Establishment of breast cancer cell line-MDA-MB-231 through down-regulating the expressions of annexin A3 gene by RNA interference techniq ue

doi:10.3699/j.issn.1002-7386.2016.20.002

Abstract：Objective To establish a breast cancer cell line-MDA-MB-231 transfected with shRNA ( small hairpin RNA) targeting at annexin A3, in order to provide a basis for further study.Methods The expression levels of ANXA3 mRNA and protein in MDA-MB-231 cells and MCF-7 cells were detected by fluorescent quantitation RT-PCR and Western Blot,respectively.The three shRNAs (shRNA1, 2 and 3) plasmids of silencing ANXA3 gene and negative control plasmid were established.MDA-MB-231 human breast cancer cells were transfered with liposome method.The interference plasmid that could silence ANXA3 gene most effectively was selected for subsequent experiment.The stable transfection cells were selected out by using puromycin, and the expression levels of annexin A7 were detected by Western Blot.Results The expression levels of ANXA3 mRNA and protein in MDA-MB-231 cells were significantly higher than those in MCF-7 cells ( P <0.05) . The three shRNAs plasmids targeting at annexin A3 gene were successfully established.The expression levels of ANXA3 mRNA in MDA-MB-231 cells after transfected with ANXA3-sh1 ~3 and negative control plasmid were 0.0196 ±0.0002, 0.0085 ±0.0002, 0.0220 ±0.0035, 0.0661 ±0.0057, respectively, however, which in MDA-MB-231 cells without transfection and in liposomes group were 0.0692 ±0.0050, 0.0652 ±0.0118, respectively.The silencing efficiency of ANXA3-sh2 on ANXA3 mRNA in MDA-MB-231 cells was the highest (87.72%), thus, ANXA3-sh2 was selected for subsequent experiment.The stable transfection cells selected by puromycin were named as MDA-MB-231-Sh cells and MDA-MB-231-NC cells. Western Blot showed that the expression levels of ANXA3 protein in MDA-MB-231-Sh cells were significantly lower than those in MDA-MB-231 cells and in MDA-MB-231-NC cells ( P <0.01 ) .Conclusion The expression levels of annexin A3 protein in MDA-MB-231 cells are higher than those in MCF-7 cells.The successful establishment of MDA-MB-231 cells of down-regulating the expressions of annexin A3 stably lays the foundation for furthere researchabout the expression and characteristics of ANXA 3.