Study On The Mechanisms Of Tumorigenesis And Recurrence Of Giant Cell Tumor Of Bone By Comparative Proteomics Analysis

Giant cell tumor of bone (GCTB) is commonly regarded as a low-grade or potential malignant tumor with unpredictable bio-behavior. The tumors show a tendency forsignificant bone destruction, high local recurrence, and occasionally lung metastasis.GCTB represents approximately 20% of all primary bone tumors in China, and the incidence rate of this tumor in China is nearly 3-5 folds higher than that in the Unite State and Europe. Surgical resection is the first choice of treatment for GCTB. However, postoperatively local recurrence rates reported in the previous study range from 27% to 55%, especially serious in the spine.The mechanisms of tumorigenesis and recurrence of GCTB remain still obscure. Formerly, the research topics of GCTB mainly focused on a minority of interesting proteins and or genes, such as RANKL and P53. The study of minority of several proteins and genes will not be sufficient to understand mechanisms of tumorigenesis and recurrence of GCTB, since the mechanisms of tumorigenesis and recurrence of GCTB may be a result of complex interaction processes of multiple genes, multipleproteins and multiple factors. There are also still no definitive tumor markers for GCTB that might be used to achieve early diagnosis and predict local recurrence. Tofully understand the molecular mechanisms that participate in the formation of GCTB, we analyzed primary tumors (ptGCTB) and recurrent tumors (rtGCTB) of GCTBcomparing with normal bone tissue by using activity-based comparative proteomics.The tissue samples were classified into 3 groups including ptGCTB, rtGCTB and normal bone tissue. 10 samples of each group were collected. After tissue specimenswere ground into powder in liquid nitrogen and lysed in lysis buffer, proteins lysates were extracted and submit to digest in solution. All the extracted peptides were desalted using a 1.3ml C18 solid phase extraction column. The peptides were driedusing a vacuum centrifuge and then re-suspended with loading buffer, separated andanalyzed by two-dimensional (2D) strong cation-exchange (SCX)/reversed-phase (RP)nano-scale liquid chromatography/mass spectrometry (2D-nanoLC/MS). The experiments were performed on a Nano Aquity UPLC system connected to an LTQ Orbitrap XL mass spectrometer equipped with an online nano-electrospray ion source. All MS/MS spectrums were identified by using SEQUEST against the human Swiss-Prot database.381 proteins were identified in the ptGCTB samples by nano 2D-LC/MS/MS. 51 proteins were found to be differentially expressed between ptGCTB and normal bone tissue, including 29 up-regulated and 22 down-regulated in the ptGCTB samples. Similarly, 38 differentially expressed proteins were also identified between rtGCTB and ptGCTB tissue, including 9 up-regulated and 29 down-regulated in the rtGCTB samples. We analysed the differentially expressed proteins among these three groups,according to Gene Ontology functional categories (including cellular component, biological process, and molecular function) and KEGG pathway by using the DAVID and KEGG database. The aboved data were also analysed by using cluster analysis. Protein-protein interaction networks were drawn.In conclusion, a reference proteome profiling of GCTB was constructed and will beserve as a basis for further studies of GCTB. The pathways of focal adhesion, ECM-receptor interaction and regulation of actin cytoskeleton may play important rolesin the mechanisms of tumorigenesis and recurrence of GCTB. The proteins screenedfrom above differentially expressed proteins, such as FN1, THBS1, DCN, GAPDH and VTN might be potential biomarkers for tumorigenesis and recurrence of GCTB.In the mean time, our study can offer technique support for the studies in the bonetumors.