| Objective To investigate the effect of Egr-1 and the expression of AQP9 in the process that the combination of arsenic trioxide（As2O3）and 3’-azido-3’-deoxythymidine（AZT）promote apoptosis of human hepatoma carcinoma cells,and explore its possible mechanism in support of theoretical basis for clinical application of the two drugs against hepatoma.Methods HepG2 cells were cultured in vitro.Cells were electrotransfected with Egr-1siRNA and treated with drugs after 24 h.Cells were divided as Mock group（the blank control）,NC group（nonspecific control group）and Egr-1siRNA group（transfected with Egr-1siRNA）.The transfection efficiency of Egr-1siRNA was detected by q-PCR.After treatment of As2O3（2μmol/L）combined with AZT（20μmol/L）,the expression levels of Egr-1,Caspase3,P53 mRNA and protein of cells in 3 groups were analyzed by q-PCR and Western blotting,respectively.HepG2 and MHCC97 H cells were cultured in vitro.The two strains of cells were treated with As2O3（2μmol/L）combined with a range of different concentrations of AZT（0,10,20μmol/L）for 48 h,and the proliferation inhibition rates were detected by MTT assay,and AQP9 mRNA/protein expression was determined by real time RT-PCR and western blotting assays,respectively.Result Transfection efficiency of Egr-1siRNA was 55.9% after transient transfection.The result of q-PCR indicated a significant increase of Egr-1,Caspase3 and P53 mRNA expression in combination group than blank control group and nonspecific control group（P<0.05）,and there was no significant difference between Mock and NC groups（P>0.05）.These results are consistent with the expression of protein by western blot assay.MTT assay showed that the proliferation inhibition rate of HepG2 and MHCC97 H cells in combination of As2O3（2μmol/L）and AZT（20μmol/L）groups were significantly higher than As2O3 alone groups.Similarly,the expression levels of AQP9 mRNAs and proteins in combination of As2O3（2μmol/L）and AZT（20μmol/L）group were higher than those of As2O3 alone groups and blank control groups,in HepG2 and MHCC97 H cells,respectively.Conclusion Our study indicated that Egr-1play a significant role in the combination treatment of As2O3 and AZT promoting apoptosis of HepG2 cells by up-regulating P53 and Caspase3,and AZT may accelerate the intake of As2O3 for HepG2 and MHCC97 H cells through increasing the expression of AQP9,so as to improve the use efficiency of As2O3 andto promote its proliferation inhibition of liver cancer cells. |
