Frozen tumor specimens were obtained from 50 patients with EOC. The median patient age was 56 years (range 38–82). The histological type and histological grade were as follows: subtype (serous, 22 cases; mucinous, 12 cases; endometrioid, 8 cases; clear cell, 8 cases), and grade (G1, 15 cases; G2, 10 cases; G3, 35 cases). Clinical stages, determined according to the FIGO system (International Federation of Gynecologists and Obstetricians) were stage I–II in 12 patients, stage III–IV in 38 patients. Twenty samples of normal ovarian tissues were obtained from patients undergoing ovarian biopsy or ovariotomy (patients suffering from uterine fibroids, dysfunctional uterine bleeding or uterine prolapse underwent hysterectomy meanwhile, pathologically precluded abnormality in ovarian tissues), which aged ranging from 42 to 68 years (median age 49). Twenty samples of benign ovary tumor were obtained from patients (15 cases of serous cystadenoma and 5 cases of mucinous cystadenoma) aged ranging from 24 to 58 years (median age 43). These samples were collected from patients admitted to the Shengjing Hospital of China Medical University (Shenyang, China). None of the patients had received chemotherapy, radiotherapy or immunotherapy. This work was approved by the ethical committee of the China Medical University. Informed consent was obtained from each patient.

Total RNAs were extracted from tissue samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The amount of RNA was quantified in duplicate using UV absorbance at 260 nm. Equal amounts of total RNA was reverse-transcribed into cDNA using the Takara RNA PCR kit (DRR037, Takara, Dalian, China) in a final volume of 20 μl following the manufacturer’s protocol. To minimize variation in the reverse transcription reaction, all RNA samples from a single experimental setup were reverse transcribed simultaneously. The PCR primers were as follows: for livin (GenBank: AF311388) specific primers discriminating between the α- and the β-variants were: sense, 5′-AGTTCCTGCTCCGGTCAAA-3′; antisense, 5′-GCACGGCACAAAGACGAT-3′, which yields two products of 347 bp and 293 bp, respectively; and for β-actin (GenBank: NM_001101) (internal control), sense 5′-GATTGGCTCAGGACATTTCTG-3′ antisense 5′-GATTGCTCAGGACATTTCTG-3′ (751 bp). The amplification conditions were as follows: denaturation at 94°C for 5 min; 32 cycles of 94°C for 40 sec, 55°C (for livin) and 52°C (for β-actin) for 1 min, 72°C for 1 min; and a final 5 min extension at 72°C. The PCR products were separated on 2% agarose gels, and the bands were visualized by ethidium bromide and photographed under UV light.

Frozen tissues were washed with ice-cold PBS and homogenized in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1 mM PMSF, 0.1% Nonidet P-40, 0.1% Triton X-100, 50 mM NaF, 0.5 mM Na₃VO₄). Lysates were centrifuged at 12,000 × g for 20 min at 4°C and supernatants collected. Protein concentrations were determined using Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of proteins were separated on 12% SDS-polyacrylamide gel electrophoresis and transfered onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was then blocked with 5% nonfat milk and immunoprobed with rabbit polyclonal anti-livin antibody (IMG-347, Imgenex, San Diego, CA, USA), followed by exposed to horseradish peroxidase-conjugated secondary antibody and visualized using ECL plus reagent (GE Healthcare, Piscataway, NJ, USA). Quantity One software (Bio-Rad) was applied for analysis of the optical density of the protein bands. The relative expression quantity of livin protein was illustrated as the percentage of the optical density (OD) of livin protein, adjusted with the corresponding β-actin OD.

Four non-overlapping segments which are located at 786–804, 647–665, 609–627 and 519–537 position in livin cDNA (NM_022161.2) were selected as candidate targets using Target Finder and Design Tool of Ambion (http://www.ambion.com/techlib/misc/siRNA_tools.html). The dsDNA sequences simultaneously aimed at both of the two variants. Another shRNA of nonspecific sequence was used as a control (Table I). BLAST search of the human genome database was carried out and found no homology with other human genes. Then, these shRNAs were subcloned into lentiviral vector pGCL-GFP (GeneChem, Shanghai, China) plasmid between the HpaI and XhoI enzyme sites and the recombinants generated were named Psi-1, Psi-2, Psi-3, Psi-4 and Psi-NC. The inserts in those recombinants were confirmed by DNA sequencing (data not presented).

