The human lung adenocarcinoma SPC-A1 cell line was acquired from Nanjing KeyGen Biotech., Co., Ltd. (Nanjing, China). The primers for livin siRNA and control siRNA were synthesized by Beijing Genomics Institute (Beijing, China). All antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco-BRL (Carlsbad, CA, USA). TRIzol reagent and Lipofectamine 2000 were obtained from Invitrogen Life Technologies (Carlsbad, CA, USA). Reverse Transcriptase SYBR Green Master mixture was acquired from Takara Bio, Inc. (Otsu, Japan). The bicinchoninic acid protein assay kit and ECL-Plus kit were purchased from Thermo Scientific (Rockford, IL, USA). The MTT cell proliferation assay kit was purchased from Sangon Biotech (Shanghai, China) and TRAIL was purchased from Merck Millipore (Darmstadt, Germany). In addition, flavopiridol was obtained from Sigma-Aldrich (St. Louis, MO, USA) and Z-VAD-FMK was purchased from R&D Systems (Minneapolis, MN, USA).

SPC-A1 cells were cultured in DMEM medium supplemented with 10% heat-inactivated FBS. All were placed in a humidified incubator, containing 5% CO₂ at 37°C. SPC-A1 cells were replated at 2×10⁵ cells/well in six-well plates once they had reached exponential phase. When the cell density reached 40–50%, cells were transfected with Lipofectamine 2000 with a pcDNA3.1 expression vector (Invitrogen Life Technologies) and cultured at 37°C and 5% CO₂ for 24 h. The clone in which the livin-siRNA was transfected was termed the livin-siRNA group, the group transfected with the negative control vector was termed the negative control (NC) group and SPC-A1 cells were termed the control (CON) group. The following RNA silencing sequences were used: siRNA-livin790, forward 5′-GAGAGGUCCAGUCUGAAAG-3′ and reverse 5′-CUUUCAGACUGGACCUCUC-3′ and siRNA-livin180, forward 5′-CCUAAAGACAGUGCCAAGU-3′ and reverse 5′-ACUUGGCACUGUCUUUAGG-3′.

Total RNA was isolated with TRIzol reagent and reverse-transcribed to synthesize cDNA. cDNA was subsequently amplified by SYBR-Green based qPCR using the following primers: Livin, forward 5′-GGAGAGAGGTCCAGTCTGAAAGT-3′ and reverse 5′-ACCTTGCACGTCCTCTCCTC-3′ and Homosapiens histone acetyltransferase (HBOA), forward 5′-ATCAAAGAAATCAGTCAGGAGACG-3′ and reverse 5′-CTCTTTGGCTATCCACTCATCAAT-3′. The 25 μl fluorescent PCR reaction mix contained: 8.5 μl ddH₂O, 12.5 μl 2X SYBR Premix Ex Taq II, 2 μl cDNA and 1.0 μl forward and reverse primers (10 μmol/l), respectively. The cycling parameters were as follows: 40 cycles, including denaturation at 95°C for 5 sec, annealing at 60°C for 30 sec and extension at 72°C for 30 sec. Melting curve analysis was used to confirm the primer specificity. The comparative cycle threshold (Ct) method was used for calculation of livin mRNA expression. The calculation methods were as follows: ΔCt=Ct (Livin) − Ct (HBOA), ΔΔCt=ΔCt (Treatment) − ΔCt (Control). ΔΔCt mean values were compared between groups. The relative mRNA expression was expressed in 2^−ΔΔCt.

SPC-A1 cells were harvested after 48 h of transfection and extracted using lysis buffer. Cell extracts were separated on 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Separated protein bands were electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Finally, 5% skimmed milk powder was used to block the PVDF membranes for 1 h. The membrane was incubated overnight at 4°C with a 1:100 dilution of mouse monoclonal anti-livin primary antibody (sc-166390; Santa Cruz Biotechnology, Inc.). The following day, goat anti-mouse Immunoglobulin G horesradish peroxidase-conjugated secondary antibodies (sc-2005; Santa Cruz Biotechnology, Inc.) were added at a dilution of 1:2,000 and the mixture was incubated for 2 h at room temperature. PVDF membranes were washed in phosphate-buffered saline four times. The ECL-Plus kit was used to visualize the immunoreactive bands. Relative protein level was normalized by β-actin concentration. Three separate experiments were performed in duplicate for each treatment.

