The Effecct Of Tumor Suppressor Gene TSLC1 On Invasion And Metastasis Of Human Osteosarcoma

Objective :1. To establish a nude mice model of human osteosarcoma spontaneous lung metastasis. 2. To establish the osteosarcoma cell sublines which stably expressing TSLC1 protein and to identify its biological characteristics. 3. The effecct of TSLC1 on invasion and metastasis of osteosarcoma cell MG63.Methods: 1. The growth of human osteosarcoma cell sublines M8 and M6 was determined by MTT assay. 2x10~7 cells were injected into the tail vein of nude mice. Mice were sacrificed started on week 4 after injection, and lung metastases were evaluated under both macroscopic and microscopic observation with HE staining and immunohistology. 2. Full length of TSLC1 cDNA was amplified from RNA of normal human lung by RT-PCR, cloned into pCI-neo expression vector. The recombinant plasmid pCI-TSLC1 was identified with restriction enzyme and sequencing, and then was stably transfected into M8 cells with Lipofectamine 2000. The positive clones were developed by selection by G418. Biological characteristics of one of the 16 cell lines, namely, the M8T were studied. 3. Human osteosarcoma cell subline M8T was stably transfected with exogenous gene TSLC1. Cell growth was analyzed with MTT assay. FACSort flow cytometry analysis was performed to assess the cell cycle distribution and apoptosis. Transwell was performed to access the ability of migration and invasion of M8T. 1×10~7cells were injected into the two flanks of nude mice. The tumor growth was monitored once a week. 2×10~7 cells were injected into the tail vein of nude mice. Mice were sacrificed started on week 4 after injection, and lung metastases were evaluated under both macroscopic and microscopic observation with HE staining.Results: 1. The growth of low-metastatic subline M6 was lower than high-metastatic sublines M8. 17 mice after injected M8 had occurred lung metastases while just 5 mice had occurred in M6 group. Moreover, the expression of Bcl-2 and cyclin E1 of lung metastasic tissue were positive. 2. The eukaryotic expression vector pCI-TSLC1 was successfully constructed and the stable cell subline highly expressing TSLC1 protein was obtained. The genetic stability and purity of the cell population were confirmed. The kinetics and genetic of M8T was stable. The DNA product amplified from total RNA of osteosarcoma cell sublines reveals that ectogenous gene TSLC1 has integrated into the genomes of the M8 cell. The cell line M8T was not contaminated by microorganisms. 3. The growth of TSLC1-transfected cells M8T was significantly suppressed in vitro, displaying that the amount of G0-G1 cells increased and the amount of S phase cells decreased significantly, which suggested a G0-G1 cell cycle arresting. The exogenous gene TSLC1 could induce cell apoptosis. The M8T cells proliferation and the ability of invasion or migration which d were significantly reduced in vitro in comparison to the control. Moreover, tumorigenicity of M8T cells was suppressed in vivo, Lung metastases of nude mice occurred later and less than the control.Conclusions: 1. A mouse model for human osteosarcoma cancer spontaneous lung metastasis can be established by injection different ability of metastasis MG63 cells into tail vein. 2. The stable osteosarcoma cell sublines highly expressing TSLC1 has successfully established, which provided a basis for further exploring the roles of TSLC1 in osteosarcoma. 3. The ability of migration and invasion of high-metastatic osteosarcoma cell subline M8 was significantly suppressed by TSLC1 both in vitro and in vivo. TSLC1 would serve as a good candidate molecular target for the treatment of osteosarcoma.