In the present study, a total of 119 clinical specimens from patients with ovarian cancer who underwent initial surgical treatment at the Department of Gynecologic Oncology of the Chinese Academy of Medical Sciences Cancer Hospital (Beijing, China) were collected from May, 2007 to January, 2013. All patients provided informed consent to participate in the study. This study was approved by the Ethics Committee of the Chinese Academy of Medical Sciences Cancer Hospital.

IHC was performed as previously described (12). USP39 expression was assessed using a rabbit monoclonal antibody (ab131244; Abcam) at a 1:50 dilution at 4°C overnight, according to the manufacturer's instructions. As negative controls, sections incubated with rabbit immunoglobulin G (1:1000; ab6721; Abcam) instead of primary antibodies were used. Following incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (OriGene Technologies, Inc.) at room temperature for 1 h, the positive signals were visualized with 3,3′-diaminobenzidine (OriGene Technologies, Inc.) as a substrate. All slides were scanned with an Aperio scanning system (Aperio Group, LLC) and Aperio Image Scope software (version 10.2.2.2317, Aperio Technologies) was employed for the quantitative analysis of USP39 protein expression. For analysis, 4-6 different areas of the slide were randomly selected. The intensity score was graded with a score ranging from 0 to 3 according to the percentage of positively stained tumor cells. When 0-10% of tumor cells were stained, a score of 0 was given; when 10-25% of tumor cells were stained, a score of 1 was given; when 25-50% of tumor cells were stained, a score of 2 was given; and when 50-100% of tumor cells were stained, a score of 3 was given. Scores of 0 and 1 were considered to represent a low expression, while scores of 2 and 3 were considered to represent a high expression. The staining results of USP39 were evaluated according to the scoring criterion reported in a previous study (13).

The human ovarian cancer cell lines ES2, SKOV3 and the 293T cells were purchased from the American Tissue Culture Collection. The human ovarian cancer cell lines were cultured in RPMI-1640 (Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific), 100 U/ml ampicillin (Life Technologies, Gibco) and 100 μg/ml streptomycin (Life Technologies, Gibco). The 293T cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS, 100 U/ml ampicillin and 100 μg/ml streptomycin. All cells were cultured at 37°C in 5% CO₂.

For the stable knockdown of USP39, two small hairpin RNA (shRNA) sequences (shUSP39#1 and shUSP39#2) targeting USP39 were respectively inserted into pSIH1-H1-Puro (Invitrogen, Thermo Fisher Scientific, Inc.), and sh-green fluorescence protein (GFP) (Invitrogen, Thermo Fisher Scientific, Inc.) was used as control. The two USP39 shRNA sequences were as follows: 5′-GTACTTTCAAGGCCGGGGT-3′ (shUSP39#1) and 5′-ACAAGCAGTACACAAGAAT-3′ (shUSP39#2). shRNA plasmids were transfected into the 293T cells together with 3 packaging plasmids (3 μg PLP1, 3 μg VSVG and 3 μg PLP2) (Invitrogen, Thermo Fisher Scientific, Inc.) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) with OPTI-MEM (Thermo Fisher Scientific, Inc.). The virus-containing media were pooled at 48 h after transfection, centrifuged under 800 × g for 10 min at 4°C and filtered by passing through a 0.45-μm filtration device (EMD Millipore). Viral supernatant was added in a 1:3 dilution to previously seeded cell lines, supplemented with 5 μg/ml of polybrene (EMD Millipore). Stably transduced cells were selected with puromycin at a final concentration of 1 μg/ml. For the stable overexpression of USP39, full-length complementary DNA of human USP39, which was generated by polymerase chain reaction (PCR) amplification, was cloned between the XhoI and XbaI sites of pLVX-IRES-Neo (Invitrogen, Thermo Fisher Scientific, Inc.) to prepare a constitutive lentiviral vector. A total of 8×10⁶ 293T cells were seeded in 100-cm² plates. pLVX-IRES-Neo or pLVX-IRES-Neo-USP39 lentiviral vectors (7.5 μg) and the corresponding packaging plasmid (6.5 μg pCMV ∆8.91, 3.5 μg VSVG and 2.5 μg PLP2) were added to a mixture of Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) with OPTI-MEM (Thermo Fisher Scientific, Inc.). Following a similar transfection procedure as the one described above, stably transduced cells were selected with G418 (Sigma-Aldrich) at a final concentration of 400 μg/ml. At 48 h following transfection, knockdown and overexpression efficiencies were evaluated by reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis.

