The 656C and 725C are two important sites in gene STGC3 for its negative regulation on cell growth

The aim of this study was to analyze the functional sites of the nasopharyngeal candidate tumour suppressor gene STGC3. Recombinant plasmid pcDNA3.1(TM)/myc-His B-STGC3 was constructed. Site-directed mutagenesis of pcDNA3.1(TM)/myc-His B-STGC3 plasmid at sites of C656G, C725T and T913G was induced by the Stratagene mutagenesis method. Recombinant plasmids with point mutations at C656G, C725T and T913G of gene STGC3 were named as STGC3-C656G, STGC3-C725T and STGC3-T913G, respectively. CNE2 cell lines stably expressing wild and mutant STGC3 genes were established. STGC3 expression was detected by Western Blotting and immunocytochemistry. Cell proliferation was analyzed by 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and trypan blue staining. Flow cytometry analysis was used to assess apoptosis of CNE2 cells. Bax protein expression was detected by Western Blotting. Proteins of wild-type and mutant STGC3 genes were expressed in the cytoplasm and nucleus of CNE2 cells. Compared with the control groups, in cells stably expressing wild-type STGC3 and STGC3-T913G genes, cell proliferation was significantly inhibited, whereas levels of apoptosis and Bax protein expression were significantly increased. However, the cell proliferation, apoptosis and Bax protein expression in cells stably expressing STGC3-C656G and STGC3-C725T genes were not significantly different from those in the control groups. Our results suggest that mutations at 656C and 725C, but not 913T, abolished the effects of the wild-type STGC3 gene on CNE2 cells and that the 656C and 725C were important sites in gene STGC3 for its negative regulation on cell growth.