The human EOC cell lines A2780 and CAOV3 were purchased from the China Center for Type Culture Collection (CCTCC, Wuhan University, China). A cisplatin-resistant subline (A2780/DDP) was generated and maintained by our laboratory (12). Cisplatin was purchased from Shandong Qilu Pharmaceutical Factory. All cell lines were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal calf serum (FCS; Gibco; Thermo Fisher Scientific, Inc.). Cells were maintained in a 37°C humidified incubator with an atmosphere of 5% CO₂. A2780/DDP cells were cultured without cisplatin for more than 1 month prior to use in this study to exclude stress reactions mediated by drug treatment.

The Dicer-shRNA and control-shRNA vectors were purchased from Shanghai GenePharma (Shanghai, China). The target sequence for Dicer-shRNA was 5′-GCCAAGGAAATCAGCTAAATT-3′. A2780 and CAOV3 cells were seeded in a six-well plate at a concentration of 5×10⁵ cells per well. Transfection was performed via lentivirus infection according to the manufacturer's instructions. Then, the cells were selected by 2 µg/ml puromycin until resistant cell colonies were obtained. The stably transfected cells were harvested 48 h after transfection and used for further assays. To verify the knockdown efficiency, mRNA and protein levels in the stably transfected cells were analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot assays.

Total RNA was extracted from the cell lines using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and cDNA was synthesized using an RT kit (Toyobo Life Science, Osaka, Japan) according to the manufacturer's protocol. Dicer and β-actin were measured by a SYBR Green (Takara Bio, Inc., Otsu, Japan) qPCR assay. The sequences of the Dicer primers were as follows: Upstream, 5′-GTGGTTCGTTTTGATTTGCCC-3′ and downstream, 5′-CGTGTTGATTGTGACTCGTGGA-3′ (NM_001195573.1). β-actin was used for normalization, and the primer sequences were as follows: Upstream, 5′-GCCAACACAGTGCTGTCTGG-3′ and downstream, 5′-GCTCAGGAGGAGCAATGATCTTG-3′. Dicer was amplified under the following qPCR reaction conditions: Initial denaturation at 95°C for 1 min, followed by 40 cycles of denaturation at 95°C for 15 sec, and annealing at 62°C for 1 min. All reactions were run on an Applied Biosystems 7500 Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The 2^−ΔΔCq method was used to calculate relative changes in gene expression (13).

Cellular proteins were lysed in NP40 Cell Lysis Buffer (Beyotime Institute of Biotechnology, Haimen, China) containing 1 mM protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 4°C for 30 min. The protein concentrations were determined using the bicinchoninic acid (BCA) method. Fifty micrograms of total protein from each sample was resolved by 8% (Dicer) or 10% (P53, P63, P73, caspase-9 and caspase-3) SDS-PAGE and then was transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Membranes were incubated with mouse anti-Dicer (1:500 dilution; Abcam, Cambridge, MA, USA), rabbit anti-P53 (1:1,000 dilution; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit anti-P63 (1:500 dilution; Abcam), rabbit anti-P73 (1:500 dilution; Abcam), rabbit anti-caspase-9 (1:1,000 dilution; Cell Signaling Technology, Inc.), rabbit anti-caspase-3 (1:2,000 dilution; Cell Signaling Technology, Inc.), rabbit anti-P21 (1:1,000 dilution; Cell Signaling Technology, Inc.) and mouse anti-β-actin (1:5,000 dilution; Wuhan Boster Biological Technology, Ltd., Wuhan, China). β-Actin was used as a loading control. The primary antibodies were detected using HRP-conjugated secondary antibody (1:5,000 dilution; Wuhan Boster Biological Technology, Ltd.). The final immunoblots were detected by using an enhanced chemiluminescence system (Pierce; Thermo Fisher Scientific, Inc.). Protein bands were quantitated after scanning using Quantity One software (Bio-Rad Laboratories, Inc.).

Chemoresistance to cisplatin was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Briefly, cells were plated in triplicate in a 96-well plate at a density of 5000 cells/well. Cells were treated with cisplatin at various concentrations ranging from 2.5 to 100 µM for an additional 48 h. After that, 20 µl of MTT (Sigma-Aldrich; Merck KGaA) at a final concentration of 0.5 mg/ml was added to each well and incubated at 37°C for 6 h. Then, the cells were dissolved in 100 µl of DMSO by incubating overnight at 37°C. The absorbance at a wavelength of 570 nm was measured using an iMark microplate reader (serial no. 10601; Bio-Rad Laboratories, Inc.). The percentage of cell survival at each dose was calculated as the absorbance ratio of treated to untreated cells. The 50% inhibitory concentration (IC₅₀) values were calculated by linear interpolation. The data shown are representative of three independent experiments.

Cells were plated in triplicate in a six-well plate at a density of 1×10⁵ cells/well and incubated for 24 h. After that, A2780 cells were treated with 40 µM cisplatin, and CAOV3 cells were treated with 30 µM cisplatin. After a 48 h incubation at 37°C, the cells were fixed in 4% paraformaldehyde for 10 min at room temperature. Finally, 1 µg/ml Hoechst 33342 (Invitrogen; Thermo Fisher Scientific, Inc.) was added for 5 min. Then, images of the six-well plates mounted with emission fluorescence were acquired with a fluorescence microscope (model IX70; Olympus Corporation, Tokyo, Japan) and Image-Pro Plus software (Media Cybernetics, Inc., Rockville, MD, USA). The Hoechst reagent was taken up by nuclei, and the nuclei of apoptotic cells exhibited bright blue fluorescence. Each experiment was repeated separately three times.

