Experssion of human FGFR2IIIc and its S252W mutant in MDA-MB-231 breast cancer cells with adenovirus vectors / 生物工程学报

ABSTRACT

To study the functions of human Fibroblast growth factor receptor 2IIIc (FGFR2IIIc) gene in cancer cells, breast cancer cells MDA-MB-231 were infected by recombinant adenoviruses containing FGFR2IIIc and its S252W mutant, respectively. FGFR2IIIc gene was amplified from an existing plasmid and its S252W mutant was obtained by overlapping extension PCR. These two genes were separately cloned into the adenoviral shuttle plasmid pAdTrack-CMV, confivmed by DNA sequencing linearized, and co-transformed into Escherichia coli BJ-5183 with the adenoviral vector pAdEasy-1. The resulting recombinant expression vectors Ad-FGFR2IIIc and Ad-FGFR2IIIcS252W were linearized and transfected into HEK293A cells to get adenoviral particles. GFP was used to verify the gene expression. The recombinant adenoviral particles were harvested, titrated, and then infected MDA-MB-231 cells. The expression of FGFR2IIIc and its S252W mutant were examined by RT-PCR and Western blotting, and the effect of these recombinant adenoviruses on MDA-MB-231 cell proliferation was analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry. The results showed the recombinant adenoviral particles could infect MDA-MB-231 cells and express the target proteins. MTT showed that both FGFR2IIIc and its S252W mutant inhibited MDA-MB-231 cell proliferation, but the mutant was more effective. Flow cytometry showed that both FGFR2IIIc and its S252W mutant arrested MDA-MB-231 cell cycle at G0/G1 phase, resulting in low cell proliferation.