Effect Of PTP4A3 Regulated By HIF-1α On Benzene-induced Bone Marrow Hematopoietic Toxicity

People exposed to benzene through the respiratory tract.Short-term inhalation of high concentrations of benzene can cause acute benzene poisoning,and prolonged exposure to low concentrations of benzene can lead to chronic benzene poisoning and affect the hematopoietic function.Hypoxia-inducible factor(HIF-1α)is a transcription factor that mainly regulates the metabolism and physiological functions of hematopoietic stem cells.HIF-1αcan help maintain hematopoietic stem cell quiescence and long-term proliferation.In this study,benzene poisoning mouse model was established to study the role of HIF-1αin proliferation,apoptosis and other changes of mouse bone marrow LSK cells after benzene exposure.Bioinformatics analysis was performed by ChIP-Seq to screen the proliferation-related gene PTP4A3 regulated by HIF-1αand further verification was performed.This provides clues for the mechanism of regulation of HIF-1αin bone marrow hematopoietic toxicity induced by benzene,and provides a scientific basis for possible biomarkers of benzene hematotoxicity.Chapter1:Regulation of HIF-1αon bone marrow hematopoietic toxicity induced by benzeneC57BL/6 mice with high expression of HIF-1α(C57BL/6-HIF-1α~+)were constructed and benzene poisoning mouse model was established with C57BL/6-HIF-1α~+and C57BL/6male mice.After exposure,body weight,organ coefficient,blood routine,and hematopoietic system changes were analyzed.The results showed that the C57BL/6-HIF-1α~+mouse model was successfully constructed.The expression level of HIF-1αprotein in C57BL/6-HIF-1α~+bone marrow cells was significantly higher than C57BL/6 mice.Mice exposure to 150 mg/(kg.d)benzene resulted in lower liver coefficient,spleen co efficient,lung coefficient,and peripheral white blood cells,red blood cells,and platelets compared with control mice group.The results of flow cytometry revealed that the LSK ratio’s reduction and increasement of early apoptosis rate in bone marrow cells of C57BL/6-HIF-1α~+mice were lower than control mice.The LSK cell proliferation of C57BL/6-HIF-1α~+mice was higher than control mice.The expression levels of HIF-1αprotein in bone marrow cells of C57BL/6 and C57BL/6-HIF-1α~+mice lower than control group after exposed to benzene.The above results indicated that the hematopoietic toxicity of C57BL/6 mice with high expression of HIF-1αis weaker than C57BL/6 mice.The mechanism may be related to the proliferation of hematopoietic stem cells maintained by HIF-1α,suggesting that the high expression of HIF-1αplays a positive role in the self-renewal and survival of bone marrow hematopoietic stem cels.Chapter 2:Screening of downstream target genes related to cell prolife ration regulated by HIF-1αin benzene-exposed miceBenzene-exposed bone marrow cells of C57BL/6 mice and control mice were used to perform ChIP,chromatin immunoprecipitation sequencing(ChIP-Seq)was used to explore the downstream target genes affected by abnormal HIF-1αexpression in benzene-exposed mice.The combined level,transcription and translation levels of HIF-1αregulated response genes were detected.ChIP-Seq results showed that there were 353 up-regulated and 245 down-regulated enrichment peaks in the benzene-exposed group in the promoter region.Among the 245down-regulated genes,the UCSC database was used to search for 42 genes containing the HIF-1αspecific binding site HRE(aCGTG/gCGTG)in the gene sequence of the differentially enriched region,and there are 25 genes were labeled in GO gene.11 out of 25 genes’s mRNA level were significantly down-regulated in benzene-exposed mice bone marrow cell compared with control group.ChIP-qPCR results showed that enrichment fold of benzene-exposed group was 0.03 times of control group.The proliferation-related gene PTP4A3 was selected in the GO gene function.WB results showed that the protein level of PTP4A3 was down-regulated in C57BL/6 mouse bone marrow cells exposed to benzene compared to the control group,in C57BL/6-HIF-1α~+mouse bone marrow cells were up-regulated compared to control mice.The above results suggested that ChIP-Seq can effectively screen out the transcription factor HIF-1αresponse genes.ChIP-qPCR results confirmed that the binding of HIF-1αto PTP4A3 was lower in the benzene exposed group than in the control group.PTP4A3increased with the increase of HIF-1α,and decreased with the decrease of HIF-1α,thus confirming that PTP4A3 is the target gene of HIF-1α.Chapter 3:Study on functional