| ObjectiveTo investigate the different expression of nuclear factor erythroid2-related factor2(Nrf2) in imatinib-resistant chronic myeloid leukemia cell line K562/G01cell lines and imatinib-sensitive K562cell lines,as well as the change of the drug resistance through Nrf2siRNA transfection.The result may provide a theoretical basis for the treatmeat of chronic myeloid leukemia.Methods(1) In CML cell lines K562and imatinib-resistant CML cell lines K562/G01, the expression of Nrf2mRNA and protein were examined by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting.(2) Nrf2-siRNA mediated by lentivirus was transfected into K562/G01cell lines, and then the transfection efficiency was detected by laser scanning confocal microscope (LSCM) and Flow Cytometry (FCM).The expression of Nrf2mRNA and protein were detected by RT-qPCR and Western blotting.(3) The sensitivity of drug resistance was detected by Cell Counting Kit-8(CCK-8) assay and the apoptosis rate was detected by FCM.Results(1) Compared with K562cells, the expressions of Nrf2protein was higher in K562/G01cells.(2) In transfected K562/G01cells, the expressions of Nrf2protein and mRNA were significantly inhibited (P<0.05).Compared with control group, the expression of Nrf2mRNA was reduced by (66.3±0.42)%, and the expression of Nrf2protein was reduced by (65.82±2.36)%.(3)As identified by RT-qPCR and Western blotting, the K562/G01cell models with decreased Nrf2expression is successfully established;(4)Compared with untreated group, the resistance index was significantly decreased in silencing Nrf2gene group.The inhibitory Concentration50of K562/G01cell, NC-GFP-LV control group and Nrf2-RNAi-LV experimental group is22.64umol/L,21.37μmol/L,14.64μmol/L.The resistance index of K562/G01cell, NC-GFP-LV control group and Nrf2-RNAi-LV experimental group is34.28,32.23,22.09. Under the same dose of imatinib,the apoptosis ratio was significantly increased in silencing Nrf2gene group. As the results of CCK-8and FCM, down-regulation of Nrf2may increase the apoptotic sensitivity of K562/G01cells (P<0.05);ConclusionThe siRNA-mediated knockdown of Nrf2might improve the sensitivity of imatinib-resistant K562/G01cells. |
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