The Effect Of PIK3CA Gene Silence On Antimyeloma Action Of Aspirin And The Underlying Molecular Mechanisms

Objective:1.The expressions levels of PIK3 CA gene and protein in human multiple myeloma cell line RPMI-8226 cells with exposure to ASA were observed to explore the potential target of PIK3 CA gene in the antimyeloma activity of ASA.2. The effects of proliferative activity and phosphorylated AKT induced by silencing PIK3 CA gene were examined in RPMI-8226 cells with exposure to ASA,to verify the effect and molecular mechanism of PIK3 CA gene in ASA against multiple myeloma.Methods:1. The proliferative inhibition of human multiple myeloma cell line PRMI-8226 was treated with 24 h,48h,72 h by different concentrations of ASA(0,2.5,5,10mmol/L).2. The levels of PIK3 CA gene m RNA and protein were determined in RPMI-8226 with exposure to ASA within 0~10mmol/L concentration.3. In RPMI-8226 cells transfected with PIK3 CA siRNA, the rate of transfection was examined in fluorescence micrcscope and the level of PIK3 CA mRNA was measured by qRT-PCR.4. The proliferative inhibition of PRMI-8226 cells transfected with PIK3 CA si RNA was examined when treated by ASA for 24 h.5. The cell apoptosis and cell cycle were assayed in PRMI-8226 cells transfected with PIK3 CA siRNA when exposed by ASA treatment for 24 h.6. The expression of AKT and phosphorylated AKT protein were determined after treatment of ASA for 24 h in RPMI-8226 cells transfected with PIK3 CA siRNA.Results:1. ASA displayed anti-proliferative action against RPMI-8226 cells in doseand time –dependent fashion within the range of 2.5~10mmol/L and 24~72h.2. 2.5~10mmol/L ASA exerted an inhibitory effect on the level of PIK3 CAmRNA and protein in human MM cell line RPMI-8226 dose- dependently.3. PIK3 CA siRNA transfection caused the significant decrease of PIK3 CA gene to 83.2% of the level prior to transfection, indicating the successful transfection.4. Compared to the NC siRNA transfected group, PIK3 CA siRNA transfection attenuated the antimyeloma action of ASA in RPMI-8226 cells without the dose-dependent manner.5. In comparison with NC si RNA transfected group, PIK3 CA siRNA transfection markedly decreased the proapoptotic rate of ASA in in RPMI-8226 cells.6. When compared with NC siRNA transfected group, PIK3 CA siRNA transfection substantially reduced the induction of cell cycle arrest in G1 phase in RPMI-8226 cells.7. ASA displayed the inhibitory effect on phosphorylated AKT protein dose-dependently in RPMI-8226 cells, but the effect failed to exisit in total AKT protien. Silencing PIK3 CA gene attenuated the inhibition of phosphorylated AKT induce by ASA in RPMI-8226 cells.Conclusions:1. ASA inhibited the expression of PIK3 CA gene and protein in dose-dependent fashion in MM cells.2. PIK3 CA gene silence markedly attenuated the anti-proliferation, proapoptotis and G1 phase arrest of ASA in MM cells.3. Silencing PIK3 CA gene significantly decreased the inhibitory effect of ASA on phosphorylated AKT in MM cells.4. ASA displayed the antimyeloma action via repression of PIK3 CA and the phosphorylation of AKT.