The Effect And Mechanism Of Hif-1α In Benzene-induced Bone Marrow Hematopoietic Toxicity

Benzene is a chemical carcinogen,and long-term chronic benzene exposure can lead to aplastic anemia,myelodysplastic syndrome,acute myeloid leukemia and malignant lymphoma.Bone marrow is an important hematopoietic organ.Hypoxia-inducible factor HIF-1αregulates the metabolism and physiological functions of hematopoietic stem cells through multiple cell signaling pathways in the hypoxic environment of bone marrow.After entering the bone marrow,benzene metabolites produce a large amount of reactive oxygen species through oxidative metabolism,causing oxidative stress,destroying the bone marrow micro-environment,and ultimately affecting the migration,development,self-renewal and cell cycle status of hematopoietic stem cells.In this study,we mainly constructed a mouse model of benzene poisoning to detect the expression of reactive oxygen species,HIF-1αand its downstream gene protein expression in bone marrow cells,and explore whether HIF-1αis involved in benzene hematopoietic toxicity and the possible pathways of it.Furthermore,HIF-1αstably and highly expressed cells and control cells were exposed to 1,4-benzoquinone,cell proliferation,cycle,apoptosis,reactive oxygen species and downstream gene expression level of which were detected to study which cellular functions are regulated by HIF-1αto affect the cytotoxicity of 1,4-benzoquinone.Finally,using ChIP-Seq technology to screen for cell function-related HIF-1αtarget genes,differing in bone marrow cells of benzene poisoning mice and control mice,to provide further clues for further study of the regulatory mechanism of HIF-1αin benzene-induced bone marrow hematopoietic toxicity.It also provides direction for finding biomarkers for chronic benzene poisoning.Chapter Ⅰ Study on the regulation of benzene exposure on HIF-1αin mouse bone marrow cellsApply different doses of benzene,such as 60mg/(kg.d),150mg/(kg.d),300mg/(kg.d),to C57BL/6 mice for 15 days to construct a benzene poisoning mouse model,and then detect mouse body weight,peripheral blood routine,organ coefficient and proportion of hematopoietic stem cells in the bone marrow.Bone marrow cells were collected and the levels of reactive oxygen species and protein expression levels of HIF-1αand downstream genes in bone marrow cells were measured.The results showed that the ratio of peripheral blood and hematopoietic stem cells were significantly lower than that of the control group,and the level of reactive oxygen species in mouse bone marrow cells increased significantly,when mice were exposed in intermediate doses of benzene(150mg/(kg.d)).When mice were exposed in high doses of benzene(300mg/(kg.d)),body weight and thymus organ coefficient on day 15 were significantly lower than in the control group.After exposure to benzene for 15 days of C57BL/6 mice,the protein expression levels of HIF-1αand BMI1 in bone marrow cells of mice exposed to intermediate-dose and high-dose benzene were significantly decreased.The protein expression levels of NOX4,VEGF and BCL2 in bone marrow cells of mice exposed to high-dose benzene were significantly increased.The above results indicate that a benzene poisoning mouse model can be successfully constructed by infecting at a dose of 150 mg/(kg.d)for 15 days.Benzene poisoning can increase the production of reactive oxygen species in bone marrow cells,inhibit the expression of HIF-1α,and then affect the levels of BMI1 and BCL2 which are the downstream genes of HIF-1αto participate in benzene-induced bone marrow hematopoietic toxicity.Chapter Ⅱ Study on the role of HIF-1αin cytotoxicity of K562 induced by 1,4-benzoquinoneIn this experiment,HIF-1αstably expressed K562 cells(K562-HIF-1α~+)and control cells(K562-NC)were constructed by lentiviral transfection,and the expression levels of HIF-1αprotein in the two cell lines were detected.Different concentrations of1,4-benzoquinone(0μmol/L,10μmol/L,20μmol/L,40μmol/L)exposure K562-HIF-1α~+cells and K562-NC cells 6 hours,24 hours or 48 hours,and the cell proliferation,cycle and apoptosis were measured,as well as intracellular reactive oxygen species levels and protein expression levels of HIF-1αand downstream genes.The results showed that the expression level of HIF-1αin K562-HIF-1α~+cell line was significantly higher than that in the control cell line K562-NC.After 1,4-benzoquinone exposure,the proliferation rates of the two cell lines decreased