To quantify cell proliferation, 100 μl stably transfected cells at a density of 1×10⁴ cells/ml (n=6) were seeded in 96-well plates. A Cell Counting kit-8 (CCK-8) assay kit was used to detect cell viability at 5 time points. The cells were cultured in 100 μl/well fresh medium mixed with CCK-8 solution (10:1) (Dojindo Molecular Technologies, Inc.) and incubated for 1 h at 37°C. The optical density values were then measured at 450 nm using a spectrophotometer (Bio-Rad).

The ES2 and SKOV3 cells were analyzed for apoptosis using an Annexin V-fluorescein isothio-cyanate (FITC)/propidium iodide (PI) kit [Multisciences (Lianke) Biotech Co., Ltd.]. Briefly, the cells were seeded at 5×10⁵ cells/well in 6-well plates in triplicate. Following 24 h of incubation with carboplatin, the cells were harvested through trypsinization and washed twice with cold PBS. The cells were centrifuged at 3,000 × g at room temperature for 5 min. The supernatant was then discarded and the pellet was resuspended in 500 μl 1X binding buffer. Finally, the cells were incubated with 5 μl FITC-conjugated Annexin V and 10 μl of PI for 5 min at room temperature in the dark. The samples were analyzed by a FACSCalibur flow cytometer (BD Biosciences).

For cell cycle analysis, the cells were harvested and fixed with 70% ethanol for 18 h at -20°C upon digestion with 0.05% trypsin. Upon washing with PBS and collecting the fixed cells by 800 × g centrifugation at 4°C for 5 min, samples were resuspended with 500 μl DNA staining solution and then incubated in 37°C for 30 min. Analysis was performed on a FACSCalibur flow cytometer (BD Biosciences).

Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The cDNA was synthesized by the FastQuant RT kit [TIANGEN Biotech (Beijing) Co. Ltd.] using 100 ng of total RNA. Quantitative analysis was performed using diluted cDNA combined with the SYBR-Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.), forward and reverse primers, and RNase-free water. The primer sequences were as follows: USP39 forward, 5′-GGTTTGAAGTCTCACGCCTAC-3′ and reverse, 5′GGCAGTAAAACTTGAGGGTGT-3′; and β-actin forward, 5′-CCGTTCCGAAAGTTGC CTTTT-3′ and reverse, 5′GAGGCGTACAGGGATAGCAC-3′. The RT-qPCR reactions were conducted using the following parameters: 95°C for 10 min, 40 cycles at 95°C for 10 sec, at 60°C for 20 sec and at 72°C for 30 sec. The relative mRNA expression in each sample was calculated using the 2^−ΔΔCq method (14).

Total protein was extracted from whole cells using ice-cold cell lysis buffer (1% Triton X-100; 10 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.25% sodium deoxycholate; 5 mM EDTA, pH 7.4) and assayed for protein concentration by a bicinchoninic acid assay (Pierce). Samples containing 15 μg of protein were separated by 8-12% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Merck KGaA). After blocking with 5% skimmed milk powder for 1 h at room temperature, the membranes were incubated with antibodies against USP39 (1:1,000; ab131244; Abcam), caspase-3 (1:1,000; #9661; Cell Signaling Technology, Inc.), poly-ADP ribose polymerase (PARP) (1:1,000; #9542; Cell Signaling Technology, Inc.) and β-actin (1:5,000; A5316; Sigma-Aldrich; Merck KGaA) overnight at 4°C, followed by incubation with a appropriate HRP-linked secondary antibody (1:2,000; #7074/#7076; Cell Signaling Technology, Inc.) at room temperature for 1 h. Samples were detected with an enhanced chemiluminescence detection system (Pierce; Thermo Fisher Scientific, Inc.).

For the colony formation assay, lentivirus-infected cells were seeded evenly in 6-well plates at an initial density of 2,000 cells/well and cultured in a 5% CO₂ incubator for 10 days at 37°C. The cells were washed with ice-cold PBS, fixed with 4% paraformaldehyde (PFA), stained with crystal violet at room temperature for 30 min and rinsed 3 times with double distilled H₂O.

The cells were plated until they reached 90% confluence in 6-well plates. Cell monolayers were scratched with 200-μl pipette tips. Upon forming wound gaps, the cells were washed with PBS twice to remove floating cells and cultured in serum-free medium. Cell migration into the wound area was observed under an inverted microscope (Leica Microsystems) at different time points. The speed of wound closure was analyzed by measuring the distance of the migrating cells from different areas for each wound.

