Ovarian cancer cell lines (OVCA429, OVCA433 and OVCAR3) and normal ovarian epithelial cells (IOSE144) were purchased from The Cell Bank of Shanghai Institutes for Biological Science. Cells were maintained in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin (Thermo Fisher Scientific, Inc.) in an incubator with 5% CO₂ at 37°C.

In total, 10⁶ cells were plated in each dish 18 h before transfection. A total of 8 µg plasmid was transfected into ovarian cancer cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacture's protocol. Cells were incubated with antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) for 3 days, and the resistant cells were pooled and used for the subsequent experiments.

In total, 30 ovarian cancer samples and paired non-cancerous tissues were collected from patients who underwent surgery at The First People's Hospital of Jining (Jining, China) between April 2009 and March 2015. No treatment was performed prior to surgery. Written informed consent was obtained prior to the surgery. The collected tissues were stored in liquid nitrogen. The present study was approved by The Ethics Committee of The First People's Hospital of Jining.

The proteins were extracted from tissues and cell lines using RIPA lysis buffer (Cell Signaling Technology, Inc.), the protein concentration was measured by bicinchoninic acid assay. In total, 20 µg protein was loaded in each lane. Proteins were separated by 8% SDS-PAGE (Sangon Biotech Co., Ltd.). Subsequently, the proteins were transferred onto a PVDF membrane (EMD Millipore). Following blocking with 5% BSA (Sangon Biotech Co., Ltd.) for 1 h at room temperature, the membranes were incubated with primary antibodies overnight at 4°C. The membranes were subsequently washed with TBS-Tween-20 (0.5%) and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (cat. no. 7074; Cell Signaling Technology, Inc.) for 1 h at room temperature. The proteins were visualized using an enhanced chemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.). Antibodies anti-CALB1 (1:3,000; cat. no. ab108404), p16 (1:3,000; cat. no. ab51243), p21 (1:3,000; cat. no. ab218311), p27 (also known as CDKN1B; 1:3,000; cat. no. ab32034), Tubulin (1:6,000; cat. no. ab210797), glutathione S-transferase (GST; 1:5,000; cat. no. ab111947), human influenza hemagglutinin (HA)-tag (1:5,000; cat. no. ab9110) and Flag (1:5,000; cat. no. ab49763) were purchased from Abcam. Anti-GAPDH (1:5,000; cat. no. 5174), myc (1:1,000; cat. no. 2276), β-actin (1:5,000; cat. no. 3700) and MDM2 (1:1,000; cat. no. 86934) were purchased from Cell Signaling Technology, Inc.

The 5-µm-thick sections were deparaffinized and rehydrated using xylene (100%) and descending ethanol series (75–100%). Each incubation was performed for 10 min at room temperature. Subsequently, 0.3% H₂O₂ for 10 min at room temperature was used to block endogenous peroxidase activity. Subsequently, the antigens were retrieved using sodium citrate (pH 6.0, 0.01 M) at 98°C for 20 min). Non-specific binding was blocked using 5% BSA (Sangon Biotech Co., Ltd.) at room temperature for 1 h. The sections were stained with CALB1 primary antibody (Abcam) at 4°C overnight and visualized with the appropriate horseradish-conjugated secondary antibody using an EnVision system (Agilent Technologies, Inc.) according to the manufacturer's protocol. Subsequently, the slides were developed with 3,3′-diaminobenzidine at room temperature for 4 min and counterstained with hematoxylin at room temperature for 4 min. The staining intensity and the protein expression levels were evaluated using the Vectra2 system (PerkinElmer, Inc.). The staining was assessed using the H-score as previously described (20), which was calculating by multiplying the percentage of positive cells by the staining intensity of the tumor cells (ranging between 0 and 3).

Cells at 80% confluence were washed twice with PBS and fixed with 4% formaldehyde for 5 min at room temperature. Subsequently, cells were incubated at 37°C with β-gal staining solution (Beyotime Institute of Biotechnology) overnight according to the manufacturer's protocol. Stained cells were visualized using an inverted phase contrast light microscope (Olympus Corporation). In total, >300 random cells were analyzed per sample, and percentages of stained cells were calculated.

