The Expression And Mechanism Of B7-H3 In Osteosarcoma Microenvironment

Object:Osteosarcoma, which is a high-grade malignant bone neoplasm, occurs mainly in adolescents and children, and it is very difficult for clinical treatment of osteosarcoma. Based on specific location in bone and unclear pathogenesis, it is not a significantly increases for cure rate. The molecular pathways contributing to osteosarcoma development and progression have recently been discovered, and various studies have been carried out to investigate the genes that are involved in osteosarcoma growth, such as SV40 T, p53, c-Fos, Rb, p62, et al. B7-H3 is one of the B7 costimulatory molecules superfamily members, which was found by Chapoval. A large number of studies have been done, which found the expression of B7-H3 in cancer was closely associated with the tumor growth and metastasis. Moreover, some researchers found that B7-H3 can mediate cell-cell and cell-extracellular matrix protein adhesion, the adhesion function of B7-H3 provides new clues of the molecules in tumor invasion and metastasis. This study aimed to explore the role of B7-H3 in osteosarcoma cancer bone metastasis, and lay the foundation for the study of B7-H3 in osteosarcoma metastasis mechanism.Method:Three parts in this study:(1) The B7-H3 over expression vector, p IRES2-B7-H3-EGFP, was transfected into the mouse osteosarcoma cell K7M2-WT to get the cell line(K7M2-WT(B7-H3)) with stable over-expression of B7-H3. And the transfected cells were then identified by the real time PCR, reverse transcription PCR(RT-PCR) and flow cytometry(FCM).(2) For bio-function investigation of K7M2-WT(B7-H3), the cell adhesion assay was employed to detect the cell adhesion capability, the CCK-8 test for 7 days were employed to detect the cell proliferation, the wound scrape assays were employed to detect the cell migration ability, transwell invasion assays were employed to detect the cell invasiveness, apoptosis assays were used to detect the cells resistance to the adverse environment, respectively.(3) The animal model of high-expressive B7-H3 was established, and we researched the osteosarcoma tumor’s size, morphology by histology(H&E), and B7-H3 content by western blot, using this animal model.Result:(1) On the levels of mRNA and protein, the B7-H3 expressions were significantantly higher in the transfected mouse osteosarcoma cell K7M2-WT than in the untransfected group(K7M2-WT), detected by real-time PCR, RT-PCR and FCM.(2) By comparing the results of the transfected and untransfected cells, we found that the abilities of K7M2-WT(B7-H3) in cell adhesion, proliferation, migration, invasion and anti-apoptotic were significantly more than the untransfected group K7M2-WT. In addition, osteogenic differentiation of K7M2-WT was not changed by regulation of B7-H3, for ALP test in 7 days.(3) In animal models, the tumor size of K7M2-WT(B7-H3) group was bigger than that the K7M2-WT group. And the expression of B7-H3 in mouse osteosarcoma was significantly higher than the normal group by immunohistochemistry and western blot.Conclusion:We successfully construct the mouse osteosarcoma cell K7M2-WT(B7-H3) with the high-expression of B7-H3. The overexpression of B7-H3 in K7M2-WT(B7-H3) can improve osteosarcoma cells’ adhesion, proliferation, migration, invasion and anti-apoptotic abilities, but not change ALP content for osteogenic differentiation. The expression of B7-H3 in mouse osteosarcoma was significantly higher than the normal group in animal models. It signified B7-H3 could suppress the tumor immunity and facilitate cancer progression.