Non-coding RNA Targeting AKT Pathway Combines Chemo-drug Surppress Breast Cancer Cell Growth In Vitro

The PI3K/AKT signaling pathway regulates many normal cellular processes including cell proliferation and survival. Small-molecule therapeutics that block AKT pathway may improve the effect of cancer treatment. In this study, non-coding RNA technology was employed to target AKT pathway to investigate the mechanism of human breast cancer MCF-7 suppression in vitro.Oligofectamine was used to transfect PI3Kp85αsiRNA to knock down the PI3Kp85αexpression level in MCF-7 human breast cancer cell line in vitro. The expression level of PI3Kp85αwas significantly knocked down with PI3Kp85αsiRNA in MCF-7 cells compare to the control and scramble cells determined by the real time PCR. Cell survival rate was dramatically suppressed in PI3Kp85αsiRNA-treated group. Cell cycle was arrested in G0/G1 phase and cell apoptosis percentage was greatly increased. The result of immuno-fluorescence shown that the level of PI3Kp85α,p-AKT,AKT-2,Ki67 and Bcl-2 were down-regulated which were also proved by western blotting.AKT1 siRNA was transfected to MCF-7 human breast cancer cells.to knock down the AKT1 expression level using oligofectamine. The expression of AKT1 was dramatically regressed in AKT siRNA-treated cells detected by RT-PCR. Data from MTT assay indicate that cell growth was delayed in AKT siRNA treated group. Cell cycle was arrested in G0/G1 phase and cell apoptosis percentage of increased significantly which were determined by flow cytometry analysis.The recombinant adenovirus vector plasmid expression vector which contained short hairpin RNA (shRNA) expression construct of AKT1,PI3KP85 was transfected into human breast carcinoma MCF-7 cells. Downstream correlators of PCNA,CyclinD1 were downregulated, while P53 was upregulated. Cell growth was inhibited over 50% as indicated by MTT assay and accompanied with G0/G1 phase arrest. Cell growth on matrigel matrix showed normal cell appearances and mix together in control group and nonsense sequence group cells, while the cells of rAd5-siAKT1-siPI3K transfection group were detached from the matrix or grew in a scattered clustering patterns, signifying poor cell growth activities in 2-D matrigel. The rAd5-siAKT1-siPI3K transfected cells formed only small aggregates as compared with the control and nonsense sequence groups in 3-D matrigel matrix growth. In the last part, PAMAM dendrimer was employed to transfect miR-21 inhibitor combine with taxol to investigate the chemo-sensitivity of human breast cancer cells. MTT showed that the drug concentration causing 50% growth inhibition (IC50) with taxol alone is 600 nmol/mL, while the combination group is 80nmol/mL. The miR-21 inhibitor significantly improved the cytotoxicity of taxol and dramatically increased the apoptosis of MCF-7 cells, while the invasion ability of the tumor cells was decreased. Western blot ananlysis proved that the expression of EGFR,p-AKT,STAT3,p-STAT3,BcL-2,Cyclin D1,MMP-2,MMP-9 were down regulated; PTEN, caspase-3 were up regulated. Thus, combination of miR-21 inhibitor and taxol could be an effective therapeutic strategy for controlling the growth of breast cancer.