The A2780/CP cell line was obtained from the Institute of Biochemistry and Cell Biology (Shanghai, China). Adenovirus-mediated SVV shRNA vector, negative control vector and virion-packaging elements were purchased from Genechem (Shanghai, China). Primers for SVV, PCNA, cleaved caspase-3 and MMP-2 were synthesized by Advanced Biotechnologies, Inc. (Columbia, MD, USA). Antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Cisplatin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Waltham, MA, USA); TRIzol reagent and Lipofectamine 2000 were from Invitrogen (Carlsbad, CA, USA); M-MLV Reverse Transcriptase was from Promega Corp. (Madison, WI, USA); SYBR-Green Master Mixture was from Takara Bio, Inc. (Shiga, Japan). A cell apoptosis kit, PI, RNase A and Annexin V-FITC were obtained from KeyGEN Biology (Nanjing, China).

A2780/CP cells were cultured in DMEM supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were placed in a humidified atmosphere containing 5% CO₂ at 37°C. Ad5-SVV and negative control adenovirus were used to transfect A2780/CP cells. Cells were subcultured at a 1:5 dilution in 300 μg/ml G418-containing medium. Positive stable transfectants were selected and expanded for further analysis. The clone in which Ad5-SVV vectors were transfected was designated as Ad5-SVV, the negative control vectors transfected were designated as NC, and A2780/CP cells were designated as CON.

To quantitatively determine the mRNA expression level of SVV in A2780/CP cell lines, real-time PCR was performed. Total RNA from each clone was extracted with TRIzol according to the manufacturer’s instructions. Reverse transcription was performed using M-MLV and cDNA amplification was carried out using a SYBR Green Master Mix kit according to the manufacturer’s instructions. The SVV gene was amplified using specific oligonucleotide primers and the human GAPDH gene was used as an endogenous control. PCR primer sequences were as follows: SVV, 5′-ACCAGGTGAGAAGTGAGGGA-3′ and 5′-AACAGTAGAGGAGCCAGGGA-3′; PCNA, 5′-CCATCC TCAAGAAGGTGTTGG-3′ and 5′-GTGTCCCATATCCGC AATTTTAT-3′; cleaved caspase-3, 5′-AGAGGGGATCGTTG TAGAAG-3′ and 5′-GTTGCCACCTTTCGGTTAAC-3′; MMP-2, 5′-GGCCCTGTCACTCCTGAGAT-3′ and 5′-GGC ATCCAGGTTATCGGGGA-3′; GAPDH, 5′-CAACGAATT TGGCTACAGCA-3′ and 5′-AGGGGTCTACATGGCAA CTG-3′. Data were analyzed using the comparative Ct method (2^−ΔΔCt). Three separate experiments were performed for each clone.

A2780/CP cells were harvested and extracted using lysis buffer (Tris-HCl, SDS, mercaptoethanol, glycerol). Cell extracts were boiled for 5 min in loading buffer and then equal amounts of cell extracts were separated by 15% SDS-PAGE. Separated protein bands were transferred onto polyvinylidene fluoride membranes and the membranes were blocked in 5% skimmed milk powder. Primary antibodies against SVV, PCNA, pro-caspase-3, cleaved caspase-3 and MMP-2 were diluted according to the manufacturer’s instructions and applied to membranes overnight at 4°C. Following this, horseradish peroxidase-linked secondary antibodies were added at a dilution ratio of 1:1,000 and incubated at room temperature for 2 h. Membranes were washed with PBS three times and the immunoreactive bands were visualized using ECL-PLUS kit according to the manufacturer’s instructions. Relative protein levels in various cell lines were normalized to GAPDH concentration. Three separate experiments were performed for each clone.

Cell proliferation was analyzed with the MTT assay. Briefly, cells infected with ad5-SVV virus were incubated in 96-well plates at a density of 1×10⁵ cells/well with DMEM supplemented with 10% FBS. Cells were treated with 20 μl MTT dye at 0, 24, 48, 72, 96 and 120 h and then incubated with 150 μl DMSO for 5 min. The color reaction was measured at 570 nm with an enzyme immunoassay analyzer (Bio-Rad, Hercules, CA, USA). Proliferation activity was calculated for each clone.

