Study On Mechanism Of FOXO3a Gene Protection Cells From Anoikis

Aim: To explore the effect of FOXO3a on Anoikis.Method: Constructed the retrovirus plasmid including LMP plasmid and FOXO3a shRNA designed by ourselves, the new plasmid was transfected into phoenix cell for virus package after colon selection, the virus was employed to transduct the MCF10a, MCF7and MDA-MB-231 cells, the cell lines with the silenced FOXO3a were set up after puromycine selection. Established the cell lines of inducible expression FOXO3a by serum starvation. Using the polyhema forced the cells in suspension in analog of the detachment from extra cellular matrix. By the caspase-3 assay, Annexin V/PI flowcyte , DNA ladder and Trypan blue staining, value the cell apoptosis and death. The level of ROS (Reactive Oxygen Species) was examined and the cell apoptosis and death were judged when addition of the ROS or NAC which was the inhibitor of ROS. The relationship between FOXO3a transcription factor and promoter of MnSOD gene and the protein expression of MnSOD in FOXO3a silenced cell were checked by GSA (Gel Shift Assay) and western blot respectively. IP (immune- Precipitation) and western blot were applied to observe whether or not there is a reaction between FOXO3a protein and E-cadherin protein and the protein expression of E-cadherin in FOXO3a silenced cell.Result: The cell lines that can silence and induce the expression of FOXO3a by shRNA and serum starvation respectively were successfully constructed. The assay for cell apoptosis and death indicated that the apoptosis rate and cell death number in suspension cells were higher than those in attachment cells; no difference was found among the attachment cells; In suspension cells, the apoptosis rate and cell death number in FOXO3a silenced cells were dramatically increased comparing to the control cells who were parent cell or transducted with virus containing LMP empty vector. Conversely when cell was induced to express the FOXO3a by deprivation of serum, the apoptosis rate and cell death number was decreased. The ROS level was obviously reduced in suspension cell comparing to the attachment cell; the significant decrease of ROS level was found in FOXO3a silenced cell by comparison to the control cell ; addition of small dose of ROS can obviously improved the cell survival, however, the NAC as the inhibitor of ROS promoted the cell apoptosis and death. The result from GST displayed that FOXO3a can bind with the its binding site in the promote of MnSOD gene and regulate the expression of MnSOD, furthermore, the expression of MnSOD in FOXO3a silenced cell was relatively higer. FOXO3a and E-cadherin can react each other which was confirmed by IP (immune- Precipitation), and the protein level of E-cadherin in FOXO3a silenced cell was decreased as well.Conclusion: FOXO3a confer the Anoikis resistance to cells by regulating the MnSOD in control of the ROS and by reacting with the E-cadherin in hand of the survival signal.