Objective: To construct a eukaryotic expression vector of stathmin gene and investigate its effect on esophageal squamous cell carcinoma (ESCC) EC9706 cell line.
Methods: Stathmin cDNA coding sequence was amplified by RT-PCR and cloned into a eukaryotic expression vector pcDNA3.1(+). EC9706 cells were transfected with this recombinant plasmid pcDNA3.1-stathmin by lipofectamine. And the stable transfectants were selected with G418 medium. Stathmin protein expression was detected with Western blot in transfected EC9706 cell lines. Morphologic change of stable transfectants was observed under microscope. The proliferation of transfected cells was measured by cell counting, MTT and in vitro formation assay of flat. Flow cytometry was used to detect the cell cycle. Nude mice were adopted to investigate the in vivo tumorigenic characteristics of the transfected cells.
Results: A 450 bp coding sequence of stathmin cDNA was amplified by RT-PCR and then cloned into pcDNA3.1(+) plasmid to harvest the recombinant plasmid pcDNA3.1-stathmin. The recombinant plasmid pcDNA3.1-stathmin and blank vector were transfected respectively into EC9706 cells. The up-regulated expression of stathmin protein was validated by Western blot (P < 0.01). Compared with control, EC9706 cells transfected with pcDNA3.1-stathmin appeared swollen and multi-nuclear with a cell mitotic arrest; doubling generation time of pcDNA3.1-stathmin transfectants was prolonged (25 - 28 h); The in vitro cell proliferation ability and clone formation rate (34.5% ± 6.9%) decreased, cell cleavage was blocked at G(2)/M phase (21.7% ± 3.4%) and the oncogenicity of inoculated cells in nude mice decreased (all P < 0.01).
Conclusions: The up-regulated expression of stathmin protein triggered by the recombinant plasmid pcDNA3.1-stathmin can inhibit the proliferation and oncogenicity of ESCC EC9706 cells. This molecule may be a promising therapeutic target in ESCC patients.