B7-H3, a co-stimulatory molecule, has been found expressed in ovarian cancer, but its role and mechanism is not clear. In this study, we further verified the expression of B7-H3 in ovarian carcinoma and normal epithelial ovarian tissues. Three ovarian cancer cell lines, A2780, SKOV3 and HO8910 were selected to explore the effects of B7-H3 on proliferation, apoptosis, migration and invasion. We found that B7-H3 was mainly located in the cytoplasm of ovarian cancer cells as determined by immunofluorescence staining. The ability of cell invasion, migration, proliferation decreased after silencing B7-H3 whereas the apoptosis increased, which was related to the upregulation of Bax, caspase-8, cleaved caspase-8 and the downregulation of Bcl-2, Bcl-xl, matrix metalloproteinase-2 (MMP2) by western blotting. In addition, B7-H3 enhanced the H08910 cell capacities in invasion, migration and proliferation. Expression of the phosphorylation signal transducer and activator of transcription 3 (pStat3) molecules and their upstream molecules phosphorylation Janus kinase 2 (pJak2) were significantly increased. In order to investigate whether B7-H3 plays a role in this pathway, we treated the overexpressed HO8910 cells with AG490 (inhibitors of Jak2). Our findings revealed that B7-H3 affect ovarian cancer progression through the Jak2/Stat3 pathway, indicating that B7-H3 has the potential to be a useful prognostic marker.
Ovarian cancer is the most lethal gynecological malignancy and the fifth most common cause of cancer death among women, 90% is epithelial ovarian cancer (EOC) (
B7-H3 is encoded by the CD276 gene, including two main isoforms, named 4Ig-B7-H3 and 2Ig-B7-H3, which were first identified in 2001 (
Ovarian cancer tissue samples were collected from 41 patients who underwent surgery from 2015 to 2016 in Qilu Hospital of Shandong University. In addition, 31 normal ovarian tissues were collected to use as control. None of the patients received preoperative chemotherapy or radiotherapy before the surgery. All patients were confirmed by pathological diagnosis. The present study was approved by the Ethics Committee of Qilu Hospital.
The human ovarian cancer cell lines A2780, SKOV3, and HO8910 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). A2780 and HO8910 were cultured in the medium of Roswell Park Memorial Institute (RPMI)-1640 (Hyclone Laboratories, Logan, UT, USA), while SKOV3 in the medium of Micro-5A (Gibco-Invitrogen, Grand Island, NY, USA). All of them were supplemented with 10% fetal bovine serum (FBS; Gibco, Sydney, Australia) and incubated at 37°C in a humidified incubator supplemented with 5% CO2.
Rabbit anti-human Jak2, Phospho-Jak2 (Tyr1007), signal transducer and activator of transcription 3 (Stat3), phospho-Stat3 (Tyr705), Bcl-2 and Bax antibodies were purchased from Abcam (Cambridge, MA, USA). Mouse anti-human Bcl-xl, caspase-8 and MMP-2 were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Rabbit anti-human B7-H3, cleaved caspase-8 and the GAPDH were purchased from Cell Signaling Technology (CST) (Danvers, MA, USA). All of the above are monoclonal antibodies. Tryphostins AG490 was from Abcam and prepared at a concentration of 100 mmol/l stock solution in dimethyl sulfoxide (DMSO).
Polink-2 Plus Polymer HRP Detection System (ZSGB-BIO, Beijing, China) was used. Paraffin tissues were cut into 4-µm sections. Section were de-paraffinized in xylene, re-hydrated through a graded ethanol series, then repaired by citric acid (microwave boiling method). H2O2 (3%) was incubated for 10 min to block endogenous peroxidase activity. PBS (phosphate-buffered saline) was used for washing three times for 3 min each. After blocking endogenous peroxidase activity, the slides were incubated with primary antibody overnight at 4°C. The next day, sections were washed in PBS three times for 5 min each. Secondary antibody was applied for 30 min at 37°C. DAB was used for coloring. Finally slides were counterstained with water rinsing, staining, dehydration, transparent and mounting. Quantification was recorded as follows: <10% positive cells, 0; 10–25%, 1; 26–50%, 2; >50% positive cells, 3. Sections with a final score of 0–1 was classified as negative, and ≥2 was considered positive.
To find the B7-H3 expression location, the A2780 and SKOV3 cells were seeded in a 24-well plate at 37°C overnight. Then, after three washes with PBS, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and permeabilized with 0.2% Triton 150–200 µl in PBS for 10 min. After that, the B7-H3 (1:200 dilution) antibody was incubated at 4°C for overnight and secondary antibody (1:200) for 1 h. Finally, cells were stained with 4,6-diamidino-2-phenylindole (DAPI) for 5 min in the dark at room temperature. The fluorescence images were observed using a fluorescence microscope (Olympus, Tokyo, Japan).
