Upregulation Of Mdrl Gene Is Related To Activation Of The MAPK/ERK Signal Transduction Pathway And YB-1 Nuclear Translocation In B-cell Lymphoma

Objective:1.To investigate the expression of Y-box binding protein-1 (YB-1) in diffuse large B cell lymphoma (DLBCL) and analyze the relation between YB-1 and drug resistance, chemotherapy response or clinical prognosis; 2.To explore the role of YB-1 protein on the induced expression of mdr1 geng in lymphoma cell line Daudi after treated with doxorubicin (DOX).3.To determine whether the induced expression of the mdrl gene was related to the activation of the MAPK/ERK signal transduction pathway, nuclear translocation of YB-1. and the DNA binding activity of YB-1 in lymphoma cells.Method:l.The YB-1. pERK and p-glycoprotein(P-gp) were measured by immunohistochemistry (Envision two-step kit) on paraffin-embedded specimens in diffuse large B cell lymphoma and reactive lymphoid hyperplasia (RLH). The relation between expression of YB-1 and P-gp, pERK or clinical parameters was analyzed.2. A plasmid pGensil-1 containing human U₆, snRNA promoter was ligated to a 19 bp reverse repeated motif of YB-1 targeted sequence or random sequence with 9 bp spacer. Enzyme digestion and DNA sequencing were used to exam whether the recombinant plasmid YBX1, YBX2, YBX3 and pGensil-1/con were correct.3.The recombinant eukaryotic expression plasmid including YB-1 shRNA and the vector-random-sequence were introduced into Daudi cells by lipofectamine mediation and positive clones were screened by G418.4. Daudi and Daudi/Y(the cells of YB-1 gene silenced) cells were treated with doxorubicin at different concentrations and times. The expression of mdrl and YB-1 genes were examined by reverse transcription-polymerase chain reaction (RT-PCR). P-gp was detected by flow cytometry. Nuclear extracts were prepared for Western blot to detect the activation of YB-1. MTT assay was applied to assess the development of chemoresistance of Daudi cells induced by doxorubicin and the cytostatic efficacy of doxorubicin. IC50 values were calculated and used to evaluate the cytostatic efficacy of chemotherapy drugs. Intracellular Rhodamine 123 (Rho123) retention assay was applied to test the function of P-gp.5.The electrophoretic mobility shift assay (EMSA) was adopted to detect the DNA binding activity of transcription factor YB-1.6.To further analyze the role of YB-1 in mdrl transcription, we performed chromatin immunoprecipitation (ChIP).7.The promoter of mdrl and the sequence with YB-1 binding sites mutants of mdrl promoter were synthesized, and then cloned into the luciferase reporter gene vector PGL3-Basic. The recombinant plasmid was transiently co-transfected into Daudi cells with control vector (β-gal by lipofectamine reagent. The mdrl promoter activity was detected after the cells being treated with DOX.8.Daudi cells were induced by doxorubicin after being pretreated with MAPK inhibitor PD98059 for 1 hour. Western blotting was used to detect the expression of ERK. pERK and YB-1 protein. RT-PCR and FCM were used to determine the expression of mdr1 mRNA and P-gp.Results:1.YB-1 nuclear expression in the DLBCL was significantly higher than that in the RLH group (P＜0.05):YB-1 and pERK. nuclear expressions inâ…¢-â…£clinical stage, ex-nodal group or marrow invasion group patients were significantly higher than that in I-II group, in-nodal group or non-invaded marrow group (P＜0.05); There was no significant difference in the total expression level of P-gp between the DLBCL and the RLH, and P-gp expression was not related to patients' age. gender, clinical stage, nodal and marrow invasion in DLBCL(P＞0.05); YB-1 nuclear expression was significantly correlated with P-gp (γ=0.950.P＜0.005) or pERK expression intensity (y=0.930,P＜0.005):The frequency of YB-1 nuclear expression in chemotherapy efficiency group was lower than that in inefficacy group (P=0.038); The pERK and P-gp expression intensity in inefficacy group was higher than those in efficiency group (P＜0.001). Nuclear expression of YB-1 and pERK were positively correlated with the increased expression of P-gp. and significantly associated with poor response to chemotherapy.2.The YB-1 shRNA expression frame and random sequence were successfully inserted into eukaryotic expression vector pGensil-1 with U6 snRNA promoter and termination signal of RNA polymeraseâ…¢. Enzyme digestion by Pstl and Sall got a 400 bp DNA fragment. The sequences of shRNA recombinant plasmid were the same as that of designed fragments.3.The mdrl gene and P-gp were highly expressed when Daudi cells were exposured to doxorubicin (0.03μg/mL and 0.05μg/mL). Incubation of Daudi cells with doxorubicin also increased the expression of YB-1 gene. Doxorubicin activated the nuclear translocation of YB-1 protein in a dose-dependent manner. YB-1 nuclear expression was significantly correlated with the expression of mdrl gene.4.The recombinant plasmid YBX1. YBX2. YBX3 and pGensil-1/con have been transfected into Daudi cells, then five cell lines were obtained as followed:Daudi. Daudi/c. Daudi/Y1, Daudi/Y2 and Daudi/Y3. The level of YB-1 mRNA and protein decreased dramatically in three positively transfected cells when compared with Daudi or Daudi/c cells. The density value of YB-1 mRNA standarded byβ-actin in those five cell lines were 0.2613±0.026,0.2395±0.011,0.1523±0.016,0.2592±0.029 and 0.0866±0.015. The three shRNA inhibited YB-1 by (41.0±0.01)%,(13.3±0.12)% and (65.7±0.09)%. respectively. The introduction of exogenous YB-1 shRNA gene into Daudi cells resulted in decreased levels of the expression of mdrl gene and P-gp induced by doxorubicin. IC50 values of Daudi/Y/A0.03 and Daudi/Y/A0.05 cells to doxorubicin. etoposide. vincristine and cisplatin were much lower than those of Daudi/A0.03. Daudi/c/A0.03. Daudi/A0.05. Daudi/c/A0.05 cells. The Daudi/Y/A0.03 and Daudi/Y/A0.05 accumulated higher levels of rhodamine 123 compared with control cells(P＜0.05).5.A few DNA-protein complexes were observed in Daudi cells, but the major DNA-protein complex was increased around 2.61-fold in Daudi/A0.01. 5.63-fold in Daudi/A0.03. and 12.52-fold in Daudi/A0.05 cells, respectively. Major DNA-protein complex levels were markedly reduced by addition of unlabeled double-stranded Y-box oligonucleotide and anti-YB-1 antibodies in a dose-dependent manner.6.YB-1 appeared to contribute to the activation of mdr1 transcription through directly interacting with the mdr1 promoter, as evidenced by its physical binding to the promoter region of the mdrl gene in chromatin immunoprecipitation. 7.Doxorubicin significantly increased the reporter activity of pMDR-luc. the plasmid bearing the non-mutated Y-box, in a dose-dependent manner, but had little effect on the luciferase activity of pMDR-Ym-luc. the plasmid bearing a mutation in the Y-box. in Daudi cells.8.The phosphorylation of ERK in Daudi cells after treated with doxorubicin detected by western blotting. When Daudi cells were pretreated with MAPK inhibitor PD98059, the phosphorylation of ERK was effectively inhibited as well as the nuclear translocation of YB-1 protein and the expression of mdr1 gene.Conclusion:1.Nuclear expression of YB-1 and pERK was positively correlated with the increased expression of P-gp. and significantly associated with poor response to chemotherapy.2.Doxorubicin can increase the expression of mdrl/P-gp through activating MAPK/ERK transduction pathway, then incresing the expression of YB-1, inducing YB-1 nuclear translocation. and enhancing DNA binding activity of YB-1.