The coding sequence of CALB1 was cloned into the expression vector pGEX-4T-1 (Clontech Laboratories, Inc.). A PCR was performed using cDNA derived from OVCA429 cells as template. The primers used to clone CALB1 were as follows: Forward, 5′-ATGGCAGAATCCCACCTGCA-3′ and reverse, 5′-CTAGTTATCCCCAGCACAGAG-3′. The PCR was performed using KOD polymerase (Takara Bio, Inc.). The thermocycling conditions of the PCR were as follows: Initial denaturation at 95°C for 5 min, followed by 35 cycles of 94°C for 30 sec, 56°C for 1 min, 72°C for 2 min, with a final extension at 72°C for 10 min. The fusion protein GST-CALB1 was purified as previously described (21). The whole cell lysate of OVCA429 cells was extracted using a lysis buffer (containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tergitol-type NP-40 and a protease inhibitor cocktail). The protein concentration was determined using the bicinchoninic acid assay. Subsequently, 5 µg GST-CALB1 fusion protein and 500 µg total protein from OVCA429 cells were incubated overnight at 4°C. In total, 50 µl Glutathione Sepharose 4B beads (GE Healthcare) were added to the samples and incubated at 4°C for 1 h to allow the binding between the beads and the GST fusion protein. Following three washes with lysis buffer, the proteins were eluted in Laemmli buffer and analyzed by SDS-PAGE as aforementioned.

Cells were lysed with RIPA buffer (Cell Signaling Technology, Inc.). Following centrifugation at 4°C for 20 min at 12,000 × g, the supernatant of the cell lysate was incubated with a primary antibody overnight at 4°C. Subsequently, the supernatant was incubated with 50 µl protein A beads (GE Healthcare) for 4 h at 4°C, and the beads were washed with RIPA buffer three times. The immunoprecipitated proteins were mixed with Laemmli (Cell Signaling Technology, Inc.) buffer and boiled for 5 min at 100°C prior to western blot analysis as aforementioned.

The coding sequence of CALB1 was amplified by PCR and inserted into the expression vector pcDNA3.1 (Clontech Laboratories, Inc.) to construct the myc-tagged CALB1. The coding sequence of MDM2 was amplified by PCR and inserted into the expression vector pCMVTag2B (Clontech Laboratories, Inc.) to construct the Flag-tagged MDM2. The coding sequence of p53 was amplified by PCR and inserted into expression vector pCMV-HA (Clontech Laboratories, Inc.) to construct the HA-tagged p53. The cDNA was synthesized using RNA extracted from ovarian cancer cells as aforementioned. The primer sequences for MDM2 and p53 were as follows: MDM2 forward, 5′-ATGTGCAATACCTACATGTCTGTACC-3′, MDM2 reverse, 5′-CTAGGGGAAATAAGTTAGCACAA-3′; P53 forward, 5′-ATGGAGGAGCCGCAGTCAGATCCTAGCG-3′ and P53 reverse, 5′-TCAGTCTGAGTCAGGCCCTTCTGT-3′. The PCR was performed using KOD polymerase (Takara Bio, Inc.). The thermocycling conditions of the PCR were as follows: Initial denaturation at 95°C for 5 min, followed by 35 cycles of 94°C for 30 sec, 56°C for 1 min and 72°C for 2 min, with a final extension at 72°C for 10 min.

To knockdown CALB1, P21 and P27, lentiviral particles (10⁸ PFU) containing short hairpin (sh)-control or sh-CALB1, sh-P21 and sh-P27 (Shanghai GeneChem Co., Ltd.) were used to infect cells at 60% confluence for 24 h, and cells exhibiting stable knockdown were selected using DMEM containing puromycin (2 µg/ml) for ≥1 week. The sequences for sh-CALB1 were as follows: sh-CALB1 #1, 5′-AATCCCACCTGCAGTCATCCC-3′; sh-CALB1 #2, 5′-AATATGATACTGACCACAGTG-3′. The sequences for the additional sh-RNAs were as follows: sh-con, 5′-ACCACATCGCGTCTACACCTC-3′; sh-P21, 5′-AACCGGCTGGGGATGTCCGTC-3′; sh-P27, 5′-AAGAGTTAACCCGGGACTTGG-3′. In the senescence assay, only one sh-RNA for each gene was used. Only the sh-P21/P27 that was used in the transfection efficiency experiment was used in the senescence assay experiments.

Cells were plated in 96-well plates at a density of 1×10³ cells/well. Cell proliferation was determined using the MTT colorimetric assay (R&D Systems, Inc.) over 1 week. Every other day, cell proliferation was determined following incubation with the MTT solution (50 µg/well) for 4 h. Following the MTT incubation, the purple formazan crystals were dissolved using DMSO (Sigma-Aldrich; Merck KGaA) and proliferation was analyzed at a wavelength of 540 nm. All experiments were performed in triplicate.

