High Levels of BMPER in Ovarian Cancer and How It Helps Tumor Cells Grow and Spread

A total of 171 paraffin-embedded ovarian tissue samples were obtained from surgical tissue from 2008 to 2014 in the Department of Gynecology of Shengjing Hospital of China Medical University. All histological sections were reviewed by pathologists in our hospital to confirm the diagnosis. The specimens were divided into four groups based on the tissue type: 94 ovarian epithelial malignant tumors (malignant group), 24 ovarian epithelial borderline tumors (borderline group), 23 ovarian epithelial benign tumors (benign group), and 30 normal ovarian tissue samples (normal group). The median age and age distribution in each group was as follows: ovarian cancer group, 53 years (19-83 years); borderline group, 48 years (26-58 years); benign group, 46 years (24-65 years); and normal group, 45 years (36-67 years). There was no significant difference in age among the groups (P > 0.05). The histological types, pathological grades, surgical pathological staging (according to the International Federation of Gynecology and Obstetrics (FIGO), 2006 standard), and lymph node metastasis statuses are shown in Table 1. All specimens were primary lesions with complete clinicopathological data, and no radiotherapy or chemotherapy was performed before surgery. The study was approved by the Ethics Committee of Shengjing Hospital affiliated to China Medical University.

The ovarian tissues of each group were serially sectioned at 5 μm, and BMPER expression was detected by the immunohistochemical streptavidin-peroxidase-linked (SP) method. BMPER antigen repair was performed by citrate buffer thermal repair. The working dilution of anti-human BMPER antibody (rabbit polyclonal to BMPER, Abcam, USA) was 1 : 75, and the staining method was carried out according to the SP kit instructions (MXB, China).

All the sections were scored under a light microscope in five randomly selected fields at 400-fold magnification. The criteria were based on the staining of the tumor cell membrane and the cytoplasm with brown-yellow granules. All the sections were scored according to the methods described in a previous study [13]. The slides were scored by two senior pathologists who reviewed the slides independently.

Human ovarian cancer cell lines (CAOV3, OVCAR3, SKOV3, and ES-2) were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). CAOV3, OVCAR3, and SKOV3 cells were incubated in 37°C and 5% CO2 incubator with RPMI 1640 medium containing 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), and ES-2 cells were cultured in the same incubator in McCoy's 5A medium containing 10% fetal bovine serum. BMPER was expressed in four ovarian cancer cell lines: CAOV3, OVCAR3, SKOV3, and ES-2. Among these cell lines, the miRNA and protein expression levels of BMPER were higher in CAOV3 and OVCAR3 cells, so these two cell lines were selected for the RNA interference experiment. CAOV3 and OVCAR3 cells were transfected with BMPER-specific siRNA and negative control (NC) using Lipofectamine 3000 (Thermo Fisher Scientific, USA). Cells were collected 72 hours after transfection, and the effects on BMPER interference were detected by RT-PCR and Western blot. The BMPER siRNA sequence (GenePharma, Shanghai, China) is as follows: sense, GGUCCUGUGUGACAGACAUTT and antisense, AUGUCUGUCACACAGGACCTT.

Total RNA from ovarian carcinoma cells was extracted using TRIzol reagent and reverse transcribed into cDNA using an RT-qPCR kit (Takara, Kusatsu, Japan) according to the manufacturer's instructions. PCR was then performed in a 20 μL reaction system containing 2.0 μL of cDNA template. The BMPER forward primer sequence was 5′-TTGTGTTCTACGCCAGTGCC3′, the BMPER reverse primer sequence was 5′-GCAGCACATTCCCAGATGCT-3′, and GAPDH served as an internal reference. An Applied Biosystems® 7500 fast PCR instrument was used for real-time PCR amplification. The reaction cycle parameters were as follows: 40 cycles of denaturation at 95°C for 30 seconds, annealing at 95°C for 5 seconds, and extension at 60°C for 30 seconds. The expression level of the target gene was calculated using the comparative 2^–ΔΔCT method.

