| Object: To study expression of enhanced GFP gene in MG63 cells and isolation and characterization of their sublines with different metastatic potential. Gene expression profile in MG63 sublines with different metastatic potentialities were investigated by cDNA microarry, in order to identify new candidate genes related to the development, progress and metastasis of osteosarcoma. To compare the expression level of MTA1 in the higher and lower metastatic sublines of human osteosarcoma cells (MG63) and explore the correlation of expression level of MTA1-EGFP with in vitro invasion of osteosarcoma cells.Methods: After MG63 cells were transfected by enhanced GFP gene in liposome-mediated gene transfer, single cell clones were isolated using limited dilution method and selected by G418. Through preliminary electrophoretic technique and spontaneous metastasis assay in nude mice, two sublines (M6, M8) of MG63 cell were selected for further observation. The morphological feature, the cellular activity, cell cycle and in vitro growth and invasion assays in nude mice were observed. Total RNA of the lower metastatic osteosarcoma and the higher metastatic osteosarcoma was extracted, purified to mRNA and then reserve transcripted to cDNA respectively. M6 subline as experimental group and M8 subline as control group were investigated by cDNA microarray containig 8064 cDNA clones. The cDNA of M6 was labeled with cy3 and the cDNA of M8 was labeled with cy5. The two sublines were hybridized with the cDNA microarray. The hybridization signals were scanned by Generation III array scanner and analyzed by software Imagequant 5.0. The expression level of MTA1 in two sublines was detected by semi-quantitative RT-PCR, and then cell invasion assayand cell proliferation assay were used to evaluate the invasive capacity in vitro in two sublines; M6 subline of MG-63 cells was transfected with MTA1-EGFP full-length cDNA expression plasmid by lipofectamine; the changes of MTA1-EGFP expression and in vitro invasion potential were evaluated after transfection. Results: We found that there was some difference on electrophoretic mobility and in vitro invasive abilities between M6 and M8 subline, and then the population doubling time was 38.4h and 23.0h respectively. But there were no obvious differences in other biological feasures among the two sublines. There were 330 differentially expressed genes between M6 and M8. M6 subline was found 152 genes happened up regulation and 178 genes happened down regulation compare to M8 subline. To analysis these genes including to major of proliferate genes of cells according to gene function classify, Mode changed to express hyperplasia on M6 subline were inhibited. Secondly a large number of genes were changed correlated with regulating and the signal transduction, the obvious difference of gene expression exists between M6 and M8 sublines. These genes were all correlated to tumor occurrence and development. M8 subline expressed significantly higher level of MTA1 than that of M6 subline by RT-PCR. The invasive potentiality of M6 subline was increased after MTA1 gene transfection. Conclusion: The results show that the MG63 cell line consists of different subpopulations of cells with different metastatic properties. These sublines transfected by EGFP may be valuable for further study on the molecular mechanisms of cancer metastasis. The gene expression profile in osteosarcoma cell lines is an important index in the search of new candidate genes related to tumor occurrence, development and metastasis. There may be a relationship between MTA1 and invasive potentiality of human osteosarcoma cell, and the mechanism of MTA1 in osteosarcoma metastasis and its possible role in associated gene therapy deserve further studies. It is helpful for the further studies of MTA1 in human osteosarcoma cell metastasis.
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