Expression And Mechanism Of BACH2 In Acute Myeloid Leukemia

Introduction:Acute myeloid leukemia(acute myelocytic leukemia,AML)is a highly heterogeneous hematological malignancy caused by dysgenesis of myeloid hematopoietic stem/progenitor cells,characterized by abnormal clonal proliferation and differentiation arrest of primitive and naive myeloid cells in bone marrow and peripheral blood.AML has complex cytogenetic and epigenetic mutations,including aberrant DNA methylation,leads to a high degree of heterogeneity in diagnosis,development,and prognosis.With the continuous study of AML,it is found that abnormal DNA methylation of special gene promoter affects the change of its own expression level,especially tumor suppressor,which leads to the occurrence and development of disease.Although the emergence of various molecular targeted therapies for AML has increased the possibility of AML treatment options,its 5-year survival rate is still very low,especially in elderly patients with high risk factors.hence,in-depth exploration of the pathogenesis of AML has important scientific significance and clinical application value for disease prevention,diagnosis,specific treatment and prognostic stratification.BACH2(Broad-complex,tramtrack and bric-a-brac and cap ‘n’ collar homology 2)belonging to the poxvirus and zinc finger domains(Poxvirus and zinc fingerdomai,POZ)(also called BTB domain)type and cap and collar(Cap’n’ collar,CNC)type alkaline region leucine zipper(basic leucine zipper,B-Zip)family transcription factors,which can form heterodimers with small Maf family members(Maf K、Maf F and Maf G)or other B-Zip family proteins to inhibit or activate the expression of target genes.During the development of B cells,BACH2 can mediate negative selection of B cell receptor checkpoints(BCR)by activating P53,which is necessary for switch-like recombination(CSR)and somatic hypermutation(SHM)of immunoglobulin genes.In addition,BACH2 become a therapeutic target for many immune system diseases by participating in the development and differentiation of T cells.In recent years,studies of BACH2 found that it also plays an important role in the occurrence and development of many kinds of tumors.However,the expression level,clinical significance and regulatory mechanism of BACH2 in AML are still unclear.Ferroptosis is a programmed death mode of cell death caused by the accumulation of lipid peroxides and reactive oxygen species dependent on iron.it is currently considered to be related to the occurrence and development of a variety of diseases,including in inhibiting tumor growth such as liver cancer,prostate cancer,breast cancer,lung cancer and so on.Ferroptosis process has a complex regulatory network,and the current research has not fully analyzed its intrinsic mechanism.The solute carrier family7 member A11(SLC7A11),as an important subunit of the transporter encoding cystine /glutamate reverse transport,is an important regulator of ferroptosis.It can promote glutathione synthesis by mediating cysteine uptake and glutamate release and protect cells from oxidative stress and prevent lipid peroxidation-induced ferroptosis.However,expression and regulatory mechanisms of SLC7A11 in AML have not been reported.To sum up,this study examined the expression of BACH2 in AML clinical samples,analyzed the correlation between its expression level and clinical characteristics,explored the clinical value of BACH2 in acute myeloid leukemia,and provided a new theoretical basis for AML clinical research.We explored the biological function of BACH2 in AML and whether BACH2 can play a role in the pathogenesis of ferroptosis pathway.Materials and Methods:1.Materials: Remaining bone marrow blood samples of AML patients and controls,AML cell lines,human embryo kidney HEK293 t cell lines.2.Methods:2.1 Bone marrow mononuclear cell extraction was used to collect bone marrow samples from 124 patients with primary AML and 36 patients with non-hematological malignant diseases.Real-time quantitative fluorescence PCR(qRT-PCR)method was used to detect the difference of BACH2 expression level in each group and to compare the correlation between BACH2 expression level and clinical characteristics.2.2 The DNA methylation extraction method was used to extract the DNA of 12 primary AML patients and 12 controls with non-hematological diseases,Methyl Target sequencing method compared DNA methylation level of two groups.2.3 The endogenous BACH2 expression of AML cells was compared by qRT-PCR method,and the m RNA expression level and DNA methylation level of AML cells before and after demethylated drug treatment were compared by methylation sequencing method.2.4 Lentiviral transfection was used to onstruct overexpressed BACH2 and its negative control AML cells.CCK-8 and flow cytometry detects the effects of overexpressed BACH2 on proliferation,apoptosis,differentiation and cycle of AML cells.2.5 RNA-seq screened differentially expressed genes in overexpressed BACH2 and their negative control AML cells,then bioinformatics enrichment analyzed related pathways,then qRT-PCR,Western blot and flow cytometry were used to verify.2.6 The changes of mitochondrial structure of overexpressed BACH2 and its negative control AML cells were observed by transmission electron microscope.The levels of reactive oxygen species,lipid reactive oxygen species and lipid peroxides in cells with overexpressed BACH2 and their negative control AML cells were analyzed by flow cytometry,fluorescence microscopy and lipid oxide detection kit.The iron ion and glutathione detection kit were used to detect the iron ion and glutathione levels in the cells of the overexpressed BACH2 and its negative control AML cells,respectively.2.7 The binding sites of BACH2 and SLC7A11 were predicted by bioinformatics software,and the transcriptional regulation of BACH2 and SLC7A11 was verified by dual-luciferase reporter experiment and chromatin immunoprecipitation experiment.2.8 