The tumor diameters were calculated during the injection period and for 4–6 weeks following the final injection.

The apoptosis of cells was detected using a TUNEL assay. The cells were seeded onto a 25 cm² plate 24 h prior to transfection, and divided into the four groups. At 48 h post-transfection, 4% paraformaldehyde was used to fixed non-adherent cells and adherent cells, and the cells were permeabilized with a solution of 0.1% Triton X-100 in 0.1% sodium citrate, followed by incubation for 1 h in the TUNEL reaction mixture at room temperature (Roche Diagnostics GmbH). The cells were washed with PBS three times, and the TUNEL-positive (FL1 >10¹) cells were analyzed by FACS analysis using CellQuest version 5.1 software following flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) (1).

To examine apoptosis in the tumor tissues, the mice were sacrificed following the final injection, and the tumor tissues were harvested and fixed in 4% paraformaldehyde in PBS and embedded in paraffin. The tumor tissues were cut into 5-µm thick tissue sections, and the sections were de-paraffinized in xylene, rehydrated in reduced grades of alcohol and then washed in PBS. The rehydrated slides were permeabilized in 0.1% Triton X-100 and 0.1% sodium citrate, and then incubated in the TUNEL reaction mixture at 37°C in humidified conditions for 1 h. The sections were then washed in PBS and observed under a fluorescence microscope. Fluorescence values were calculated from three randomly selected fields per section and expressed as the mean ± standard deviation.

The data are presented as the mean ± standard deviation and were analyzed by one-way analysis of variance with a Newman-Keuls post hoc test using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

In order to evaluate the effects of Livin shRNA and/or Survivin shRNA transfection on mRNA levels, fluorescent RT-qPCR was used to evaluate the relative mRNA expression levels of Livin and Survivin. Following transfection for 48 h, the relative mRNA expression of Livin was 0.18±0.02 in the Survivin shRNA and Livin shRNA co-transfection group, which was lower than that in the Livin shRNA transfection group (0.72±0.17; Fig. 1A). The relative mRNA expression of Survivin was 0.21±0.02 in the Survivin shRNA and Livin shRNA co-transfection group, which was lower than that in the Survivin shRNA transfection group (0.75±0.15; Fig. 1B), indicating that shRNA transfection decreased the mRNA expression levels of Livin and Survivin. Co-transfection was more efficient than single transfection with either Survivin shRNA or Livin shRNA alone (Fig. 1).

Western blot analysis was performed to measure the protein expression levels of Livin and Survivin. Following transfection for 48 h, the relative protein expression of Livin was 0.32±0.03 in the co-transfection group, which was lower than that in the Livin shRNA transfection group (0.69±0.18; Fig. 2A). The relative protein expression of Survivin was 0.35±0.04 in the co-transfection group, which was lower than that in the Survivin shRNA transfection group (0.67±0.19; Fig. 2B), indicating that shRNA transfection decreased the protein expression of Livin and Survivin. Co-transfection was more efficient than single transfection with either Survivin shRNA or Livin shRNA alone (Fig. 2).

As shown in Fig. 3, transfection with Livin shRNA and/or Survivin shRNA inhibited cell proliferation in the transfected A549 cells. The proliferation of cells in the co-transfection group exhibited a higher rate of growth inhibition, compared with that in either the Livin shRNA or Survivin shRNA transfection group (P<0.05).

In order to evaluate the antitumor effects of the shRNAs in vivo, the nude mice, which were implanted with tumor xenografts established using A549 cells, were injected with shRNA plasmids. As shown in Fig. 4, shRNA plasmid injection inhibited tumor growth significantly in the mice, compared with that in the control group, and double injection with Livin ShRNA and Survivin shRNA exerted a more marked effect, compared with that in the groups transfected with Livin shRNA or Survivin shRNA alone.

As shown in Fig. 5A, after 48 h, transfection with Livin shRNA and/or Survivin shRNA enhanced cell apoptosis. The apoptotic rate was 55.40±5.48% in the co-transfection group, which was higher than that in either the Livin shRNA transfection group (37.80±4.11%) or the Survivin shRNA transfection group (42.61±3.99%; P<0.05). This indicated that Livin and Survivin shRNA co-transfection was more effective in promoting apoptosis.

As shown in Fig. 5B, the injection of Livin shRNA and/or Survivin shRNA induced apoptosis in the tumor tissues, and the number of apoptotic cells in the Livin shRNA and Survivin shRNA double injection group was higher, compared with the number of apoptotic cells in either the Livin shRNA injection group or the Survivin shRNA injection group (P<0.05). This suggested that Livin shRNA and Survivin shRNA double injection was more effective than either Livin shRNA or Survivin shRNA in inducing apoptosis in the tumor tissues.