The Effect Of Bmi-1 Expression On CPT Chemothrapy Drugs Sensitivity Of K562 Cells And Its Mechanism

Objective: To explore the effect of Bmi-1 expression on the sensitivity of K562 cells to the chemotherapy drug CPT and its mechanism.Methods:1.The pGenesil-2-Bmi-1 1 siRNA,p-MSCV-Bmi-1 and their blank plasmid were transfected into K562 cells by transient transfection method,and the expression of Bmi-1 was detected by RT-PCR and Western blot.2.MTT tested the anti-proliferative effect of K562 cells by different concentrations of CPT for 1-4 days,and found the optimal time and concentration.3.MTT assay analysed for 1-5 days to observe the effect of Bmi-1 expression on CPT sensitivity of K562 cells.4.Immunofluorescence assay was used to find the number of ?-H2 AX foci in cells to observe the extent of DSBs.5.Apoptosis was detected by flow cytometry to clarify the effect of Bmi-1 on CPT-induced the apoptosis of K562 cells.6.The changes in mitochondrial membrane potential was tested by JC-1 probe.7.The expression of the major apoptosis proteins Cytochrome C,Caspase-3,Bax and Bcl-2 in the mitochondrial apoptosis pathway were detected by Western blot.Results:1.The expression of Bmi-1 was reduced by transiently transfected of the pGenesil-2-Bmi-1 1 siRNA compared to the blank control,and overexpressed by transiently transfected of the p-MSCV-Bmi-1.2.The effect of CPT on K562 cells was time and concentration dependent.72 h was selected as the optimal time according to the inhibition rate curve,and the IC50 was calculated to be 0.1 ± 0.07?M.3.Silencing Bmi-1 enhanced the sensitivity of K562 cells to CPT,and overexpression of Bmi-1 attenuated the sensitivity of K562 cells to CPT.4.Immunofluorescence assay showed that silencing of Bmi-1 CPT-induced the number of ?-H2 AX foci was increased.Overexpression of Bmi-1 CPT-induced the number of ?-H2 AX foci was decreased.The results of flow cytometry showed that silencing of Bmi-1 CPT-induced the apoptosis of K562 cells was increased by 12.06%,and overexpression of Bmi-1 CPT-induced the apoptosis of K562 cells was decreased by 10.09%.5.JC-1 probe assay clarified that CPT-induced the mitochondrial membrane potential of K562 cells was decreased by knockdown of Bmi-1,and was increased when Bmi-1 was over-expressed.6.Western blot analysis further detected the expression of mitochondrial apoptosis pathway related proteins.Silencing Bmi-1 increased the expression of pro-apoptosis protein Cytochrome C,Caspase-3 and Bax,and decreased the expression of anti-apoptotic protein Bcl-2.Overexpression of Bmi-1 decreased the expression of pro-apoptosis protein Cytochrome C,Caspase-3 and Bax,and increased the expression of anti-apoptosis protein Bcl-2.Conclusion:1.Bmi-1 may be involved in DNA damage response in K562 cells.2.Bmi-1 may be related to the sensitivity of K562 cells to CPT.Silencing Bmi-1 enhanced the sensitivity of K562 cells to CPT,and overexpression of Bmi-1 attenuated the sensitivity of K562 cells to CPT.And its mechanism may be associated with its involvement in the regulation of mitochondrial apoptosis pathway and its related apoptosis proteins.