The Mechanism Of Nrf2 Regulating OGG1 Mediated Acute Myeloid Leukemia Resistance To Cytarabine

Objective:Nuclear factor E2 related factor 2(Nrf2)is a key factor in oxidative stress system.Studies have shown that Nrf2 plays an important role in the process of drug resistance of many kinds of tumors.Acute myeloid leukemia(AML)is a malignant disease of hematological system with abnormal proliferation of myeloid primordial immature cells.Drug resistance in patients with AML is the main cause of treatment failure and the main challenge in clinical treatment of AML.AML patients are easy to cause DNA damage during chemotherapy,which initiates the DNA repair pathway,which mediates that AML patients are insensitive to chemotherapeutic drugs,resulting in drug resistance.However,the mechanism of whether Nrf2 promotes AML drug resistance by affecting DNA damage repair pathway remains unclear.The purpose of this study was to explore the effect of Nrf2 on the repair pathway of DNA damage and the potential molecular mechanism of AML drug resistance.Methods:1.Bone marrow blood was collected from normal healthy donors and patients with complete remission and relapse of AML.Bone marrow mononuclear cells were extracted from normal healthy donors,AML patients with complete remission and relapse with Ficoll separation solution.The protein sample was extracted by RIPA lysate.Total RNA was extracted by Trizol and reverse transcribed into c DNA.Western Blot,PT-PCR and immunocytochemical(ICC)methods were used to analyze the expression of Nrf2 and OGG1 in normal healthy donors,AML patients with complete remission and relapse.According to the median of Nrf2 m RNA expression in PT-PCR results,the clinical samples of AML were divided into Nrf2 high expression(Nrf2-High)group and Nrf2 low expression(Nrf2-Low)group.2.Drug resistant AML cell lines were induced by low concentration drug culture,and IC50 values of drug resistant and sensitive cell lines were detected by CCK-8method.At the same time,the expression of 8-hydroxyguanine DNA glycosidase(OGG1)in sensitive and drug resistant AML cell lines was detected by immunofluorescence(IF).Western Blot and PT-PCR were used to detect the expression of Nrf2 in sensitive and drug resistant AML cell lines.3.AML cell lines with over-expression and silence of Nrf2 were constructed by lentivirus transfection.The transfection efficiency of lentivirus was observed by immunofluorescence microscope,and the expression of OGG1 after regulation of Nrf2 was detected by Western Blot.The sensitivity of AML cell lines to Ara-C was detected by flow cytometry(FCM)after regulating Nrf2.Meanwhile,the apoptosis rate of AML cells co-cultured with cytarabine(Ara-C)and OGG1 inhibitor for 24 h was detected.4.Detection of interaction between Nrf2 and OGG1 promoters by Chromatin immunoprecipitation(Ch IP).5.The expression levels of Nrf2 and OGG1 in paired normal samples and AML tumor samples were analyzed by GEPIA database,and the relationship between Nrf2,OGG1,AKT signaling pathway and AML drug resistance was analyzed by Gene Mania protein database and Western Blot.Furthermore,AKT signaling pathway inhibitor MK-2206(2 μM)was used to pretreat the cells for 24 h to detect the expression of related pathway proteins by Western Blot.6.non-obese diabetic/severe combined immunodeficiency(NOD/SCID)male mice aged 4-6 weeks were selected to establish xenograft tumor model.AML model mice were established by subcutaneous injection of AML cells.The growth status of mice was observed every other day.When the tumor could be touched,the mice were immediately treated with the chemotherapeutic drug Ara-C.When the mice were sacrificed,the subcutaneous tumor was removed and the expression of Nrf2 and OGG1 in the tumor tissue was detected by immunohistochemistry(IHC),and the effect of regulating Nrf2 on the survival of mice in vivo was verified.7.The data were analyzed by SPSS 19.0 software,the graphics were drawn by Graph Pad Prism software,and the gray values of protein bands were analyzed by Image J software.Independent sample t-test was used for analysis between the two groups,and single factor analysis of variance was used for multiple groups.The experimental data are expressed as mean ± standard deviation.The meaning of P value is: *P<0.05,**P<0.01,***P<0.001.Among them,P < 0.05 was considered to be statistically significant.Results:1.In clinical samples,we found that the high expression of Nrf2 was closely related to the relapse of AML.Further study found that the high expression of Nrf2 is closely related to the base excision excision repair(BER)pathway in the DNA damage repair pathway.The expression of BER excision repair pathway genes(OGG1,APE1,POL-β,XRCC1)was detected in Nrf2-High and Nrf2-Low groups.The results showed that the expression of OGG1 was significantly increased in Nrf2-High group.Western Blot results showed that the expression of OGG1 protein was significantly increased in Nrf2-High group,while OGG1 protein level was significantly decreased in Nrf2-Low group.2.The expressions of Nrf2 and OGG1 were significantly increased in drug resistant cell lines,and the drug resistant cell lines were less sensitive to Ara-C.3.After lentivirus transfection into AML cells,it was found that the expression of OGG1 protein increased and the sensitivity of cells to chemotherapeutic drug Ara-C decreased in the up-regulated Nrf2 group,which could protect AML cells in vitro,while in the down-regulated Nrf2 group,the expression of OGG1 protein decreased,the sensitivity of AML cells to chemotherapeutic drug Ara-C increased,and the apoptosis increased significantly.When TH5487,an inhibitor of OGG1,was used to inhibit the expression of OGG1,the sensitivity of AML cells to Ara-C was increased in up-regulated Nrf2 group.The results of Ch IP experiments showed that the combination of Nrf2 and OGG1 promoters further enhanced the function of Nrf2-OGG1 axis.4.Over-expression of Nrf2 in AML cells activates AKT signaling pathway and promotes the expression of OGG1 protein.When AML cells were co-cultured with AKT signaling pathway inhibitor for 24 h,it was found that the expression of OGG1 protein decreased in Nrf2 over-expression group.At the same time,Ch IP results suggested that the binding of Nrf2 and OGG1 promoters decreased significantly after the use of AKT pathway inhibitors.5.The results of in vivo study showed that the tumor tissue of AML model mice grew slowly and the survival time was longer after down-regulation of Nrf2.Meanwhile,the expression of OGG1 in Nrf2 down-regulation group was lower,which had a certain protective effect on xenografted tumor mice.Conclusions:Over-expression of Nrf2 promoted the expression of OGG1 and decreased the sensitivity of AML cells to Ara-C.Further studies showed that over-expression of Nrf2 activated AKT signaling pathway to promote OGG1 expression and mediate drug resistance of AML cells to Ara-C.