| Osteosarcoma is the most common primary malignant bone tumors in addition to plasma cell tumors. It has a high degree of malignancy and could be occur metastasis in the earlier stage. Among them, the pulmonary metastasis rate is highest, and osteosarcoma has a high mortality rate and seriously affected the prognosis of adolescent patients. Therefore, understanding the molecular biological characteristics of osteosarcoma, and finding a new molecular marker will be helpful to properly assess the biological behavior of osteosarcoma, provide the basis for individualized treatment of osteosarcoma, and be of great significance to improve the survival rate of osteosarcoma patients. SNAT1is a neutral amino acid transporter protein coupled by Na+ions, which is mainly mediated by small molecules such as glutamic acid, alanine, proline and glutamine. SNAT1is expressed in the human brain and spinal cord neurons, but did not be found in other substantive organs such as liver, kidney, and spleen.Recent researchs have been shown that SNAT1was overexpressed in several malignant tumors, indicating that SNAT1is an independently cancer risk factor. But there are many arguments about the biological characteristics of SNAT1in tumor field. It is still not clear about the mechanism of SNAT1in the regulation of physiological functions, gene expression, and tumor metastasis. Furthmore, it has not been reported that the expression and function of SNAT1in osteosarcoma. In the present study, we investigated that the expression of SNAT1protein levels in osteosarcoma tissues, and the relationship between SNAT1expression and clinicopathological parameters. Meanwhile the function and possible mechanism of SNAT1expression in osteosarcoma cell lines were been studied from in vitro and in vivo expriments. Our study will provide a new marker for prognosis and metastasis of osteosarcoma, and a new target for the clinical biological therapy of osteosarcoma.Part I The expression and clinical significance of SNAT1in human osteosarcoma tissueObjective:To study the expression of SNAT1in human osteosarcoma and its correlation with the clinicopathology, and explore the biological significance of SNAT1expression in human osteosarcoma.Methods:Forty osteosarcoma patients and ten osteochondroma patients were randomly selected from Shanghai Changzheng Hospital and Changhai Hospital from January2006to December2009. The specimens were obtained by surgical resection and confirmed by pathological diagnosis. Then, immunohistochemistry was applied to detecte the exprssion levels of SNAT1protein. Combined with clinical data, statistical analysis on the experimental results will explore the clinical significance of SNAT1expression in human osteosarcoma.Results:1. The expression of SNAT1protein in the tissue of osteosarcoma was significantly increased compared to osteochondroma. The positive staining ratio of SNAT1in primary osteosarcoma tissue was85%(34/40), while it was40%in osteochondroma.2. The expression of SNAT1in osteosarcoma was associated with distant metastasis and prognosis, but not with the size of tumor, age and sex of patients.3. The expression of SNAT1was negatively correlated with survival period of patients. The prognosis of patients with higher expression of SNAT1was worse than those with lower expression of SNAT1. SNAT1may be an independent prognostic factor for the prognosis of osteosarcoma.Conclusions:SNAT1overexpression may play an important role in the development of osteosarcoma, which is closely associated with the prognosis of patients with osteosarcoma.Part â…¡ Functional studies ofSNAT1in cell proliferation of osteosarcomaObjective:To explore the function of SNAT1expression in the cell proliferation of osteosarcoma.Method:1. Design a shRNA sequence specifically targeted to SNAT1gene,which was cloned to a lentiviral vector. After identification with restriction digestion and sequencing, the lentiviral supernatant was obtained by the packaging in293T cells. Then the osteosarcoma cell line MG-63-2was infected with lentiviral and the infection efficiency was indentified under fluorescence microscope.2. Construct successfully osteosarcoma cell lines with shSNATl or shNon. The interference effect of shSNATl was identified using real-time PCR and western blotting.3. The cell proliferation regulated by shSNAT1was detected using MTT assay in MG-63-2cells.Results:1. The lentiviral carrying shRNA specifically targeted to SNAT1was established, which could downregulate the expression of SNAT1.2. The downregulation of SNAT1suppressed the cell proliferation in osteosarcoma cells, suggesting that SNAT1could promote cell proliferation in tumors. Conclusion:Downregulation of SNAT1mediated by shRNA could suppress the proliferation of tumor cells, suggesting that SNAT1promotes cell proliferation as an oncogene.Part m The study of SNAT1in the tumorigenesis of osteosarcoma cells in nude miceObjective:To investigate the effects of SNAT1expression in the tumorigenesis of osteosarcoma cells in nude mice.Methods:The osteosarcoma cells MG-63-2with stable expression of shSNAT1or shNon were inoculated into nude mice, and the growth of tumor was recorded. The tumor tissues were isolated for immunohistochemistry assay at the terminal stage.Results:1. The tumorigenesis of osteosarcoma cell transfected with shSNAT1were significantly reduced in nude mice, and the tumor volume was also less than the control group (shNon).2. Downregulation of SNAT1expression inhibited tumor formation in nude mice by immunohistochemistry assay. Several pathways may be involved in the function of SNAT1in in tumorigenesis and development of osteosarcoma.Conclusion:Downregulation of SNAT1expression inhibits the tumorigenesis of osteosarcoma in nude mice, suggesting that SNAT1plays an important role in the tumorigenesis and development of osteosarcoma. SNAT1will be a new target for the molecular diagnosis and gene therapy. |
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