Total protein was extracted from cells of each group using RIPA buffer. Denatured proteins were separated by SDS-PAGE through a 10% gel and then transferred to a PVDF membrane. The membrane was blocked with 5% milk or BSA for 2 hours and incubated with the appropriate primary antibody at 4°C overnight. The working concentrations of each antibody were as follows: anti-BMPER (1 : 500; Abcam, USA); anti-mechanistic target of rapamycin (mTOR), anti-phospho-mTOR, anti-LC3A/B, anti-Beclin-1, anti-PCNA, and anti-phospho-MEK 1/2 (1 : 1,000; CST, USA); anti-ERK1/2 (1 : 500; Bioss, Beijing); anti-phospho-ERK 1/2 (1 : 300; Bioss, Beijing); anti-MEK1/2 (1 : 300; Santa Cruz Biotechnology Inc.); anti-Bcl2, anti-Bax, anti-MMP2, anti-MMP9 (1 : 1,000; Proteintech, Rosemont, PA, USA); and anti-GAPDH (1 : 2,000; ZSGB- BIO, Beijing, China). Goat anti-rabbit or goat anti-mouse secondary antibody (1 : 3,000; ZSGB-BIO) was separately added and incubated for 2 hours after the membranes were rinsed with TBST. The protein bands were detected on a GDS8000 gel electrophoresis image analyzer (Thermo Fisher Scientific) using an enhanced electrochemiluminescence (ECL, Thermo Fisher Scientific) detection kit.

Cell proliferation was monitored using the thiazolyl blue tetrazolium bromide assay (MTT). Cells were seeded at a density of 2,000 cells/well in 96-well culture plates. The cell proliferation was monitored every 24 hours from 0 hour to 96 hours. The cells from each well were treated with MTT reagent at 20 μL/well 4 hours prior to harvest. The medium in all wells was removed, and 150 μL of DMSO was added to each well. After 10 minutes of shaking, the OD at 490 nm was measured using a microplate reader.

Each group of cells was digested with EDTA-free trypsin. After digestion and washing with cold PBS, cells were suspended in 500 μL of Annexin V Binding Buffer (Annexin V, FITC, Dojindo, Japan). Next, 5 μL of Annexin V-FITC solution and 5 μL of PI solution were added to the cell suspension and incubated for 15 minutes in the dark. Apoptotic cells were detected by flow cytometry.

Cells were seeded into a six-well culture plate, cultured, and then transfected. A 200 μL pipette tip was used to make a linear scratch wound on the well plate. After washing twice with PBS, cells were cultured with FBS-free medium. The width of the scratches was observed under a microscope at 0, 24, and 48 hours after scratching. The area of the scratches was measured with ImageJ, and the wound healing rate was calculated.

For Transwell invasion assay, Matrigel (BD Biosciences, USA) was diluted 1 : 8 with serum-free medium and added to the upper chamber of a Transwell chamber (80 μL/chamber, Corning Incorporated, Corning, NY, USA) on ice. After the Matrigel solidified, 200 μL (cell concentration: 2 × 10⁵ cells/mL) of cells suspended in serum-free medium was added to each upper chamber, and 500 μL of medium containing 10% fetal bovine serum was added to the lower chamber. After the cells were incubated for 24 hours, the membranes were fixed with 4% paraformaldehyde for 30 minutes and stained with crystal violet for 30 minutes. The upper chamber was wiped with a cotton swab. The number of invading cells in 5 fields was counted under a microscope, and the mean value was obtained.

All data were analyzed using the SPSS 24.0 statistical software. Unpaired two-tailed Student's t-test and chi-square test were used for comparisons between two groups, and comparisons of differences among more than two groups were analysed by one-way ANOVA. The Kaplan-Meier test was used to produce the survival curve, and the Cox model was used to analyze the relationship of key variables with prognosis. P < 0.05 was considered statistically significant.

BMPER was mainly stained in cytoplasm and intercellular space, and a small number of nuclei were colored with brown or brownish yellow granules. The positive rate and high expression rate of BMPER in ovarian epithelial malignant tumors were 90.43% and 73.91%, respectively, which were significantly higher than the corresponding values in ovarian epithelial borderline tumors (73.91%, 39.13%), ovarian epithelial benign tumors (41.67%, 16.67%), and normal ovarian tissue (30.00%, 10.00%) (P < 0.01). Meanwhile, the rate of positive BMPER expression in ovarian epithelial borderline tumors was higher than that in ovarian epithelial benign tumors and normal ovarian tissues (P < 0.05), and its high expression rate was higher than that in normal ovarian tissues (P < 0.05) (Table 2 and Figures 1(a) and 1(b)).