with the increase of the concentration of the drug,and when the concentration of 1,4-benzoquinone was 10μmol/L and 20μmol/L,the relative proliferation rate of K562-HIF-1α~+cells was lower than that of K562-NC cells after 24 hours of exposure;The proportion of G0/G1 phase in normal cultured K562-HIF-1α~+cells was lower than that in K562-NC cells,with the higher ratio of G2/M phase,and when the concentration was 10μmol/L,the ratio of S phase of K562-HIF-1α~+cells was higher than that of K562-NC cells;The apoptosis rates of both cell lines increased after exposure,and when the concentration was 40μmol/L,the ratios of early apoptosis and late apoptosis in K562-HIF-1α~+cells were lower than those in K562-NC cells;When the concentration was 40μmol/L,the levels of reactive oxygen species in the two cells were significantly increased,and the levels of reactive oxygen species in K562-HIF-1α~+cells were higher than those in K562-NC cells.The expression levels of PGK1,HIF-1α,PFKL and PKM2 proteins in K562-NC cells increased after exposure,and the expression levels of BCL2and PKM2 proteins in K562-HIF-1α~+cells increased.The results indicated that this experiment successfully constructed a stable and highly HIF-1αexpressed K562 cell and control cell model.Under 1,4-benzoquinone exposure,HIF-1αmay regulate cell cycle and apoptosis by regulating the genes PGK1 and PFKL which are involved in cellular energy metabolism.Chapter Ⅲ Study on target genes specifically regulated by HIF-1αin bone marrow cells exposed to benzeneApply Chromatin Immunoprecipitation Sequencing technology to analyze the genome-wide binding profile of the transcription factor HIF-1αin bone marrow cells of benzene-exposed and control mice.Screening for hematopoietic function-related target genes and pathways in the regulation of HIF-1αin the process of hematopoietic toxicity caused by benzene exposure.The mRNA levels of HIF-1αtarget genes in bone marrow cells of benzene poisoning mice were detected,and the results were compared with ChIP-Seq bioinformatics results.The ChIP-Seq results were verified at the mRNA level,which can improve the accuracy of screening HIF-1αtarget genes.The results showed that compared with the control group,in the benzene-exposed group,there were 353 differential genes up-regulated and 245 differential genes down-regulated in the promoter region.Compared with the control group,there were 101 GO Terms in the biological process part of the down-regulated genes by GO analysis in the benzene-exposed group.Pathway analysis showed that the Jak-STAT signaling pathway is the down-regulation pathway with the highest enrichment score.Among the differentially expressed genes in the benzene-treated group,the promoter regions of 42 genes contained HIF-1αspecific binding sites,and 23 of them were found to be related to biological processes with reference to GO analysis results.The mRNA levels of GRB2 and PIK3CA genes in bone marrow cells of benzene poisoning model mice were decreased.The results indicated that the target genes of HIF-1αin bone marrow cells of benzene poisoning mice and control group were successfully screened by ChIP-Seq technology.HIF-1αmay regulate the function of bone marrow cells in benzene poisoning mice by regulating the expression of GRB2 and PIK3CA genes in the Jak-STAT pathway.ConclusionThis study found that the level of HIF-1αprotein in bone marrow cells of benzene poisoning mice decreased with the increase of reactive oxygen species,indicating that reactive oxygen species may inhibit the expression of HIF-1αprotein in bone marrow cells.The levels of BMI1 and BCL2 protein in bone marrow cells of benzene-infected mice decreased with the decrease of HIF-1αprotein expression level,indicating that HIF-1αmay regulate benzene-induced bone marrow hematopoietic toxicity by regulating BMI1 and BCL2levels.Studies on the cytotoxicity of HIF-1αstably and highly expressed K562 by1,4-benzoquinone indicate that HIF-1αmay be involved in the regulation of cell cycle and apoptosis,which may regulate cell function through genes PGK1 and PFKL which are involved in cellular energy metabolism.ChIP-Seq technology was used to screen out the HIF-1αtarget genes GRB2 and PIK3CA,which have different expression in bone marrow cells of benzene poisoning mice and control group and related to biological processes,and they may be involved in the regulation of bone marrow cell function in benzene poisoning mice under the control of HIF-1α.