title: The 656C and 725C are two important sites in geneSTGC3for its negative regulation on cell growth.
abstract: The aim of this study was to analyze the functional sites of the nasopharyngeal candidate tumour suppressor geneSTGC3. Recombinant plasmid pcDNA3.1TM/myc-His B-STGC3was constructed. Site-directed mutagenesis of pcDNA3.1TM/myc-His B-STGC3plasmid 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 geneSTGC3were named asSTGC3-C656G,STGC3-C725T andSTGC3-T913G, respectively. CNE2 cell lines stably expressing wild and mutantSTGC3genes 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 mutantSTGC3genes were expressed in the cytoplasm and nucleus of CNE2 cells. Compared with the control groups, in cells stably expressing wild-typeSTGC3andSTGC3-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 expressingSTGC3-C656G andSTGC3-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-typeSTGC3gene on CNE2 cells and that the 656C and 725C were important sites in geneSTGC3for its negative regulation on cell growth.
authors.affiliation: Cancer Research Institute, University of South China , Hengyang , Hunan , P.R. China.
authors.forename: Suyun
authors.initials: S
authors.lastname: Li
authors.affiliation: Department of Pathology, Daye People's Hospital , Daye , Hunan , P.R. China.
authors.forename: Lili
authors.initials: L
authors.lastname: Wang
authors.affiliation: Cancer Research Institute, University of South China , Hengyang , Hunan , P.R. China.
authors.forename: Yuanjie
authors.initials: Y
authors.lastname: Xie
authors.affiliation: Cancer Research Institute, University of South China , Hengyang , Hunan , P.R. China.
authors.forename: Xiusheng
authors.initials: X
authors.lastname: He
authors.affiliation: Cancer Research Institute, University of South China , Hengyang , Hunan , P.R. China.
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Zhang
keywords: STGC3; nasopharyngeal; site-directed mutagenesis