All 94 patients with ovarian cancer in this study had histologically confirmed ovarian epithelial malignancies. High BMPER expression was associated with lymph node metastasis, and the proportion of patients with high BMPER expression in the lymph node metastasis positive group was higher than that in the lymph node metastasis negative group (P < 0.05). In terms of pathological types, the rate of high BMPER expression in patients with clear cell carcinoma was higher than that in other pathological types, but it was not statistically significant. There was no difference in BMPER expression among the different FIGO stages and tumor differentiation grades (Table 1).

There was no significant difference in demographic among the groups. All 94 patients with ovarian cancer were followed up until September 2018. The Kaplan-Meier survival analysis showed that the BMPER expression level was closely related to the overall survival (OS) of patients. The survival rate of patients with high BMPER expression was significantly lower than that of patients with low BMPER expression. Cox regression analysis showed that high BMPER expression was an independent risk factor for poor prognosis in patients (P = 0.026). Additionally, the results showed that FIGO stage and lymph node metastasis were also associated with poor prognosis, while tumor differentiation was not correlated with the patient survival (Figure 1(c) and Table 3).

The expression levels of BMPER in four ovarian cancer cell lines CAOV3, OVCAR3, SKOV3, and ES-2 were detected by real-time PCR and Western blot. The results showed that the mRNA and protein expression levels of BMPER in CAOV3 and OVCAR3 cells were higher than those in SKOV3 and ES-2 cells, with ES-2 cells exhibiting the lowest levels of BMPER expression (Figure 2(a)). BMPER siRNA was transfected into CAOV3 and OVCAR3 cells, and BMPER expression was detected after transfection for 72 hours. The results showed that the mRNA and protein expression levels of BMPER in the transfected siRNA group were significantly lower than those in the control and untransfected groups (Figures 2(b) and 2(c)).

After transfection of BMPER-specific siRNA into the ovarian cancer cell lines CAOV3 and OVCAR3, the changes in cell proliferation, migration, invasion, apoptosis, and cell cycle progression were further examined. The expression of proliferating cell nuclear antigen (PCNA), the apoptosis-related indicators B-cell lymphoma 2 (Bcl-2) and Bax, and the invasion- and migration-related indicators matrix metallopeptidase 2 (MMP2) and matrix metallopeptidase 9 (MMP9) were detected by Western blot. The results showed that the proliferation, invasion, and migration abilities of cells transfected with BMPER siRNA were significantly reduced compared with those of the control group, while the apoptosis rate increased (Figure 3); however, the cell cycle distribution did not change significantly (results not shown). In addition, compared with the control group, the siRNA transfection group exhibited decreased expression of PCNA, decreased expression of Bcl-2, increased expression of the proapoptotic protein Bcl-2 family activating protein-X (Bax), and decreased expression of MMP2 and MMP9 (Figure 4).

To further understand the mechanism by which downregulation of BMPER causes changes in the biological behavior of ovarian cancer cells and to determine the possible signaling pathway through which BMPER may play a role, siRNA transfection was used to inhibit BMPER expression in CAOV3 and OVCAR3 cells. Subsequently, the expression levels of the MAPK signaling pathway molecules ERK, p-ERK, MEK, and p-MEK and the autophagy-related pathway molecules mTOR, p-mTOR, Beclin 1, and microtubule-associated proteins 1A/1B light chain 3 (LC3) were detected by Western blot analysis. The results showed that after BMPER expression was downregulated, the levels of p-ERK, p-MEK, and p-mTOR proteins decreased while Beclin 1 levels and the LC3II/I ratio increased, indicating that the MAPK signaling pathway was inhibited after interference with BMPER expression but that autophagy-related pathways were promoted upon BMPER knockdown (Figures 5(a) and 5(b)).

The data used to support the findings of this study are available from the corresponding author upon request.