Silencing or overexpression of B7-H3 sequences were designed by GenePharma Co, Ltd. (Shanghai, China). The cells were seeded in 6-well plates, 24 h after the cell attachment, the virus liquid was mixed in the culture medium. The cells were screened with puromycin dihydrochloride (2 µg/ml; Amresco, Solon, OH, USA) 72 h later. After screening for 5–7 days, the cell line with stable overexpression of B7-H3 (HO8910-B7-H3-EGFP) and their control cell lines (HO8910-NC) were obtained. The A2780 and SKOV3 cells were seeded in a 6-well plate with 3×104/ml cell per well, 24 h later, cells were transfected with 50 nM of the Sh-B7-H3 or control sequences using Lipofectamine-2000 (Invitrogen Life Technologies). The transfected cells were harvested 48 h post-transfection for the follow-up experiments, with A2780-NC, A2780-sh-B7-H3, SKOV3-NC and SKOV3-sh-B7-H3.
The cells were washed 3 times with PBS and then lysed on ice for approximately 30 min. The pyrolysis solution is composed of radio immunoprecipitation assay buffer (RIPA), phenylmethylsulfonyl fluoride (PMSF) and NaF. The cells were then lysed with ultrasound. Cells were centrifuged at 12000 rpm at 4°C, then supernatant were drained, loading buffer was added and heated for 5 min in metal bath. The protein concentrations were measured by using the BCA Protein Assay kit (Beyotime, Jiangsu, China). Total protein (30–50 µg) was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in 10% gels (Invitrogen) and transferred to PVDF membranes (ImmobilonP; Millipore, Bedford, MA, USA). After blocking with 5% skim milk for 2 h, the membranes were cut into strips and incubated with the indicated primary antibodies overnight at 4°C. The next day, the membranes were washed 3 times with TBS-T (20 mM Tris, pH 7.4, 137 mM NaCl, 0.05% Tween-20) and then indicated with secondary antibodies at room temperature for 1–2 h. Ultimately, the immunoreactive protein bands were detected by enhanced chemiluminescence (ECL) using ImageQuant LAS 4000 (GE Healthcare Life Sciences, Logan, UT, USA). The results were analyzed by ImageJ software (NIH, Bethesda, MD, USA).
The cell proliferation was evaluated by MTT assay. In brief, cells were seeded in 96-well plates at 2000 cells per well. At indicated time-points, the cells in the 96-well plate were incubated with 20 µl MTT at 37°C for 4 h. The cell growth was monitored every 24 h for up to 5 days. Absorbance was measured for each well at a wavelength of 490 nm using a microplate reader (Infinite 2000; Tecan, Männedorf, Switzerland).
For the
To analyze the effect of B7-H3 on the different phases of the cell cycle and apoptosis, flow cytometry was used. Cells were harvested from each sample then fixed with cold 75% ethanol at 4°C overnight. The cells were washed 3 times and stained for 30 min in propidium iodide (PI) staining solution in the dark. The cell cycle was detected by FACSCalibur flow cytometer (both from BD Biosciences, Franklin Lakes, NJ, USA) and analyzed by ModFit LT software. For apoptosis, cells were harvested and processed as described in the Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit I manual (BD Transduction Laboratories, BD Biosciences) and analyzed by flow cytometry (BD LSR II).
The data were analyzed using GraphPad Prism version 5.01 (GraphPad Software Inc., San Diego, CA, USA). The experiment was performed a minimum of three times, and all data are shown as means with standard deviations (SDs). The data were analyzed for statistical significance using an unpaired Student's t-test or a Chi-squared test. P-value at <0.05 difference was considered to be statistically significant.
In order to find out the expression of B7-H3 in ovarian cancer and the location of B7-H3, we used immunohistochemistry and immunofluorescence methods. Immunohistochemistry was performed to detect the expression of B7-H3 in 41 cases of ovarian cancer tissues and 31 cases of normal ovarian tissues. Although no expression or low expression of B7-H3 can be found in ovarian cancer and there are some weak expression in normal tissues (
The relationship between clinicopathological factors and B7-H3 expression in patients with ovarian cancer is shown in
To characterize the role of B7-H3 in A2780 and SKOV3 cell growth we measured the cell proliferation rate
To verify the effect of B7-H3 on cell apoptosis after B7-H3 interference, we performed flow cytometry. The extent of apoptosis was investigated by measuring the amount of Annexin V stained cells, a marker for early stage apoptosis. The amount of all reagent-positive cells, which reflect the late stage apoptosis, were also measured. We found that when B7-H3 affected the early stage apoptosis of A2780 and SKOV3 cells were increased, and the late stage apoptosis although increased, was P>0.05 (
We next further assessed the influence of B7-H3 on ovarian cancer cell migration and invasion by transwell assays. As shown in the results, compared with the control groups, the number of cells in migration passing through the chamber was significantly reduced in A2780-sh-B7-H3 group and SKOV3-sh-B7-H3 group (
We described above the effect of silencing B7-H3 in ovarian cancer cells. In order to further verify the role of B7-H3 in ovarian cancer cells, HO8910 cell line was overexpressed by lentivirus. Using cell cycle assay, we observed that the growth of HO8910-B7-H3-EGFP was increased compared to NC cells (
The role of B7-H3 in ovarian cancer cells is described above, therefore, we investigated which signaling pathway was involved in this process. The Jak2/Stat3 pathway has been reported to be the key in cell migration, invasion and metastasis, and inhibition of Jak2/Stat3 signaling induced CRC cell apoptosis, cell arrest and reduced tumor cell invasion (
B7-H3, as an immunoregulatory molecule, playing different roles in different types of human cancers (
In the past studies, only few scattered stromal cells in non-neoplastic ovarian tissues expressed B7-H3 (
The Jak2-Stat3 pathway plays a significant role in biological function on various human cancers. Zhang
In conclusion, results of this study identified that B7-H3 is related to tumor progression in ovarian cancer and probably can be used as an indicator in clinic in the future. The limitations are that we did not conduct animal experiments in this research due to the lack of experimental funds and the limitations of laboratory conditions. We consider this aspect well worth studying. As mentioned above, the specific role of B7-H3 and its mechanism need further studies.
