The Regulation Of YB-1 Protein On The Induced Expression Of Mdr1 Gene In K562 Cells

Objective:To explore the role of YB-1 protein on the induced expression of mdr1 geng in K562 cells after treated with DOX.Then,to study the effect of MAPK/ERK transduction pathway on YB-1 nuclear translocation and the expression of mdr1 gene induced by DOX.Method:1.K562 cells were treated with doxorubicin at different concentrations and times.The expression of mdr1 and YB-1 genes were examined by reverse transcription-polymerase chain reaction(RT-PCR). P-glycoprotein(P-gp)was detected by flow cytometry.Nuclear extracts were prepared for Western blotting to detect the activation of YB-1.2. The recombinant eukaryotic expression plasmid including YB-1 shRNA and the vector-random-sequence were introduced into K562 cells by lipofectamine mediation and positive clones were screened by G418. RT-PCR and western blotting were employed to detect the expression of YB-1 mRNA and protein in K562 cells.Then,the cells of YB-1 gene silenced were treated with doxorubicin at different concentrations and times.RT-PCR and FCM were employed to detect the expression of mdr1 mRNA and P-gp.3.K562 cells were pretreated with MAPK inhibitor PD98059 for 1 hour,and then added doxorubicin.Western blotting was used to detect the expression of ERK,P-ERK and YB-1 protein.RT-PCR and FCM were used to detect the expression of mdr1 mRNA and P-gp.Results:1.When K562 cells were exposured to doxorubicin (0.03μg/mL),the mdr1 gene was highly expressed as well as its corresponding P-glycoprotein.Incubation of K562 cells with doxorubicin also increased the expression of YB-1 gene.Doxorubicin activated the nuclear translocation of YB-1 in a dose-dependent manner.YB-1 nuclear expression was significantly correlated with the expression of mdr1 gene. 2.The recombinant plasmid pGensil-1/YBX11,pGensil-1/YBX12, pGensil-1/YBX13 and pGensil-1/HK have been transfected into K562 cells,and positive clones had been screened by G418 for 2 weeks.Then four cell lines were obtained as followed:K562/HK,K562/Y1,K562/Y2 and K562/Y3;The corresponding transfected effects were (73.48±1.19)%,(52.46±1.23)%,(62.67±1.16)%,(57.25±1.31)%.The level of YB-1 mRNA and protein decreased dramatically in K562/Y1 and K562/Y3 when compared with K562 or K562/HK cells.The density value of YB-1 gene standarded byβ-actin in those five cell lines were 0.2613±0.026,0.2395±0.011,0.1523±0.016,0.2233±0.021 and 0.0866±0.015。The inhibitory effects of three different shRNA sequences targeting YB-1 gene were(41.0±0.01)%,(13.3±0.12)%and (65.7±0.09)%.The introduction of exogenous YB-1 shRNA gene into K562 cells resulted in decreased levels of the expression of mdr1 gene and P-gp induced by doxorubicin.3.The phosphorylation of ERK increased in K562 cells after treated with doxorubicin detected by western blotting.When K562 cells were pretreated with MAPK inhibitor PD98059,the phosphorylation of ERK was effectively inhibited aswell as the nuclear translocation of YB-1 protein and the expression of mdr1 gene induced by doxorubicin.Conclusion:Doxorubicin could activat MAPK/ERK transduction pathway,and then increse the expression of YB-1,induce YB-1 nuclear translocation.The nuclear translocation of YB-1 could up-regulate the expression of mdr1 gene.Our study revealed a new machamism of the induced expression of mdr1 gene in K562 cells.