Conceived and designed the experiments: ZW LM GQ YW. Performed the experiments: ZW LM DC DH YD. Analyzed the data: ZW LM DC DH YD GQ YW. Contributed reagents/materials/analysis tools: DH YD. Wrote the paper: ZW LM YW.
The authors have declared that no competing interests exist.
BMI-1 is a member of the polycomb group of genes (PcGs), and it has been implicated in the development and progression of several malignancies, but its role in osteosarcoma remains to be elucidated.
In the present study, we found that BMI-1 was overexpressed in different types of osteosarcomas. Downregulation of BMI-1 by lentivirus mediated RNA interference (RNAi) significantly impaired cell viability and colony formation
These findings suggest a crucial role for BMI-1 in osteosarcoma pathogenesis.
Primary osteosarcoma is the most common bone tumor that predominantly develops in adolescents and young adults
BMI-1 is a member of the polycomb group family of transcriptional regulators that was originally identified as an oncogenic partner of c-Myc in murine lymphomagenesis
Although BMI-1 has been implicated as an oncogenic player, very little is known about the exact function of BMI-1 in osteosarcoma growth and progression. In this study we have investigated whether BMI-1 functions as an oncogene in osteosarcoma. Our findings confirm that BMI-1 is highly expressed in osteosarcoma cells, and it promotes tumor growth
As elevated BMI-1 expression was found in several types of human cancers, we first determine the expression of BMI-1 protein in osteosarcoma through immunohistochemical analysis. BMI-1 expression was not detected in 10 patients with noncancerous bone tissues. In contrast, positive expression of BMI-1 was observed 18/32 in osteosarcoma, 9/22 in chondrosarcoma, 3/9 in Ewing's sarcoma and 5/27 in osteochondroma (a benign neoplasm) (
BMI-1 immunoreactivity was localized in both the nucleus and cytoplasm. Images of positive BMI-1 staining in nucleus of osteosarcoma, osteochondroma and chondrosarcoma, and in cytoplasm of Ewing's sarcoma were shown (×400). Negative BMI-1 staining in a non-cancerous tissue sample served as control.
To suppress BMI-1 expression in osteosarcoma cells, short harpin RNA (shRNA) targeting BMI-1 gene was designed and inserted into the recombinant lentivirus plasmid. To verify that the effects of RNA interference (RNAi) are specific, we prepared a BMI-1 construct bearing triple-point mutation in the 19-bp sequence that served as target for small interfering RNA (siRNA)-mediated knockdown. The mutation conserved the amino acid sequence of the BMI-1 protein but rendered its expression insensitive to inhibition by the siRNA. Effeciency of lentivirus infection was more than 90% as evidenced by GFP expression 3 days after infection (
(A) Representetive graphs of SAOS-2 cells infected with indicated lentivirus at MOI of 10 were shown (×400). Following infection of cells with indicated lentivirus for 5 days, BMI-1 mRNA levels were measured with real-time PCR (B), and protein levels were detected by Western blot analysis (C). *:
To elucidate the role of BMI-1 in osteosarcoma proliferation and tumorigenesis, the growth of each lentivirus infected SAOS-2 cells were first examined by MTT assay.