Cell viability was assessed using an MTT assay. Cells were cultured in 24 well plates at a concentration of 5x10⁴ cells per well and allowed to adhere. SPC-A1 cells were termed the CON group. The group transfected with the negative control vector was termed the NC group. The group transfected with the livin-siRNA vector was termed the livin-siRNA group. The group initially transfected with livin-siRNA and then treated with 1.2 μg/ml cisplatin was termed the livin-siRNA+cisplatin group. The FP group was treated with 100 nmol/l flavopiridol and the T group was treated with 100 ng/ml TRAIL. The cisplatin treatment group was treated with 1.2 μg/ml cisplatin. The F+T treatment group was treated with 100 nmol/l flavopiridol and 100 ng/ml TRAIL. The FP+T+cisplatin treatment group was treated with 100 nmol/l FP, 100 ng/ml TRAIL and 1.2 μg/ml cisplatin. The Z-VAD-FMK group was treated with flavopiridol, 50 μmol/l Z-VAD-FMK and TRAIL. Following treatment for 24 h, 100 μl MTT (0.5 mg/ml) was added to the cells and the mixture was incubated for 4 h at 37°C. Subsequently, the supernatant was removed, dimethyl sulfoxide was used to dissolve the resultant formazan crystals and the absorbance value was read at 570 nm using a Smartspec 3000 spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

SPSS 13.0 (SPSS, Inc., Chicago, IL, USA) was used for the data analysis. All experiments were repeated three times and data are presented as the mean ± standard deviation. Differences between groups were analyzed using one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

The protein expression of livin was investigated using western blot analysis in several cell lines (SPC-A1, A549, Hale and MCF-7). As shown in Fig. 1, livin protein expression was only observed in SPC-A1 cells.

The knockdown efficiency of two candidate siRNA (siRNA-livin180 and siRNA-livin790) was evaluated using RT-qPCR. The results revealed that the livin mRNA level in cells transfected with siRNA-livin790 and siRNA-livin180 were significantly decreased to 52 and 64.4%, respectively (Fig. 2). The most significant silencing effect was observed with siRNA-livin790, therefore, the siRNA-livin790 transfection group was used in the follow-up experiment.

The synergistic inhibitory effect between flavopiridol and TRAIL on livin protein expression was evaluated by western blot analysis. Compared with either flavopiridol or TRAIL alone, combining flavopiridol with TRAIL significantly decreased the protein expression level of livin (Fig. 3). No significant difference was identified between flavopiridol and TRAIL treatment groups.

The result of western blot analysis demonstrated that the synergistic inhibitory effect between flavopiridol and TRAIL may effectively inhibit the protein expression of livin, while the caspase inhibitor Z-VAD-FMK reversed this inhibitory effect. As shown in Fig. 4, expression of livin protein in the T+FP+Z-VAD treatment group was significantly higher than the T+FP group and no significant difference was identified between the control group and T+FP+Z-VAD treatment group.

To elucidate the effect of livin inhibition on tumor growth in SPC-A1 cells, an MTT assay was used to evaluate the tumor cell proliferative activities. The survival rate of each group was as follows: Flavopiridol group, 84.30±1.34%; TRAIL group, 93.40±1.56%; F+T combination group, 48.02±1.35%; siRNA-livin group, 50.88±1.14%; cisplatin group, 19.30±0.89%; siRNA-livin+cisplatin group, 14.37±0.81%; FP+T+cisplatin group, 10.86±0.87% and the Z-VAD-FMK group, 88.16±1.64%. The data were normalized to the control group (Fig. 5). As illustrated in Fig. 5, suppression of livin resulted in a significant decrease in the proliferation rate of SPC-A1 cells at 48 h and this suppression effect unexpectedly enhanced the sensitivity of cells to the chemotherapy drug cisplatin.