verification of PTP4A3 regulation on cytotoxicity of K562 induced by 1,4-benzoquinonThe low expression of PTP4A3 lentivirus and negative control lentivirus was infected K562 cells to construct K562 cell line with low expression of PTP4A3(K562-PTP4A3~-)and negative control cells(K562-NC).Treated with 1,4-benzoquinon,PTP4A3 mRNA and protein expression of K562-PTP4A3~-and K562-NC cells were detected.Moreover,cell proliferation inhibition,cell DNA damage,cell apoptosis rate,cell cycle distribution and changes in intracellular ROS levels were tested.RT-PCR results showed that the mRNA level of PTP4A3 in K562-PTP4A3~-cells wasreduced by 90%compared with K562-NC cells,indicating that the K562-PTP4A3~-cell model was successfully constructed.Under the treatment of 0μmol/L,10μmol/L and20μmol/L 1,4-benzoquinone,the relative proliferation rate and cell proliferation activity of K562-PTP4A3~-cells were significantly lower than K562-NC cells.The early apoptosis rate of K562-PTP4A3~-cells exposed to 20μmol/L 1,4-benzoquinone was higher than K562-NC cells.The cell cycle results revealed that K562-NC cell cycle arrested at S phase while K562-PTP4A3~-cell cycle arrestd at G2/M phase after exposuring to 1,4-benzoquinone.The treatment of 1,4-benzoquinone increased the ROS content and Olive tail moment in K562-NC and K562-PTP4A3~-cells compared with the control group.The results of WB showed that the PTP4A3 protein and PI3K/AKT pathway protein were decreased in K562-PTP4A3~-and K562-NC cells treated with 0μmol/L,10μmol/L,and 20μmol/L 1,4-benzoquinone.The above results demonstrated low expression of PTP4A3 can increase proliferative damage and apoptosis of K562 cells induced by 1,4-benzoquinone.The changes of key protein level suggested that PI3K/AKT pathway were inhibited in cytotoxicity induced by1,4-benzoquinone,which prompted low expression of PTP4A3 may regulate cell proliferation though inhibition of PI3K/AKT pathway.Chapter 4:Expression of PTP4A3 in bone marrow and peripheral blood in chronic benzene poisoning miceC57BL/6 mice exposed to benzene at doses of 0 mg/(kg.d),6 mg/(kg.d),30 mg/(kg.d),150 mg/(kg.d),the general toxicity of chronic benzene exposure in mice was observed though body weight,blood routine indicators.The expression of PTP4A3 were examined in bone marrow cels,LSK cells and peripheral blood leukocytes.The results exhibited that compared with the control group,mice exposed benzene at 6mg/(kg.d),30mg/(kg.d),and 150mg/(kg.d)showed a slower increasement in body weight,which was significantly lower than control group while the white blood cell count,red blood cell count and hemoglobin content were lower than control group,espacially in 150mg/(kg.d)benzene exposure group.The blood cell count of mice showed a dose-related decrease after exposing to benzene.RT-PCR results showed that the mRNA levels of PTP4A3 in benzene-exposed mice bone marrow cells,LSK cells and peripheral blood leukocytes were lower than control mice,150 mg/(kg.d)benzene exposure group reduced significantly.The above results elucidated that the chronic benzene poisoning mouse model was established successfully.The expression of PTP4A3 in benzene-exposed mice bone marrow cells,LSK cells and peripheral blood leukocytes was lower than control group,suggesting that PTP4A3 may be a potential biomarker of chronic benzene poisoning.Conlusions:1.Hematopoietic toxicity of C57BL/6 mice with high expression of HIF-1αis weaker than C57BL/6 mice.The mechanism may be related to the proliferation of hematopoietic stem cells maintained by HIF-1α,suggesting that the high expression of HIF-1αis a positive protectable factor from benzene hematopoietic toxicity.2.ChIP-Seq was successfully used to screen out the HIF-1αresponse gene.The relationship between HIF-1αand its response genes was multiplely verified.ChIP-qPCR results confirmed that the binding of HIF-1αto PTP4A3 was lower in the benzene exposed group than in the control group.The expression of PTP4A3 decreased with the decreasement of HIF-1αand increased with the increasement of HIF-1α,which confirming PTP4A3 was the proliferation-related target gene regulated by HIF-1α.3.Low expression of PTP4A3 can increase proliferative damage and apoptosis of K562 cells induced by 1,4-benzoquinone.The changes of key protein level suggested that PI3K/AKT pathway were inhibited in cytotoxicity induced by 1,4-benzoquinone,which prompted PTP4A3 may regulate cell proliferation by involving in PI3K/AKT pathway.4.PTP4A3 may be a potential biomarker of chronic benzene poisoning.