For the soft agar assay, 12-well plates were coated with solidified DMEM-agarose [0.5% agarose (Sangon Biotech Co., Ltd.) in DMEM supplemented with 10% FBS]. Subsequently, 2×10³ cells/well suspended in a DMEM-agarose liquid solution (0.35% agarose in DMEM supplemented with 10% FBS) were added in the pre-coated wells. Following 14 days of incubation, the colonies were counted and measured using a light microscope (magnification, 20×). All experiments were performed ≥3 times.

GEPIA (http://gepia.cancer-pku.cn/) is a tool used to deliver fast and customizable functionalities based on TCGA and GTEx databases (22), including differential expression analysis, profiling plotting, correlation analysis, patient survival analysis and similar gene detection, as previously described (19). In the present study, GEPIA was used to investigate the association of CALB1 with disease-free survival rates of patients with ovarian cancer. Patients were grouped into high expression group and low expression group according to the median value of gene expression.

Data are presented as the mean ± SEM. Each experiment was repeated three times. Two-tailed Student's t-test was performed to compare data between two groups. One-way ANOVA followed by Student-Newman-Keuls post hoc test was used to compare multiple groups. Kaplan-Meier analysis and log-rank tests were used for the survival analyses. Data were analyzed using the Prism software 5.0 (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

To examine the expression of CALB1 in ovarian cancer, the protein expression level of CALB1 was assessed in healthy and ovarian cancer cells. The present results demonstrated that the protein expression level of CALB1 was increased in cancer cells compared with the normal cell line (Fig. 1A). Subsequently, the protein expression level of CALB1 was analyzed in ovarian cancer samples and adjacent non-cancerous tissues using western blot analysis and IHC. The present results suggested that the protein expression level of CALB1 was increased in cancer tissues (Fig. 1B and C). Furthermore, the analysis of the Gene Expression Profiling Interactive Analysis database (gepia.cancer-pku.cn/detail.php?gene=CALB1) demonstrated that an increased expression of CALB1 was associated with a reduced survival rate (Fig. 1D). The present results suggested that the protein expression level of CALB1 is upregulated in ovarian cancer cell lines and tissues, and its expression level may be associated with poor prognosis in patients with ovarian cancer.

To examine the biological functions of CALB1 in ovarian cancer, CALB1 was overexpressed in OVCA429 and OVCA433 cells (Fig. 2A). MTT and soft agar assays were used to examine the effects of CALB1 overexpression on the proliferation of ovarian cancer cells. Overexpression of CALB1 increased the proliferation of cancer cells in liquid medium after 4 days and the anchorage-independent cell proliferation in soft agar, as assessed by MTT and colony formation assay, respectively (Fig. 2B and C). Notably, the β-gal staining results suggested that the number of control cells positive for β-gal was higher than cells overexpressing CALB1 (Fig. 2D), suggesting that CALB1 may inhibit senescence in ovarian cancer cells.

To further investigate the biological functions of CALB1 in ovarian cancer cells, the expression of CALB1 was silenced using two sh-RNA sequences targeting CALB1 in OVCA429 and OVCA433 cells (Fig. 3A). Knockdown of CALB1 inhibited the proliferation of ovarian cancer cells in liquid culture after 3 days in OVCA429 cells and after 4 days in both cell types (Fig. 3B). Additionally, knockdown of CALB1 decreased the colony formation of ovarian cancer cells in soft agar (Fig. 3C). Furthermore, following transfection with sh-CALB1, the number of senescent cells increased, as assessed by β-gal staining (Fig. 3D). Collectively, the present data suggested that CALB1 promoted proliferation and inhibited senescence of ovarian cancer cells.

The protein expression levels of various regulators of senescence were examined. In particular, the protein expression levels of p16, p21 and p27 were assessed by western blot analysis. Knocking down the expression of CALB1 significantly upregulated the mRNA expression levels of p16, p21 and p27 (Fig. 4A). Conversely, overexpression of CALB1 decreased the protein expression level of p16, p21 and p27 (Fig. 4B). Moreover, knocking down the expression of p21 and p27 abolished the senescence induced by sh-CALB1, as assessed by β-gal staining (Fig. 4C). The present data suggested that p21 and p27 were responsible for the increase in senescence induced by CALB1 knockdown.

Numerous previous studies have demonstrated that p21 and p27 are downstream target genes of p53 (23). Therefore, the interaction between CALB1 and other members of the p53 signaling pathway was investigated in the present study. In the GST pull down assay, the fusion protein GST-CALB1 interacted with endogenously expressed MDM2 (Fig. 5A), as confirmed by the co-immunoprecipitation assay (Fig. 5B and C). Furthermore, CALB1 enhanced the interaction between p53 and MDM2 (Fig. 5D), suggesting that CALB1 may promote the degradation of p53 via MDM2.