(A) The monolayer growth rates of SAOS-2 cells from different groups were determined by MTT assay. (B) Downregulation of BMI-1 inhibited colony-forming ability. Photographs of plates and representative colonies were shown. Histogram was the average number of colonies in each plate. (C) Downregulation of BMI-1 reduced the tumorigenicity of SAOS-2 cells. The change of tumor volume during 5 weeks of inoculation was measured. Representative pictures of tumor bearing mice and tumors were shown (n = 10). Values represent the mean (standard error of the mean) from at least three separate experiments. *:
We also examined the role of BMI-1 in osteosarcoma cell migration. SAOS-2 cells treated with BMI-1 siRNA-expressing lentivirus demonstrated decreased ability to migrate through 8-µm-pore-size membranes that were not coated with Matrigel (
(A) Statistical plots of haptotactic migration assay. Columns, mean of three individual experiments; bars, SD;*:
It has been reported that knockdown of BMI-1 makes nasopharyngeal carcinoma cells more sensitive to 5-FU treatment and that depletion of BMI-1 enhances 5-FU-induced apoptosis
After infection, SAOS-2 cells were treated with 10 µg/ml cisplatin for 24 h and subjected to Annexin V-PI Apoptosis analysis (A) and measurement of caspase-3 and caspase-9 activities (B). (C) Western blot analysis of p-AKT, AKT, BCL-2 and Bid protein. *:
We subsequently explore the possible molecular mechanism by which BMI-1 protected osteosarcoma cells from cisplatin induced apoptosis. Caspases are important regulators of apoptosis. Therefore, the involvement of caspase-3 and caspase-9 in cisplatin-induced apoptosis was investigated. In the BMI-1 knockdown cells, after treatment with cisplatin (10 µg/ml), caspase-3 and caspase-9 activities were observed to be increased by 2.4-fold and 2.7-fold, respectively (
Further more, expression levels of total-AKT, phospho-AKT (p-AKT), Bid and BCL-2 were examined in BMI-1 knockdown cells, BMI-1 rescued cells and control cells. Immunoblot analysis showed that the knockdown of endogenous BMI-1 led to significant reduction in the levels of phospho-AKT and BCL-2, whereas pro-apoptotic protein Bid was elevated in BMI-1 knockdown cells. Nevertheless, wobble mutant of BMI-1 rescued the expression of phospho-AKT, BCL-2 and Bid protein in SAOS-2 knockdown cells (
In the present study, we found that BMI-1 was highly expressed in malignant osteosarcoma, and it is essential for cancer cell proliferation, migration and
Recently, it is reported that osteosarcoma might originate from somatic stem cells
Due to the therapeutic advantages, such as high efficiency, mild side effects and easy administration, cisplatin is still one of the most common used agents in chemotherapy. However, resistance to this drug is also often observed, and therefore enhancing the sensitization of cancer cells to cisplatin-induced apoptosis has become an important strategy for chemotherapy. We found that the depletion of BMI-1 in SAOS-2 cells, in which BMI-1 is highly expressed, resulted in an increased sensitivity of these cells to cisplatin. Pro-caspase-3, an effector caspase of apoptosis, is one of the effector pro-caspases activated by caspase-9
It is well known that p53 induces apoptosis in response to DNA damage, and SAOS-2 cells have a wild-type p53. Therefore we hypothized that p53 might be responsible for increased apoptosis after cisplatin treatment in BMI-1 knockdown cells. However, mRNA and protein levels of p53 and its effecter p21 were downregulated upon BMI-1 knockdown (data not shown). We assumed that the apoptosis in the SAOS-2 cells reported herein was p53-independent. Activation of the PI3K/AKT pathway enhances resistance to apoptosis is observed in a wide variety of cancers
In summary, our results demonstrated that BMI-1 functions as an oncogene in osteosarcoma and that it promotes tumorigenicity and resistance to chemotherapy. Our findings suggest that the combination of cisplatin treatment and BMI-1 depletion could be a therapeutic strategy for osteosarcoma.
Ninety patients who were histologically confirmed as having osteosarcoma, chondrosarcoma, Ewing sarcoma, osteochondroma, and normal bone tissues were enrolled Peking Union Medical College Hospital, China. The clinicopathologic variables such as gender, age, the histologic type and the status of the resection margin were retrospectively reviewed on the basis of the medical records. All patients received no treatment before surgery. The samples were used with the written informed consent from patient and the approval of the ethic committee of Peking Union Medical College Hospital, China.
Human embryonic kidney 293T (HEK293T) cell line and the osteosarcoma cell line (SAOS-2) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured in Dulbecco's modified Eagle's medium (Gibco RL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin.
