The Expression Of PBF In Osteosarcoma And Its Effort In Proliferation And Metastasis Of Osteosarcoma Cell Line

BackgroundOsteosarcoma is a common primary malignant bone cancer, mainly occurring inchildren and teenagers. This disease has a high mortality，40%of the patients appeardistant organ metastases after surgery and adjuvant therapy. In precious study, we haveidentified a novel tumor-associated antigen called PBF by SEREX method.Papillomavirus binding factor (PBF) is a nuclear-cytoplasmic shuttling protein containing513amino acid which can encode one zinc finger structure. Moreover, as aligand-dependent transcription factor, it can antagonist compete with transcriptioninhibitor E-2to combine with HPV P2site, which regulate the transcription of genes bytranscription response elements. This experiment is designed to clarify the expression ofPBF in osteosarcoma and its effect on the biological function of osteosarcoma cells. ObjectiveTo investigate the expression of PBF in osteosarcoma and its clinical indicationsignificance; Constructed PBF expression vector; Up-regulated the PBF level in U2OSthrough transfection. To explicit the effects of PBF in proliferation and metastasis inosteosarcoma cell line in order to provide a theoretical and experimental basis for PBF asa new tumor antigen in early diagnosing, prognostic prediction and targeted therapy ofosteosarcoma.Methods1. Using immunohistochemistry to detect the expression of PBF in70cases ofosteosarcoma tissue specimens and30cases of osteochondroma tissue specimens.Analyzed its relationship and significance with osteosarcoma cells developmentstatistically.2. Construct PBF expression vector, and tranfect it into osteosarcoma cell line U2OS.Use Western-blot to test the differential expression of PBF in the cells tranfectedwith PBF expression vector.3. Test modified osteosarcoma cells biological function: To investigate the effect ofPBF overexpression on the proliferation in U2OS by MTT assay. To investigatethe effect of PBF overexpression on the invasion and migration in U2OS byTranswell experiment.Results1. Immunohistochemical results: The positive expression rate of PBF inosteosarcoma was89％(62/70), which was significantly higher than that in theosteochondroma(10％,P<0.01). The expression of PBF was a positivecorrelation to clinical Ennecking stages and metastasis of osteosarcoma.2. Constructed PBF expression vector. After transfected PBF expression vector intoU2OS, measured the result by Western-blot. The results revealed that PBFexpression was upregulated in cells of the experiment group compared with cellsof the control group.3. MTT results: upregulation of PBF increased the proliferation of U2OS cells. 4. Transwell experiment results: upregulation of PBF increased the invasion andmigration of U2OS cells.Conclusion1. PBF presented a positive higher expression in osteosarcoma, and the expressionlevel was related with the malignant degree and metastases.2. Upregulation of PBF expression significantly increased proliferation, metastasisin osteosarcoma cell line U2OS.3. PBF may play as a target antigen in the diagnosis and prognosis of osteosarcoma,its mechanism has yet to be studied.