The present study was funded by the National Natural Science Foundation of China (NSFC; 81572559), the Science and Technology Developing Planning of Shandong Province (2014GH218029) and the National Science and Technology Project of China (2015BAI13B05).
B7-H3 expression in ovarian tissues and its expression location. B7-H3 immunostaining in ovarian cancer tissues (A) Weak positive, moderate positive, strong positive (magnification, ×200). (B) B7-H3 was stained in the cytoplasm of cells, while DAPI was stained in the nucleus (magnification, ×200).
Effect of silencing B7-H3 on cell proliferation. (A and B) The cell viability of sh-B7-H3 group and NC group was tested at 24 h, 2, 3, 4 and 5 days by MTT assay. (C and D) A flow cytometry was used to detect the cell cycle of cells treated with stable silencing and the percentage of the S and G1/G2 phases in the cell cycle is used to indicate cell proliferation; *P<0.05, **P<0.01, ***P<0.001 compared with the control groups.
Effect of B7-H3 on cell apoptosis. (A and B) Knockdown of B7-H3 by siRNA induced apoptosis of the A2780 cell line and SKOV3 cell line. (C and D) Early apoptosis was used to express the apoptosis rate that is shown in (A and B).
The changes of related protein molecules after silencing of B7-H3. (A and B) Western blot analysis for B7-H3 and after silencing B7-H3 the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl and the pro-apoptotic protein Bax and caspase-8, cleaved caspase-8 were measured by western blotting. (C and D) Quantification of the protein expression levels as shown in (E and F); *P<0.05, **P<0.01, ***P<0.001 compared with the control groups.
Silencing B7-H3 effects cell migration and invasion. (A and B) Silencing B7-H3 reduces the migration and invasion potential of A2780 and SKOV3 cell lines. (C and D) Quantification of the images of (A and B); *P<0.05, **P<0.01, ***P<0.001 compared with the control groups.
Effect of overexpression of B7-H3 on HO8910 cell line invasion, migration and proliferation. (A) Overexpression of B7-H3 in the HO8910 cell line increased the growth of cells that was analyzed by flow cytometry. (B) Quantification of the cell cycle. (C and E) Overexpression of B7-H3 effect on A2780 and SKOV3 cell migration and invasion by transwell chamber assay. (D and F) Quantification of the images of (D and E) (magnification, ×200); *P<0.05, **P<0.01, ***P<0.001 compared with the control groups.
Overexpression of B7-H3 affects the expression of related protein molecules. (A) Western blot analysis of related proteins after B7-H3 overexpression. (C) Comparison of relative protein levels from (A). (B) Expression of Jak2-Stat3 pathway proteins and apoptosis regulator proteins from the negative control cells (HO8910-NC), the B7-H3 overexpressing cells (HO8910-B7-H3-EGFP) to the AG490 treated B7-H3 overexpressing cells (HO8910-B7-H3-EGFP+AG490) were performed by western blotting. (D) Quantification of the expresssion of relative proteins; *P<0.05, **P<0.01, ***P<0.001 compared with the control groups.
Relationship between B7-H3 expression on tumor cells and clinicopathological factors.
Expression of B7-H3 | ||||
---|---|---|---|---|
Factors | No. | Negative | Positive | P-value |
B7-H3 | ||||
Ovarian cancer tissue | 41 | 11 | 30 | |
Normal ovarian tissue | 31 | 24 | 7 | |
Age (years) | 0.195 | |||
≤50 | 16 | 2 | 14 | |
>50 | 25 | 9 | 16 | |
Size (cm) | 0.903 | |||
≤5 | 18 | 5 | 13 | |
>5 | 23 | 6 | 17 | |
Histology | 0.920 | |||
Serous CA | 28 | 7 | 21 | |
Endometrioid CA | 7 | 1 | 6 | |
Vascular Invasion | 0.300 | |||
− | 36 | 11 | 25 | |
+ | 5 | 0 | 5 | |
Distant metastasis | ||||
− | 14 | 7 | 7 | |
+ | 27 | 4 | 23 | |
Differentiation | 1.000 | |||
Low | 2 | 0 | 2 | |
Moderate/high | 33 | 8 | 25 | |
Clinical stage | 0.057 | |||
I/II | 13 | 6 | 7 | |
III/IV | 28 | 5 | 23 |
Values in bold, P<0.05.