Transwell filters were coated with matrigel (3.9 μg/μl, 60–80 μl) on the upper surface of a polycarbonic membrane (diameter, 6.5 mm; pore size, 8 μm). Following incubation at 37°C for 30 min, the matrigel solidified and served as the extracellular matrix for analysis of tumor cell invasion. Harvested cells (1×10⁵) in 100 μl DMEM were added into the upper compartment of the chamber. A total of 200 μl conditioned medium derived from NIH3T3 cells was used as a source of chemoattractant and was placed in the bottom compartment of the chamber. Following 24 h incubation at 37°C with 5% CO₂, the medium was removed from the upper chamber. The non-invaded cells on the upper side of the chamber were scraped off with a cotton swab. Cells that had migrated from the matrigel into the pores of the inserted filter were fixed with 100% methanol, stained with hematoxylin and mounted and dried at 80°C for 30 min. The number of cells invading through the matrigel was counted in three randomly selected visual fields from the central and peripheral portion of the filter using an inverted microscope (x200 magnification). Each assay was repeated three times.

To detect cell apoptosis, A2780/CP cells treated with ad5-SVV virus were trypsinized, washed with cold PBS and resuspended in binding buffer according to the manufacturer’s instructions. FITC-Annexin V and PI were added to the fixed cells for 20 min in the dark at room temperature. Following this, Annexin V binding buffer was added to the mixture prior to fluorescence measurement by FACsort flow cytometer. Cell apoptosis was analyzed using CellQuest software (BD Biosciences, CA, USA). Three separate experiments were performed for each clone.

Results of each experiment were presented as the mean ± SD, where applicable. Statistically significant difference in each assay was determined by SPSS version 11.5. Difference in each group was tested for significance using the ANOVA analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

To examine whether SVV was knocked down by ad5-SVV and its effect on PCNA, cleaved caspase-3 and MMP-2 mRNA expression in A2780/CP cells, the expression levels of SVV, PCNA, cleaved caspase-3 and MMP-2 mRNA were measured by real-time PCR. As is evident in Fig. 1A, expression levels of SVV, PCNA and MMP-2 mRNA were decreased, while the expression level of cleaved caspase-3 was increased in ad5-SVV compared with the NC and CON groups (each P<0.01). Western blot analysis of SVV protein expression, revealed a marked downregulation in the ad5-SVV group compared with the NC and CON groups (P<0.01; Fig. 1B and C).

The effect of SVV knockdown on proliferation and cisplatin sensitivity to A2780/CP cells by MTT was investigated. The results demonstrate that SVV knockdown was identified to significantly inhibit the proliferation of A2780/CP cells in a time-dependent manner, compared with NC and CON groups (Fig. 2A). PCNA is important for a number of essential cell processes, tumor progression and the outcome of anticancer treatment. To determine whether SVV knockdown suppressed endogenous PCNA expression through translational repression, the expression of PCNA protein was examined by western blot analysis, which revealed a decreased amount of PCNA protein in the ad5-SVV compared with the NC and CON groups (P<0.01; Fig. 2B and C).

Following this, we pretreated A2780/CP cells in ad5-SVV, NC and CON groups with various concentrations of cisplatin (0.01, 0.1, 1, 10 and 100 μg/ml) and investigated the effect of SVV knockdown on cisplatin sensitivity to A2780/CP cells. Knockdown of SVV markedly enhanced the inhibitory effect and chemotherapeutic sensitivity of cisplatin on A2780/CP cells (each P<0.01) (Table I).

To determine the effect of SVV knockdown on A2780/CP cell apoptosis, flow cytometric analysis with FITC/PI-Annexin V staining was performed. As demonstrated in Fig. 3A, compared with NC and CON, the number of apoptotic cells significantly increased and morphological changes, including nuclear condensation, nuclear debris, foam and apoptotic body formation, were identified in the ad5-SVV group. In addition, the apoptotic index of A2780/CP cells in the ad5-SVV group was markedly higher than the NC and CON groups (P<0.01; Fig. 3B and C). Western blot analysis was performed to investigate the effect of SVV knockdown on the endogenous expression of pro-caspase-3 and cleaved caspase-3 protein. As shown in Fig. 4, pro-caspase-3 and cleaved caspase-3 expression was identified as significantly elevated in the ad5-SVV group, compared with the NC and CON groups (each P<0.01).

To determine the effect of SVV knockdown on A2780/CP cell invasion, a Transwell assay was performed. Invasive and metastatic potential in the Transwell assay was determined on the basis of the ability of cells to invade a matrix barrier containing laminin and type IV collagen, the major components of the basement membrane. Representative micrographs of Transwell filters are shown in Fig. 5A. The invasive potential of A2780/CP cells was markedly decreased in the ad5-SVV group, compared with the NC and CON groups (P<0.01; Fig. 5C). Western blot analysis was performed to investigate the effect of SVV knockdown on the endogenous expression of MMP-2 protein. As demonstrated in Fig. 5B and D, MMP-2 expression was identified as significantly reduced in the ad5-SVV group, compared with the NC and CON groups (P<0.01).