Cells were plated in triplicate in a six-well plate at a density of 1×10⁵ cells/well and incubated for 24 h. After that, A2780 cells were treated with 40 µM cisplatin, and CAOV3 cells were treated with 30 µM cisplatin. After a 48 h incubation at 37°C, the cells were harvested, trypsinized and washed with cold PBS. Subsequently, the cells were stained with APC Annexin V and propidium iodide (cat. no. 640932; BioLegend, Inc., San Diego, CA, USA). The stained cells were immediately analyzed by an LSR flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), and the data were analyzed using ModFit LT software (Verity Software House, Inc., Topsham, ME, USA).

Data are presented as the mean ± standard deviation (n≥3). Statistical comparisons between groups were estimated by two-tailed Student's t-tests or one-way analysis of variance followed by Dunnett's post hoc test. Data analyses were carried out using the SPSS v.13.0 statistical software package (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To investigate whether the expression of Dicer is affected by cisplatin, we treated A2780 cells with 20 µM cisplatin for different periods of time. Western blot analyses showed that the protein expression levels of Dicer were dramatically decreased after cisplatin treatment for 12 and 24 h (Fig. 1A). To identify the role of Dicer in cisplatin resistance, first, the cisplatin sensitivities of parental A2780 cells and A2780/DDP were assessed using MTT assays. As demonstrated in Fig. 1B, the A2780/DDP cells dose-survival curve was shifted to the right, and the cells became more resistant to cisplatin. The cisplatin IC₅₀ value for the A2780/DDP cells was 3.38-fold higher than that of A2780 cells (49.31 vs. 14.61 µM; P<0.001; Fig. 1C). Then, the expression of Dicer in A2780 and A2780/DDP cells was analyzed by RT-qPCR and western blot assays. The results showed that Dicer mRNA and protein levels were significantly lower in A2780/DDP cells than in parental A2780 cells (Fig. 1D and E).

To study the effects of Dicer knockdown on cisplatin sensitivity, first, A2780 and CAOV3 cells were transfected with a shRNA plasmid against Dicer (shDicer) or a scrambled control plasmid (shNC). Compared with those in the shNC cells, Dicer mRNA levels were decreased by 77.3 and 73.09% in A2780/shDicer and CAOV3/shDicer cells, respectively (P<0.01; Fig. 2A). Similarly, western blot analyses confirmed Dicer knockdown after shDicer transfection (Fig. 2B). Then, the sensitivities of parental control, shNC and shDicer cells to cisplatin were assessed by MTT assays. The results showed that the dose-survival curves in the A2780/shDicer and CAOV3/shDicer cells were shifted to the right, and the cells became more resistant to cisplatin than the shNC cells (Fig. 2C and D). In addition, compared with those of the corresponding shNC cells, the IC₅₀ values for cisplatin were increased by 2.37- and 1.68-fold in the A2780/shDicer (14.42 vs. 34.2 µM; P<0.001, Fig. 2D) and CAOV3/shDicer cells (20.34 vs. 34.2 µM; P<0.001, Fig. 3D), respectively.

Cisplatin is primarily believed to kill caner cells by inducing cell apoptosis. To explore the effects of Dicer on cisplatin-induced cancer cell apoptosis, A2780 and CAOV3 cells were treated with cisplatin for 48 h at concentrations of 40 and 30 µM, respectively, and Hoechst 33342 staining was then performed. According to fluorescence microscopy images, the shDicer cells had significantly less nuclear fragmentation than the parental control cells and the shNC cells in both A2780 ((5.22±0.41), (13.14±0.84) and (13.33±0.69)% apoptosis rates for shDicer, parental control and shNC cells, respectively, P<0.001) and CAOV3 cells ((5.14±0.46), (12.17±0.79) and (12.39±0.73)% apoptosis rates for shDicer, parental control and shNC cells, respectively, P<0.001; Fig. 3A and B). Consistently, Annexin V/propidium iodide dual staining followed by flow cytometric analyses showed that compared with the shNC cells, the proportion of apoptotic shDicer cells was decreased by 1.88-, 3.11- and 6.95-fold in A2780 cells after treatment with cisplatin at concentrations of 0, 20 and 40 µM, respectively (Fig. 3C and D); the proportion of apoptotic shDicer cells was decreased by 2.26-, 4.27- and 10.17-fold in CAOV3 cells after treatment with cisplatin at concentrations of 0, 15 and 30 µM, respectively (Fig. 3C and E).

To investigate whether Dicer affects the expression of apoptosis-related proteins in cancer cells exposed to cisplatin, P73, P63, P53, cleaved caspase-9, cleaved caspase-3 levels and P21 were measured in ovarian cancer cells that were treated with cisplatin for 48 h. The treatment concentrations of cisplatin were 0, 20 and 40 µM for A2780 cells and 0, 15 and 30 µM for CAOV3 cells. Western blot analyses showed that the cisplatin-mediated increases in the expression levels of P73, P63, P53, P21, cleaved caspase-9 and cleaved caspase-3 were concentration-dependent in both shNC and shDicer cells; however, these expression levels were generally lower in the shDicer cells, especially the active fragments of caspase-9 and caspase-3 (Fig. 4).