In accordance with GenBank, the common sequences of isomers (α, NM_139317 and β, NM_022161) of human Livin (AF311388) designed and synthesized by Shanghai GenPharma Company (Shanghai, China) were sense, 5′-GAGAGAGGUCCAGUCUGAATT-3′ and antisense, 5′-UUCAGACUGGACCUCUCUCTT-3′. Non-targeting homologous genes were excluded through comparison with BLAST. Non-human complementary siRNA sequences (sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-ACGUGACACGUUCGGAGAATT-3′) were used as negative controls and labeled with FAM fluorescence (negative control FAM).

EJ human bladder carcinoma cells (Nanjing KeyGen BioTech. Co. Ltd., Nanjing, China) were cultured in a 5% CO₂ incubator at 37°C with RPMI-1640 medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. EJ cells from the logarithmic phase were inoculated in 6-well or 96-well plates containing RPMI-1640 medium. When the confluence reached 40–60%, the cells were assigned into different groups according to treatment: blank control group (without treatment), intervention group [with the same volume of siRNA and Lipofectamine® 2000 reagent (Invitrogen Life Technologies, Carlsbad, CA, USA)], chemotherapy group [24 h treatment with 0.1 mg/ml MMC (Sigma-Aldrich, St. Louis, MO, USA)], or combination group (24 h siRNA transfection and 24 h treatment with 0.1 mg/ml MMC). Cells were transfected with negative siRNA or with Lipofectamine 2000 reagent alone. The transfection rate of cells was observed under inverted fluorescence microscopy after 24–48 h (Fig. 1). Our preliminary experiments revealed that the transfection rate in the 6-well plate reached 70% under conditions of 3×10⁵ cells/well, 100 pmol/well siRNA and 5 µl/well Lipofectamine 2000 reagent. Similarly, the transfection rate in the 96-well plate reached 70% under conditions of 4×10³ cells/well, 5 pmol/well siRNA and 0.25 µl/well Lipofectamine 2000 reagent.

After 24 h of transfection, the cells were rinsed with phosphate-buffered saline (PBS) twice, then 1 ml TRIzol was added to the lysate cells. The total RNA was extracted according to the manual and its quantity was measured using a UV spectrophotometer (Beijing ComWin Biotech Co., Ltd., Beijing, China). cDNA was synthesized under conditions of 42°C for 40 min and 85°C for 5 min, and used as a template for the amplification of the Livin gene with pre-denaturation at 94°C for 2 min, 30 cycles of denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 30 sec, then final extension at 72°C for 2 min. The sense and antisense primers of Livin and β-actin genes were 5′-GTGGATGGGCAGATCCTG-3′ and 5′-CCTTGTCCTGATGGCCTG-3′ (resulting in the production of 214 bp), and 5′-AGGTGACAGCAGTCGGTTGG-3′ and 5′-CGAAGGCTCATCATTCAAAA-3′ (resulting in the production of 300 bp), respectively, synthesized by Beijing AuGCT DNA-SYN Biotechnology Co. Ltd. (Beijing, China). Following the reaction, the PCR production was identified with electrophoresis in 1.5% sucrose and analyzed with a SensiAnsys gel-imaging system (LI-COR Biosciences, Lincoln, NE, USA). The expression of Livin mRNA was semi-quantitatively calculated using the optical density ratio of Livin and β-actin. The experiment was repeated three times.

Following 48 h of transfection, the EJ cells were removed from the medium, rinsed twice with pre-cooled PBS, mixed with 100 µl RIPA lysis buffer and 10 µl PMSF in an ice-bath for 10 min, and centrifuged at 12,000 rpm at 4°C for 10 min. The total cellular protein was collected and quantified with the bicinchoninic acid assay. Then 30 µg protein was loaded onto 12% polyacrylamide gel for electrophoresis to isolate the protein, which was transferred to a polyvinylidene fluoride membrane using the semi-dry method and blocked with Tris-buffered saline and Tween-20 (TBST) solution containing 5% skimmed milk at room temperature for 2 h. Primary Livin antibody (1:500) and β-actin antibody (1:1000; both from Beijing ComWin Biotech Co., Ltd.) were added for incubation at 4°C overnight. The membrane was rinsed with TBST three times and horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit 1:2000; Beijing ComWin Biotech Co., Ltd.) were added for incubation at room temperature for 1 h. After washing, the membrane was developed with the enhanced chemiluminescence method and exposed under a photographic system. The optical density of Livin protein was compared with that of β-actin as an internal reference. The experiment was repeated three times.

The cells were inoculated in 96-well plates at 100 µl/well with five repetition wells for each group, and cultured in a 5% CO₂ incubator at 37°C. Then the medium was replaced by 10 µl CCK-8 (Shanghai Yeasen Biotech Company, Shanghai, China) for 2 h incubation and the absorption (D) was measured at 450 nm to calculate the relative inhibition rate of cell proliferation according to the formula: inhibition rate (%) = [(D in control group - D in experimental groups) / D in control group] ×100%. The experiment was repeated three times.

EJ cells were inoculated in six-well plates as above. Following treatment, the cells were digested with ethylenediaminetetraacetic acid-free trypsin and centrifuged at 2,000 rpm at room temperature for 5 min. Then the cells were re-suspended with 1X PBS (4°C) and centrifuged. After removal of the supernatant, the cells were mixed with 300 µl 1X binding buffer and 5 µl Annexin V-FITC (Beijing Jiamay Biotech, Beijing, China), gently agitated and incubated at room temperature in the dark for 45 min. Then the cell suspension was filtered with a 300-mesh nylon net into the flow tube. A total of 5 µl propidium iodide and 200 µl 1X binding buffer was added 5 min prior to and immediately before loading. The apoptosis was measured by BD FACSAria-III (Beijing Jiamay Biotech) and analyzed with Modfit LT3.3 (Verity Software House, Topsham, ME, USA). The experiment was repeated three times.

All data were expressed as the means ± standard deviation. SPSS 19.0 (IBM SPSS, Armonk, NY, USA) was used for analysis with one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

After 24 h of transfection with siRNA Livin, the expression intensity of Livin mRNA in the blank control, negative control, liposome and intervention group (siRNA Livin) was 0.271±0.032, 0.301±0.043, 0.292±0.036 and 0.024±0.011, respectively, indicating that siRNA Livin significantly inhibited the expression of Livin mRNA (P<0.01). There was no significant difference between the blank control, negative control and liposome group (P>0.05, Fig. 2A).

Western blot analysis further indicated that the Livin protein in EJ cells was significantly decreased following the decrease of gene transcription. After 48 h of transfection, the relative expression of Livin protein in the blank control, negative control, liposome and intervention group (siRNA Livin) was 0.515±0.038, 0.539±0.047, 0.521±0.043 and 0.070±0.016, respectively. The statistic analysis indicated that Livin protein in EJ cells was significantly decreased following transfection of siRNA Livin (P<0.01). There was no significant difference between the blank control, negative control and liposome group (P>0.05, Fig. 2B).

Measurement of CCK-8 indicated that the proliferation of EJ cells was inhibited by siRNA Livin. The inhibition in the combination group was significantly higher than in the single intervention and single chemotherapy group (P<0.01, Table I).

The flow cytometry assessment indicated that apoptosis was promoted in the intervention group (15.29±1.64%), chemotherapy group (17.57±0.82%) and combination group (40.19±1.53%), when compared with the blank control group (1.18±0.47%; P<0.01). Furthermore, the apoptotic rate in the combination group was significantly higher than in the single intervention and single chemotherapy group (P<0.01, Fig. 3).