The siRNA sequence for BMI-1 (
Total RNA was prepared using Trizol reagent (Gibco RL, Grand Island, NY, USA) according to the manufacturer's instruction. Five µg of total RNA was used to synthesize the first strand of cDNA using SuperScript II RT 200 U/µl (Invitrogen, Carlsbad, CA, USA). BMI-1 mRNA expression was evaluated by real-time PCR on an ABI Prism® 7300 (Applied Biosystems, Foster City, CA, USA) with SYBR Green PCR core reagents. β-actin was applied as the input reference. The following primers were used: BMI-1:
The slides were deparafinized, rehydrated, then immersed in 3% hydrogen peroxide solution for 10 min, heated in citrate buffer, pH 6.0, at 95°C for 25 min, cooled at room temperature for 60 min. The slides were blocked by 10% normal goat serum at 37°C for 30 min, and then incubated with rabbit polyclonal antibody against BMI-1(1: 200, ProteinTech Group, Chicago, IL, USA) for overnight at 4°C. After washing with PBS, the slides were incubated with biotionylated second antibody (diluted 1: 100) for 30 min at 37°C, followed by streptavidin-peroxidase (1: 100 dilution) incubation at 37°C for 30 min. Immunolabeling was visualized with a mixture of DAB solution. Counterstaining was carried out with hematoxylin. Samples were scored positive when more than 10% of the cells reacted with the anti-BMI-1 antibody and presented cytoplasm staining.
Proteins were separated by SDS–PAGE, transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Blots were blocked and then probed with antibodies against BMI-1(1: 500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), AKT (1: 1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-AKT (Ser473; 1: 400 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bcl-2 (1: 500 dilution; Cell Signaling Technology Inc., Beverly, Massachusetts, USA), Bid (1: 500 dilution; Cell Signaling Technology Inc., Beverly, Massachusetts, USA), Mouse anti-GAPDH, (1: 5000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies and visualized by super ECL detection reagent (Applygen, Beijing, China).
Briefly, cells from different groups were seeded at an initial density of 5×104 cells/ml in 96-well plates for 24 h. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was added into each well at a final concentration of 5 mg/ml for 4 h. DMSO was then added to stop the reaction and measured with an ELISA reader (Bio-Rad, Hercules, CA, USA) at a wavelength of 570 nm. Viability of cells was expressed relative to theoretical absorbance (A).
To assay monolayer colony formation, 200 infected cells were plated in 6-well plates. After 2 weeks, cells were fixed with methanol and stained with Giemsa. The number of colonies was counted.
To assay tumor formation in nude mice, cloned pools of BMI-1 knockdown cells, scrambled RNAi cells, BMI-1 rescued cells, and parental SAOS-2 cells were trypsinized, counted, and resuspended in 1×PBS. One hundred microlitre of 1×PBS containing 106 cells was then infected into 4-to 5-week-old female nude mice which were purchased from Weitonglihua Company (Beijing, China). The mice were examined for subcutaneous tumor growth and sacrificed 5 weeks after injection. Animal study protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Peking Union Medical College Hospital, China [the permit number: SCXK (Jing) 2007-0001].
For haptotactic cell migration assay, 1×104 cells from different groups were seeded on a fibronectin-coated polycarbonate membrane insert (6.5 mm in diameter with 8.0 µm pores) in a transwell apparatus (Costar, Cambridge, MA, USA) and cultured in DMEM. FBS was added to the lower chamber. After incubation for 12 h at 37°C in a CO2 incubator, the insert was washed with PBS, and cells on the top surface of the insert were removed by wiping with a cotton swab. Cells that migrated to the bottom surface of the insert were fixed with methanol and stained by Giemsa and then subjected to microscopic inspection. Cells were counted based on five field digital images taken randomly at ×200.
After lentivirus infection and cisplatin (10 µg/ml) treatment, cells in each well were harvested and cell apoptosis was determined by Annexin V-FITC/PI staining method. Tests were performed in triplicate for each sample, and analyses were performed by FAC-Scan flow cytometer (Becton Dickinson, San Jose, CA, USA) in accordance with the manufacturer's guidelines.
The activation of caspase-3 and caspase-9 were determined in SAOS-2 cells with the colorimetric kit (Nanjing kaiji Bio-Tek Corporation, China). Cells (3×106) were harvested and washed twice with PBS. After the cells were lysed, 50 µl of 2x reaction buffer was added followed by the additional 5 µl of caspase-3 or caspase-9 substrate and incubated at 37°C for 4 h. The plate was then read with an ELISA reader (Bio-Rad, Hercules, CA, USA) at 405 nm.
All statistical analyses were performed using SPSS13.0 software. The differences between groups were compared using Student's t-test, and data were expressed as mean ± SD. Statistical difference was accepted at