For the cell migration assay, cells (2×10⁴), which has been resuspended in 100 μl serum-free medium, were added to the upper chambers of a Transwell plate (Corning Inc.). The lower chambers were filled with 600 μl RPMI-1640 complete medium containing 10% FBS. Following 24 h of incubation, the cells were fixed with 4% PFA for 30 min and stained with 0.1% crystal violet at room temperature for 1 min. After washing gently with water, the cells were imaged and counted under a microscope (Leica Microsystems). For the cell invasion assay, Matrigel (Sigma-Aldrich; Merck KGaA) was diluted from 5 mg/ml to 1 mg/ml by serum free-cold RPMI-1640. The upper chambers of the Transwell plates were pre-coated with 100 μl of the diluted Matrigel. The subsequent steps of the procedure were the same as those for the migration assay.

Pathogen-free female BALB/c nude mice (age, 5-6 weeks; weight, 18-22 g, 6 mice per group) were provided standard laboratory chow and allowed free access to water, unless otherwise stated, in a climate-controlled room with a 12-h light/12-h dark cycle at the Animal Experiment Center of Cancer Hospital, Chinese Academy of Medical Sciences. Animal studies were approved by the Animal Care and Use Committee of Cancer Hospital, Chinese Academy of Medical Sciences. All procedures involving animals were conducted according to the guidelines for the care and use of laboratory animals. A total of 5×10⁵ stably transfected ES2 cells were resuspended in 100 μl normal saline and injected subcutaneously into the flank of each mouse with a 28G syringe. At 3 days post-inoculation, carboplatin was administered to the mice by intraperitoneal injection at a dose of 50 mg/kg every 3 days for 3 consecutive times as described previously (15). Tumors were measured using an electronic caliper twice per week, and tumor volume was calculated using the formula: Volume = (length x width²)/2. All mice were sacrificed by cervical dislocation at 21 days post-injection. Tumor samples were fixed in formalin for paraffin embedding and sectioned into 4 μm slides for IHC analyses. The sections were incubated with antibodies against USP39 (1:50; ab131244; Abcam) and cleaved caspase-3 (1:50; #9579; Cell Signaling Technology, Inc.) to measure the expression of USP39 and cleaved caspase-3.

Luciferase-expressing control or USP39-overexpressing ES2 cells were cultured, harvested and suspended in RPMI-1640. Subsequently, 5×10⁵ cells were injected intraperitoneally into the female BALB/c nude mice (3 mice per group). At 3 days post-inoculation, carboplatin was administered to the mice by intraperitoneal injection at a dose of 50 mg/kg. Mice were housed for 10 days and injected with 200 μl 15 mg/ml D-luciferin PBS solution 10 min prior to imaging. The whole animals were imaged using an IVIS Lumina XRMS In Vivo Imaging System (PerkinElmer, Inc.).

Statistical analyses were conducted using Prism software 6 (GraphPad Software, Inc.). The associations between USP39 expression and the patient clinicopathological data were assessed using a Chi-square test. All data are presented as the means ± standard error of the mean of ≥3 independent experiments. For 2-group comparison, analyses were performed with a Student's unpaired t-test (two-tailed). For multiple comparisons, analyses were performed with one-way ANOVA with Tukey's post hoc test. All experiments other than histological examinations were repeated ≥2 times. P<0.05 was considered to indicate a statistically significant difference.

USP39 expression was analyzed in carboplatin-sensitive and carboplatin-resistant human ovarian cancer tissues by IHC. The results revealed that USP39 was primarily expressed in the nucleus, but rarely in the cytoplasm (Fig. 1). In the carboplatin-resistant group, 60.71% of the specimens presented a strong positive expression vs. 41.27% in the carboplatin-sensitive group (P= 0.034), determined by a Chi-square test (Table I). Subsequently, we analyzed whether the expression of USP39 can influence the clinicopathological characteristics of patients with ovarian cancer. As shown in Table I, USP39 expression was not associated with age, residual tumor size, level of cancer antigen 125 in primary tumors, International Federation of Gynecology and Obstetrics stage or tumor grade. These results indicate that high expression levels of USP39 in ovarian cancer tissues may be closely associated with the development of carboplatin resistance.

To verify the role of USP39 in ovarian cancer cells, the ES2 and SKOV3 cell lines were transduced with shUSP39s or shGFP lentiviruses. The inhibitory effects of shUSP39s on its endogenous expression in the ES2 and SKOV3 cells were determined by RT-qPCR and western blot analysis (Fig. 2A and B). To examine the effects of USP39 on cell proliferation, a CCK-8 assay was performed. As shown in Fig. 2C, the proliferative rate of the ES2 and SKOV3 cells was significantly lower in shUSP39s-transduced cells compared with the shGFP control cells. To further characterize the effects of USP39 on the cell proliferative capability, a colony formation assay was employed. As depicted in Fig. 2D, upon the knockdown of USP39, the capacity of colony formation of the ES2 and SKOV3 cells was substantially reduced. To elucidate the mechanisms through which USP39 modulates cell proliferation and colony formation, cell cycle distribution analysis was employed. The cell percentage in the G2/M phase was increased by USP39 knockdown in the SKOV3 cells, suggesting that cell growth suppression may be associated with increased G2/M phase arrest (Fig. 2E). Cell cycle-associated protein analysis also revealed that the expression of cyclin B1 was suppressed following USP39 knockdown (Fig. 2F). The overexpression of USP39 in the ES2 cells indicated that USP39 significantly increased the cell proliferation and colony formation capacity of ES2 cells (Fig. 3). Taken together, these data support the important role of USP39 in regulating the proliferation of ovarian cancer cells.