Ovarian cancer cell lines SKOV3 cells (purchased from Cancer Institute, Chinese Academy of Medical Sciences) were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS) and seeded in 6-well plates and allowed overnight growth to reach 70–80% confluence. Cells were then transfected with each of the siRNA recombinants using Lipofectamine™ 2000 reagent (Invitrogen). For confirmation of downregulation of livin gene, after 72 h, cells were harvested and analyzed for reduction of livin expression by real-time RT-PCR and immunoblotting. The lentiviral vector giving maximal knockdown was selected and packaged by the 293T packaging cell and the vector particles were concentrated. During the transfection protocol, the most suitable MOI (multiplicity of infection) of lentivirus is MOI 20. Controls consisted of either Lipofectamine-treated (untreated) or control siRNA-treated cells (MOI 20) alone.

Total RNA was extracted from treated cells and reverse transcripted into cDNA as mentioned above. The real-time PCR primers were as follows: Livin: sense, 5′-GCGTCTGGCCTCCTTCTATG-3′ antisense, 5′-AAGCACCTCACCTTGTCCTG-3′ (108 bp), which did not discriminate between the two livin isoforms. β-actin: sense, 5′-GTGGACATCCGCAAAGAC-3′; antisense 5′-AAAGGGTGTAACGCAACTA-3′ (302 bp). Untreated group values were used as calibrator. The relative level was calculated using the 2^−ΔΔCt method after normalization with β-actin as a housekeeping gene (25).

Treated cells were harvested and protein was extracted, separated and transferred onto PVDF membrane as described earlier. Filters were probed with either rabbit polyclonal anti-active caspase 9 antibody (ab25759, Abcam, Cambridge, MA, USA), anti-active caspase 7 antibody (ab2323, Abcam) or anti-active caspase 3 antibody (ab2302, Abcam) followed by secondary antibody and developed by ECL staining.

Cell proliferation was investigated by colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). In brief, SKOV3 cells were plated in a 96-well flat-bottomed plate at 5×10³ cells/well in triplicate and cultured in medium for 24 h. Then the cells were treated with lentiviral livin shRNA or control siRNA for 24, 48, 72 and 96 h. On each experimental point, 20 μl MTT (5 mg/ml in PBS) was added to each well, and the cells were incubated at 37°C for additional 4 h. Then the reaction was stopped by lysing the cells with 150 μl DMSO for 5 min. The absorbance of each well were measured at 570 nm using a microtitre plate reader (Labsystems MK3, Finland). The data were normalized to the untreated group. All experimental points were set up in three replicate wells and independently performed three times.

Cell apoptosis assay were performed by flow cytometry after 72 h since the cells were transfected with siRNAs. Briefly, cells were seeded in 6-well plates in triplicate at 1×10⁵ cells/well and transfected as described earlier. At 72 h post-transfection, the cells were harvested, washed twice with cold PBS, suspended in 1X binding buffer [10 mM HEPES (pH 7.4), 150 mM NaCl, 2.5 mM CaCl₂, 1 mM MgCl₂, 4% BSA] at a concentration of 1×10⁶ cells/ml. Cells were incubated in the dark for 15 min at room temperature with 5 μl Annexin V-FITC (BD Biosciences Pharmingen, Allschwill, Switzerland). Before analysis, 2.5 μl of 7-AAD was added to the samples in a final volume of 200 μl. A total of 10,000 cells were accumulated with a BD FACSCaliburH apparatus and data were analyzed using the CellQuest software. Apoptotic cells were determined by Annexin V-positive and 7-AAD negative cells.

Cells were treated with lentiviral siRNA for 48 h, and then with seven concentration gradient of DPP in PBS (100, 50, 25, 12.5, 6.25, 3.13, 1.56 mg/l) for 24 h; the effects on cell growth were examined by MTT assay as described earlier, the growth inhibition was calculated according to the following formula: Inhibitory ratio (%) = [1− (OD of the lentiviral siRNA/OD of the untreated) ×100%]. After treated with siRNA plus DPP (5 mg/l), flow cytometry was performed to detect cells apoptosis, and active caspase 9, 7, 3 were determined as indicated above.

All experiments were repeated in triplicate and data were expressed as mean ± standard deviation of the mean (SD). Statistical significance was assessed by one-way ANOVA followed by Bonferroni multiple comparison post-tests. Statistical analyzes were performed using SPSS 13.0 package (SPSS, Chicago, IL, USA). p<0.05 was considered significant.

