Establishment of breast cancer cell lines with the centromere protein-H stably silenced

Objective To establish breast cancer cell lines with the centromere protein-H (CENP-H) stably silenced by applying the retroviral expression system.Methods The interference sequences targeting CENP-H gene were synthesized and insetted into the retroviral vector pSUPE-Rretro-puro.The recombinant plasmids were transfected into the packing cells 293FT with calcium phosphate and the retroviral supernatant was collected to infect MCF-7 and MDA-MB-435 breast cancer cells.After selection by puromycin and culture expansion,the stable cell clones were obtained.The levels of CENP-H mRNA and protein in the selected clones were detected by Real-time PCR and Western blot.Results Two CENP-H shRNA retroviral vectors were constructed successfully.After infection,CENP-H-knocked down breast cancer stable cell lines were successfully established.The mRNA and protein expression levels of CENP-H were significantly down regulated in CENP-H-knocked down cells compared to control.Conclusion The establishment of breast cancer stable cell lines in which the expression of endogenous CENP-H is knocked down will provide an original route for further exploring the mechanism of CENP-H in human breast cancer. Key words: Breast neoplasms;  Centromere protein-H;  RNA interference