Previous studies have reported that USP39 is a potential target for several types of cancer (5,6). To explore the possibility that USP39 activity may be involved in the metastatic behaviors of ovarian cancer cells, migration and invasion assays were performed in vitro. USP39 knockdown significantly suppressed the migration and invasion of ES2 cells (Fig. 4A and B). Similarly, the capacity of wound healing was markedly decreased in the SKOV3 cells in which USP39 was silenced and increased in the USP39-overexpressing ES2 cells compared with their respective control cells (Fig. 4C and D). These results indicate that USP39 plays a potential role in the induction of cell migration and invasion in ovarian cancer.

To ascertain the regulatory mechanisms of action of USP39 as regards the tumorigenesis of ovarian cancer, multiple signaling pathways were analyzed in the ES2 cells upon altering USP39 expression. As shown in Fig. 5, USP39 knockdown markedly decreased the expression of EGFR, phosphorylated (p)-ERK and p-AKT. On the contrary, the expression levels of EGFR, p-ERK and p-AKT were increased upon the overexpression of USP39.

Based on the aforementioned histological and clinical analyses, USP39 may play a role in the regulation of patient's response to carboplatin treatment. Thus, to clarify the possible mechanisms involved in the expression level of USP39 in regulating the apoptosis induced by carboplatin in ovarian cancer cells, flow cytometry was performed and the expression of apoptosis-associated proteins was examined. USP39 deficiency significantly potentiated the apoptotic rate induced by carboplatin in the SKOV3 cells, whereas USP39 overexpression markedly inhibited the apoptotic rate of the ES2 cells (Fig. 6A and C). Consistent with the effects observed on the cell apoptotic rate exerted by USP39, higher levels of cleaved PARP and cleaved caspase-3 were observed in the ES2 cells in which USP39 was silenced than in the control cells (Fig. 6B). By contrast, USP39 overexpression in the SKOV3 and ES2 cells significantly suppressed carboplatin-induced apoptosis, as demonstrated by the marked decrease in cleaved PARP and cleaved caspase-3 levels (Fig. 6D). These data indicate that USP39 plays an important role in mediating the sensitivity of ovarian cancer cells to carboplatin.

Rescue assays were conducted to prove the function of USP39 in the chemosensitivity of ES2 cells to carboplatin, and its effects on migration and invasion. As shown in Fig. 7A and B, shUSP39#1 partly attenuated the promoting effects of USP39 on the chemosensitivity of the cells to carboplatin, as evidenced by the increased cell apoptotic rate, and the increased expression of cleaved PARP and cleaved caspase-3 in the shUSP39#1-transduced cells. Moreover, cell migration and invasion promoted by USP39 was partially recovered by shUSP39#1 (Fig. 7C). Taken together, these findings demonstrated USP39 promoted the chemoresistance of ovarian cancer to carboplatin, as well as migration and invasion.

To further validate the in vitro results, the present study examined the effects of USP39 on the chemosensitivity of ES2 and SKOV3 cells in vivo. Mice were randomly divided into 4 groups (n=6/group). The results revealed that the ectopic expression of USP39 significantly increased tumor size. Carboplatin treatment markedly suppressed the growth of ES2 cells in the animal model. The overexpression of USP39 led to a more rapid growth rate with carboplatin treatment (Fig. 8A-C). Mice were randomly divided into 6 groups (n=8/group). The results revealed that the knockdown of USP39 reduced tumor burden and carboplatin treatment markedly suppressed the growth of SKOV3 cells in the animal model (Fig. 9). IHC revealed a lower level of cleaved caspase-3 in the USP39-overexpressing cells than in the control cells treated with carboplatin

(Fig. 8D). These results suggested that USP39 attenuated the chemosensitivity to carboplatin administration. To further investigate the role of USP39 in ovarian cancer metastasis, an experimental murine peritoneal metastasis model was used. The overexpression of USP39 in the ES2 cells promoted the formation of peritoneal metastases (Fig. 8E and F). Taken together, these results demonstrate that USP39 promotes the malignant phenotype of ovarian cancer cells.