CCK-8,qRT-PCR and flow cytometry were used to detect the cell proliferation,SLC7A11 gene expression level,intracellular reactive oxygen species level and lipid reactive oxygen species level in overexpressed BACH2 and its negative control AML cells transfected with overexpressed SLC7A11 and its empty plasmid,respectively.2.9 Cell proliferation,gene expression,intracellular reactive oxygen species levels and lipid reactive oxygen species level of overexpressed BACH2 and its negative control AML cells treated with or without drugs were analyzed by CCK-8,qRT-PCR,flow cytometry and fluorescence microscopy.CCK-8 detection of cell proliferation after combined treatment with Decitabine(DAC),DNR and Erastin.Results:1.Expression and clinical significance of BACH2 in AML.1.1 qRT-PCR detection found that the expression of BACH2 in primary AML bone marrow samples was significantly lower than that in patients with non-hemorrhagic malignant diseases controls and CD34 positive cells(P<0.05).1.2 The expression level of BACH2 in AML was related to clinical characteristics:in AML patients with low BACH2 expression,peripheral blood leukocyte count and bone marrow primordial cell count were significantly increased(P<0.05),and the ratio of CEBPA double mutation was higher in BACH2 low expression group(P<0.05).1.3 Higher expression level of BACH2 patients were more likely to achieve complete remission(P<0.05).Compared with AML patients,the expression level of BACH2 in the complete remission group was increased(P<0.05),and compared with complete remission group,the expression level of BACH2 in the replase patients was decreased(P<0.05).Survival analysis showed that patients with low expression of BACH2 had a poor prognosis.1.4 DNA methylation sequencing showed an increase of BACH2 DNA methylation levels in AML patients compared to normal controls(P<0.05).1.5 qRT-PCR results showed that the expression levels of BACH2 were the lowest in the THP1 and U937 cell lines(P<0.05).1.6 qRT-PCR results showed that the expression level of BACH2 was significantly increased after the treatment of AML cell lines with demethylated drugs(P<0.05).1.7 DNA methylation sequencing showed that the DNA methylation level of BACH2 was significantly reduced after the treatment of AML cell lines with demethylated drugs(P<0.05).2.BACH2 affects the proliferation and drug sensitivity of AML cells via SLC7A11 mediated ferroptosis.2.1 The results of CCK-8 proliferation experiments showed that overexpressed BACH2 inhibited AML cell proliferation(P<0.05)。2.2 Flow cytometry analysis showed that BACH2 had no effect on apoptosis,cell differentiation and cell cycle.2.3 RNA-seq results screened 232 differentially expressed genes(FC≥2,P<0.05)in overexpressed BACH2 THP1 cells and negative controls,enrichment analysis showed that BACH2 was involved in the regulation of ferropptosis(P<0.05).2.4 Overexpressed BACH2 promote ferroptosis in AML cells: compared with the negative control group,transmission electron microscopy showed specific changes in ferroptosis in mitochondria of overexpressed BACH2 AML cells;flow cytometry analysis showed an increase in intracellular reactive oxygen species and lipid reactive oxygen species(lipid peroxides)in overexpressed BACH2 AML cells(P<0.05);The intracellular glutathione level of overexpressed BACH2 AML cells decreased(P<0.05)and intracellular iron level of overexpressed BACH2 AML cells increased(P<0.05).2.5 The bioinformatics software predicts four binding sites for BACH2 and SLC7A11 promoter regions:-116～-129bp(site 1),-307～-320bp(site 2),-1364～-1377bp(site 3)and-1635～-1648bp(site 4),and the dual luciferase reporter assay and chromatin immunoprecipitation results showed BACH2 direct targeting through site 1 and site 2 to inhibit SLC7A11 transcription(P<0.05).2.6 The overexpressed BACH2 and its negative control AML cells were transfected with overexpressed SLC7A11 and control plasmid,using qRT-PCR and flow cytometry to verify the transfection efficiency,the results showed that the SLC7A11 expression level increased significantly(P<0.05),CCK-8 and flow cytometry showed that overexpressed SLC7A11 could restore the inhibitory effect of BACH2 on AML cell proliferation,intracellular reactive oxygen species(ROS)and lipid reactive oxygen species of AML cells(P<0.05).2.7 CCK-8 proliferation assay showed that overexpressed of BACH2 could increase the drug sensitivity of AML cells to the Erastin of ferroptosis inducers.2.8 CCK-8 proliferation experiments showed that overexpressed BACH2 could increase the drug sensitivity of AML cells to DNR(P<0.05),and flow cytometry and fluorescence microscopy showed thatoverexpressed BACH2 could increase the intracellular reactive oxygen species level of AML cells treated with DNR drugs(P<0.05).qRT-PCR results showed that the SLC7A11、HMOX-1 and FTH1 expression levels of ferroptosis related genes in AML cells were increased after treatment with DNR drugs(P<0.05),and overexpressed BACH2 could inhibit the expression of these genes(P<0.05).2.9 The results of CCK-8 proliferation experiments showed that the combination of ferroptosis inducers and demethylated drugs could increase the drug sensitivity of AML cells to DNR(P<0.05).3、Conclusions:1.DNA methylation levels of BACH2 increased and expression of BACH2 decreased in AML,and BACH2 expression levels were associated with clinical characteristics,therapeutic responses and prognostic trends.2.BACH2 inhibit AML cell proliferation,but have no effect on cell apoptosis,cell differentiation and cell cycle.3.BACH2 can promote ferroptosis in AML cells.4.BACH2 act as a transcription factor to directly target inhibition of SLC7A11 gene transcription.5.BACH2 inhibits the expression of SLC7A11,promoting ferroptosis and and inhibiting cell proliferation.6.Up-regulation of BACH2 expression can increase drug sensitivity of AML cells to DNR and the combination of ferroptosis inducers and demethylation drugs can increase drug sensitivity of AML cells to DNR.