Overexpression of BMI-1 promotes cell growth and resistance to cisplatin treatment in osteosarcoma

Abstract

Background: 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.

Methodology/principal findings: 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 in vitro and tumorigenesis in vivo of osteosarcoma cells. BMI-1 knockdown sensitized cells to cisplatin-induced apoptosis through inhibition of PI3K/AKT pathway. Moreover, BMI-1-depletion-induced phenotype could be rescued by forced expression of BMI-1 wobble mutant which is resistant to inhibition by the small interfering RNA (siRNA).

Conclusions/significance: These findings suggest a crucial role for BMI-1 in osteosarcoma pathogenesis.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Immunohistochemical staining of BMI-1.

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.

Figure 2. Targeted depletion of BMI-1 through lentivirus-mediated siRNA.

(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). *: P<0.01, compared to control cells. Con: non-infected, NC: non-silencing, KD: BMI-1 knock down, Rescue: BMI-1 wobble mutant.

Figure 3. Effects of BMI-1 siRNA on the in vitro proliferation, colony formation, and in vivo tumorigenicity of osteosarcoma cells.

(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. *: P<0.01, compared to control cells. Con: non-infected, NC: non-silencing, KD: BMI-1 knock down, Rescue: BMI-1 wobble mutant.

Figure 4. Downregulation of BMI-1 suppresses osteosarcoma cell migration.

(A) Statistical plots of haptotactic migration assay. Columns, mean of three individual experiments; bars, SD;*: P<0.01, compared to indicated cells. (B) Representative photos of haptotactic migration assay. Con: non-infected, NC: non-silencing, KD: BMI-1 knock down, Rescue: BMI-1 wobble mutant.

Figure 5. BMI-1 depletion sensitized cells to cisplatin treatment through PI3K/AKT pathway.

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. *: P<0.01, compared to control cells. Con: non-infected, NC: non-silencing, KD: BMI-1 knock down, Rescue: BMI-1 wobble mutant, Annexin V−/PI−: viable cells, Annexin V+/PI−: cells in early apoptosis, Annexin V+/PI+: cells in late apoptosis, Annexin V−/PI+: cells in necrosis.