shRNA silencing of CDC25B causes altered expression of downstream proteins in MCF-7 breast cancer cells

Background: Cell division cycle 25 (CDC25) family of proteins are highly conserved dual specificity phosphatases. In mammalian cells, three isoforms of CDC25 have been identified including CDC25A, CDC25B and CDC25C. All three isoforms play a role in the control of the G1-S and G2-M transitions by CDKs. Cdc25B causes transition from G2 to M phase and its overexpression has been reported in various cancers including breast cancer. In this study, first we silenced CDC25B gene expression by shRNA and then the alterations in the expression pattern of CDC25B downstream genes was studied at the protein level.Methods: The human breast cancer cell line, MCF-7, was cultured and transfected with CDC25B specific shRNA. The effects of this silencing at the RNA and protein levels were evaluated by Real-time PCR and western blotting respectively. Following this step, the alterations in the expression pattern of CDC25B downstream proteins were analyzed by 2-D gel electrophoresis.Results: The results of Real-time PCR and western blotting showed decreased expression of CDC25B at the RNA and protein levels, respectively. The results of 2-D electrophoresis determined 12 spots whose expressions were significantly altered. Of these, 2 proteins increased and 10 proteins decreased after treatment.Conclusion: CDC25B gene silencing caused alterations in the expression pattern of CDC25B downstream proteins. This finding suggests further studies for investigating the associations between CDC25B and other genes in order to achieve better understanding of tumorigenesis and cell proliferation. Moreover, these results provide a rationale for further studies of CDC25B-based gene therapy for breast cancer.

Keywords:

CDC25B , shRNA , cell cycle , breast cancer