Down-regulation Of S100A9 Inhibits Osteosarcoma Cell Growth Through Inactivating MAPK And NF-κB Signaling Pathways

Background and Objective:Osteosarcoma(OS) is well-known for poor prognosis due to its high incidence of proliferation and metastasis. Researches have provided valuable insights into the tumorigenesis of S100A9 in some cancers. We aimed to understand the expression level, functions and mechanisms of S100A9 in human osteosarcoma for the first time.Methods:1. The clinical pathological data was collected.2. Tumor tissue microarrays and real time PCR were used for analyzing the clinical pathologic specimens.3. Knockdown of S100A9 was used in three human OS cells with siRNA interference.4. The level of mRNA and protein about S100A9 were tested by western blotting and PCR in the OS cells after knockdown of S100A9.5. CCK8 was used to examine the cell proliferation after knockdown of S100A9 in vitro.6. Flow cytometry analysis was used to test the cell cycle distributions after knockdown of S100A9.7. Flow cytometry analysis was used to test the cell apoptosis after knockdown of S100A9.8. Transwell cell migration assay was used to examine the changes in the ability of cell migration.9. Transwell cell invasion assay was used to examine the changes in the ability of cell invasion.10. Xenograft tumor model was established to examine the ability of cell proliferation in vivo.11. The proliferation index(PCNA and Ki67) were tested in xenograft tumor.12.MAPK signaling pathway and NFκB signaling pathway were tested by western blotting and enzyme activity assay after knockdown of S100A9.13. p21, p27, CDK2, CDK4 were tested by western blotting and enzyme activity assay knockdown of S100A9.Results:1. A total of 120 osteosarcoma(OS) patients who came from the First Affiliated Hospitals of Chongqing Medical University, Second Affiliated Hospitals of Chongqing Medical University, Children’s Hospital of Chongqing Medical University(Chongqing, China) and Tumour hospital of Guizhou(Guizhou, China) between 2005 and 2014. The patients weredivided into I, II and III grades according to the Enneking staging system(G-Histologic Grade, T-Anatomic site, M-Metastasis). The data confirmed S100A9 was over-expression in OS and the high-grade tissues presented a higher expression level of S100A9 than low-grade tissues according to the GTM staging system.2. 95% of the OS samples(114 of 120) were positively stained for S100A9. There were no statistical significances in distribution of S100A9 staining, gender, age, sites according to the staining results. The results about PCR also agreed with the immunohistochemistry.3. Three OS cell lines(U2OS, MG63, 143B) were transfected with S100A9-siRNA. Compared with cells transfected with empty vectors groups and blank control groups, the expression levels of S100A9 protein and mRNA were apparently reduced in the siRNA-S100A9 vectors groups according to the results of western blot and real time PCR.4. CCk-8 assays demonstrated that down-regulation of S100A9 reduced the proliferation of the three OS cell lines in 1, 2, 3 and 4 days(p<0.05).5. Flow cytometric analysis was used for searching the reason why down-regulation of S100A9 could inhibit OS proliferation. The percentage of G0/G1 and S phase cells in each group was shown in Figure.2D. It was revealed that knockdown of S100A9 could contribute to accumulation of OS cells in G0/G1 phase in comparison with empty vectors groups and blank control groups(p<0.05).6. Knockdown of S100A9 had no effect on cell apoptosis(p>0.05).7. The number of cells migrated across the polycarbonate membrane was also reduced after knockdown of S100A9(p<0.05).8. The number of cells invaded across the polycarbonate membrane was also reduced after knockdown of S100A9(p<0.05).9. We established the effects of S100A9 on OS growth in vivo using a xenograft model. Down-regulation of S100A9 in OS cells significantly decreased tumor sizes, compared with empty vectors groups(p<0.05).10. The proliferating cell nuclear antigen(PCNA) and ki67 proliferation index for these solid tumor masses were calculated after 28 days implantation, the proliferation index of the tumors obtained from siRNA-S100A9 vectors groups was lower than that from the empty vectors groups(p<0.05).11. Western blot analysis revealed that the protein levels of phosphoERK1/2 MAPK, phospho-p50 NF-κB and phospho-p65 NF-κB in siRNA-S100A9 vectors groups were lower than those in empty vectors groups and blank control groups in OS cell lines. Enzyme activity assay also confirmed the above conclusions. However, the protein level and enzyme activity of phosphor-p38 MAPK in siRNA-S100A9 vectors groups presented no substantial changes compared with the other two groups.12. Western blot quantification revealed an increasing expression of p21 and p27 in siRNA-S100A9 vectors groups. In parallel, the enzyme activityassay of cyclin dependent kinase 2(CDK2) and cyclin dependent kinase4(CDK4) were suppressed in the three OS cells transfected with siRNA-S100A9Conclusion:S100A9 might be a significant role for predicting osteosarcoma prognosis and down-regulation of S100A9 could be used as a potential target for gene therapy.