Livin mRNA was detected in 32 of 50 (64%) EOC tumor specimens, 3 of 20 (15%) benign tumors, 2 of 20 (10%) normal ovary tissues, respectively (χ²=24.448, P<0.01) (Fig. 1A). The expression levels of total livin differed greatly among the 32 positive tumor samples; with no obvious difference between two isoforms (data not shown) (Fig. 1B). The mean livin mRNA value among the 32 positive cases was set as 1.0. High expression levels (defined as values greater than the mean positive tumor level) were observed in 16 tumors. These included 4/12 (33%) patients with advanced stage (III and IV) disease, 12/38 (32%) patients with localized stage (I and II) disease. No obvious link was observed between the expression levels with tumor stage or tumor grade for the limited numbers of tissue samples investigated here. Generally, comparable changes in livin protein concentrations were also observed. Thirty-two of the 50 (64%) tumor specimens assessed by immunoblotting were positive for livin protein; all of these specimens were also positive by RT-PCR (Fig. 1C). Conversely, all the specimens negative by RT-PCR were also negative for livin protein.

SKOV3 cells were transfected with different shRNA constructs to screened effective targets of livin and obtained the optimum concentration for lentivirus transfection. The knockdown efficiency of four candidate shRNA was evaluated using western blotting. The results disclosed that the best knockdown effect was with Psi-4 siRNA at MOI 20 (data not shown). At 72 h post-transfection with Psi-4 siRNA more than 90% of the survived cells were GFP-positive (Fig. 2A). Real-time RT-PCR analyses performed at 72 h post-transfection showed that livin mRNA levels were significantly reduced by 70% when compared with control transfections (Fig. 2B). To correlate the decrease in livin mRNA expression with livin protein levels, immunoblotting analysis was performed at 72 h after siRNA treatment. The protein levels were reduced, thereby confirming efficient knockdown (Fig. 2C).

We first tested the effect of livin siRNA on SKOV-3 cell proliferation by a colorimetric assay using MTT, and the inhibition rate was calculated with the method above. The results showed that silencing of livin gene has substantially effect on SKOV3 cells proliferation compared with control groups (both P<0.05) (Fig. 3A).

The cell apoptosis was measured by flow cytometry at 72 h after transfection; apoptosis cells were determined by Annexin V-positive and 7-AAD-negative cells (Fig. 3B and C). The results demonstrated that the apoptosis ratio of livin siRNA cells obviously increased compared with control groups (both P<0.05).

We further investigated the cleavage of several molecular markers of mitochondrial apoptotic signaling pathway, including caspase 9, caspase 3, and caspase 7 by immunoblotting after SKOV-3 cells were stably transfected with livin siRNA for 72 h. As shown in Fig. 4, the results showed that in the livin siRNA cells, cleavage of caspase 9, caspase 3 and caspase 7 was remarkably increased, compared with control groups (all P<0.05).

Livin siRNA- or control siRNA-transfected cells were treated separately with DPP for various concentrations and the growth inhibition curves are shown in Fig. 5A. The results showed that the silencing of livin expression resulting in strikingly higher cell growth inhibition at different drug concentrations. The IC50 for untreated and control-siRNA were 13.96 and 14.58, but the IC50 for livin-siRNA dropped to 6.67 (P<0.05).

Cell apoptosis were determined by Annexin-V/7AAD assay (Fig. 5B). In SKOV3 cells, treatment with livin siRNA plus DPP (5 mg/l) resulted in a significantly higher apoptosis proportion (29.89±3.29) compared to control siRNA plus DPP (7.62±0.95) or DPP alone (6.66±0.69) (both P<0.05). These results suggest that livin inhibition by lentiviral shRNA resulted in remarkable enhancement in chemosensitivity of DPP for SKOV3 cells.

Before livin inhibition, treatment with DPP alone resulted in activation of apoptosis signaling pathway with increased cleavage of caspase 9, caspase 3 and caspase 7 in SKOV3 cells. Targeted inhibition of livin gene existed synergistic effect on induction of apoptosis in SKOV3 cells, as evidenced by a dramatical enhancement of caspase 9, 7 and 3 cleavages compared with control siRNA-treated cells after treatment with DPP (Fig. 6).