The Affect Of TAK1Gene Silencing On The Apoptosis Of Kasumi-1Cells Induced By Aresenic Trioxide

part one Detecting kasumi-1cells proliferation treated by As₂O₃using CCK-8Objective:This study aims to screen out the most optional concentration of As₂O₃acted onkasumi-1cells, providing a basis for the latter experiment.Methods:The kasumi-1cells were treated by As₂O₃in different concentrations such as0μmol/L,0.5μmol/L,1μmol/L,2μmol/L,5μmol/L,10μmol/L,20μmol/L and at different time-durationssuch as0h,2h,6h,12h,24h,48h,72h. The kasumi-1cells proliferation after As₂O₃treatment was analyzedby CCK-8method. To draw the curve of the cell growth and calculate the IC50value. This can provide abasis for the later experiment with an appropriate concentration of drug action.Results: The cell inhibition rate was in a concentration-dependent manner, there was no obviousrelation with time-durations. The50%inhibitory concentration was3.5μmol/L.Conclusions: The As₂O₃concentration for latter experiment is2μmol/L.part two The optimizing siRNA transfection efficiency ofkasumi-1cells by Liposome-mediated method.Objective: The purpose of the experiment was to screen out the most effective siRNA sequencesof silencing targeted gene.Methods: The TAK1was regarded as the target gene, and four siRNA sequences were designed.The effects of silencing were using real-time quantitative PCR and western blot to detect the TAK1mRNAand protein levels.Results:With TAK1siRNA transfection kasumi-1cells in the condition of serum-free and serum,the transfection efficiency was （75.48±1.07）%and （61.66±0.74）%, P=0.000. The difference wasstatistically significant.When siRNA was20pmol,Lipofectamine2000was1.0μl, and transfection time were1h,2h,4h,6h,8h,12h,the transfection efficiency were （28.37±1.01）%,（47.71±0.93）%,（55.42±1.26）%, （51.68±1.28）%,（52.42±0.91）%,（33.31±1.25）%. Comparison of6h and8h,the P value was0.433.Thedifference was not statistically significant. Other groups,P <0.007, the difference was statisticallysignificant. The highest transfection efficiency was Liposome/siRNA20pmol/1ul, P <0.05, the differencewas statistically significant.Conclusions:The results showed: SiRNA3made the best silencing effect for the transfectionduration for6or8hours in serum-free condition, proved by the PCR and western blot results.part three The study of silencing TAK1gene to strengthenarsenic trioxide proapoptosis effect on kasumi-1.Objective: This study aims to explore the mechanism of TAK1silencing promoting apoptosis ofkasumi-1cell induced by As₂O₃.Methods: Kasumi-1cells was treated with four groups which contain treatment-free group,TAK1siRNA transfection group, As₂O₃treated group and a combination of TAK1siRNA andAs₂O₃. FCMdetected the apoptosis rate. Methylcellulose semi-solid culture detected the colony forming efficiency.Western blot detected the downstream protein expression level.Results:After the kasumi-1cells were treated with arsenic trioxide, the expression level ofP-JNK in kasumi-1was enhanced, especially when treated at the concentration of2μmol/L in30min. Theapoptosis rates of kasumi-1cells without any treatment, TAK1siRNA interference alone group, As₂O₃alone group and the combined group were （5.02±1.13）%,（6.18±0.28）%,（48.33±2.70）%,（86.07±2.21）%,and the colony formation rates were （73.83±2.78）%,（76.03±1.46）%,（55.07±1.50）%,（22.20±1.15）%, apoptosis rate of TAK1siRNA group and the untreated group has no significant difference（P value0.052）; colony formation rate comparison between TAk1siRNA group and the untreated group has nosignificant difference,（P value0.179）,other groups P value0.000.Conclusions:Thus, we conclude that TAK1silencing can enhance arsenic trioxide inducedapoptosis and anti-proliferative effect. This indicated that P-JNK pathway is a negative feedback one.Molecules or drugs targeting kinases may be a new way to treat leukemia.