Sabah Mayahi - Ph.D in Molecular Medicine, Mazandaran University of Medical Sciences, Sari, Iran
Masood Golalipour - Ph.D in Molecular Genetics, Golestan University of Medical Sciences, Gorgan, Iran
Ahad Yamchi - Ph.D in Genetic Engineering. Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran
Gagan Deep Jhingan - Ph.D in Biotechnology, New Delhi, India
Majid Shahbazi - Ph.D in Gene Therapy, Golestan University of Medical Sciences, Gorgan, Iran

Background and aim: The forkhead box O3 (FOXO3) and p27Kip1 are two important genes in breast cancer progression. In the present study we analyzed the effect of simultaneous FOXO3 silencing and p27Kip1 activation on breast cancer cell survival and the potential targets of thesechanges in cancer molecular pathways. Methods:The present study involved the cloning of FOXO3a shRNA and p27Kip1 genes under the control of the bidirectional survivin promoter to down- and up-regulate FOXO3 and p27Kip1 genes, respectively. After transfection of the recombinant expression vector into the breast cancer cell line, the inhibition of cell growth was assessed by MTS and flow cytometry assays. Following the extraction of total mRNA and protein, the expression of target genes was evaluated by qPCR and Western blotting in both treatedand untreated cell lines. Then, the downstream protein responses were examined by 2D electrophoresis. The differentially expressed proteins werealso identified by mass spectrometry. Results: Rates of cell proliferation were significantly inhibited in the transfected cell line 72 h posttransfection. Proteomic profiling of the cell line resulted in the identification of seven novel protein markers in breast cancer responsive to these changes in expression of FOXO3 and p27Kip1. The changes in expression of these markers suggested that certain signalingpathways contribute to the development of breast cancer.Implications for practice: Simultaneous silencing of FOXO3 and activation of p27Kip1 inMDA-MB-231 cells caused alterations in the expression level of several genes involved in apoptosis, cell proliferation, cell cycle control, tissueinvasion, drug resistance, and metastasis. It seems that the identified genes might serve as useful biomarkers for breast cancer .

cancer, cell signaling, FOXO3a, gene therapy, P27Kip1, proteomics.