The Effect Of N-acetyltransferase 10 In Acute Myeloid Leukemia And Its Related Mechanisms

Objective: Acute myeloid leukemia(AML)is a group of malignant hematological diseases that originated from hematopoietic stem cells.It is characterized by over-proliferation apoptosis inhibition,and impaired differentiation of hematopoietic stem cells.Till now,great progress has been made in the diagnosis and treatment of AML.With advances in cytogenetics and molecular biology techniques,we get deeper understanding of pathogenesis of leukemia.Noval chemotherapy regimens,allogeneic hematopoietic stem cell transplantation and second generation sequencing technologies are convenient for medical stuff and are benefit to patients.However,the overall survival of AML remains to be unsatisfying and AML remains to be a incurable disease.Here we give an insight to N-acetyltransferase 10(NAT10).NAT10 is a member of the GCN5-related N-acetyltransferase(GANT)family and was first discovered in the promoter region of h TERT.NAT10 has been reported with the functions of DNA damage recovery,histone acetylation,r RNA acetylation and microtubules acetylation,thus stabilizing the nuclear structure and regulating the cell cycle.Studies have shown that NAT10 might be an oncogene in solid tumor such as liver cancer,colorectal cancer and melanoma and high NAT10 high-expression is always associated with poor prognosis.There are no relevant reports in leukemia.Through the bioinformatics analysis,we found that NAT10 may also be highly expressed in acute myeloid leukemia cells.By analyzing the related pathways of the proteins interacting with NAT10,we found that NAT10 may be associated with telomerase maintenance,cell cycle regulation and differentiation of myeloid cells.In view of these,we designed a series of experiments to detect the expression of NAT10 in acute myeloid leukemia and explore the roles and relevant mechanism of NAT10 in acute myeloid leukemia cells,hoping to provide a new idea for the diagnosis and treatment of acute myeloid leukemia.Methods: We collected bone marrow liquid from 48 cases of acute myeloid leukemia(40non-M3 patients and 8 M3 patients)and 20 cases of non-hematologic malignancies in bone marrow fluid.Mononuclear cells were extracted using lymphocyte separation fluid.Real-time q PCR was used to detect NAT10 m RNA expression.Clinical data was obtained from inpatient medical records.AML patients were stratified to good,medium,and poor prognosis groups by cytogenetics and molecular genetics criteria.Induction chemotherapy regimens included daunorubicin and cytarabine or idarubicin and cytarabine.Curative effect was evaluated by complete remission(CR),partial remission(PR),and nonremission(NR)after one-time induction chemotherapy.Progression-free survival(PFS)was the time from diagnosis to any type of disease relapse.Overall survival(OS)was the time from diagnosis to death.Expression of NAT10 m RNA was detected in myeloid leukemia cell lines NB4,HL-60,KG1α,K562 and U937.The NAT10 m RNA expression levels in NB4 and U937 cells were higher than the others,so we chose NB4 and U937 cells to carry out the following experiments.We used Remodelin as a chemical inhibitor of NAT10.RT-q PCR and western blot were used to verify the expression level of NAT10 in Remodelin-treated NB4 and U937.CCK-8 assay was used to detect the effect of NAT10 on the proliferation of NB4 and U937 cells.Annexin V-FITC/PI staining flow cytometry was used to detect apoptosis rates.Remodelin with or without caspase inhibitor Z-VAD-FMK was used to treat NB4 and U937 cells to detect apoptosis by flow cytometry.The effect of NAT10 on MMP was detected by rhodamine 123 staining flow cytometry.Western blot was used to detect the expression of caspase 3,cleaved caspase 3,caspase 7,caspase 9,Bcl-2,BAX and BAK proteins to explore the specific mechanism of apoptosis.The effect of NAT10 on cell cycle of NB4 and U937 was deteected by PI staining flow cytometry.We used western blot to detect the expression of cell cycle regulatory factors p21,cyclin D1,cyclin E,CDK2 and CDK4 protein.Western blot was used to detect endoplasmic reticulum stress-related proteins GRP78,PERK,e IF2α,cleaved caspase12 and CHOP to explore the role of endoplasmic reticulum stress in NAT10-mediated apoptosis.NB4 and U937 were treated with Remodelin combined with PI3 K activator IGF-1 and inhibitor LY294002 and PI3 K,Akt and p-Akt protein expression were detected by western blot assay to explore the role of PI3 K / Akt pathway in NAT10-mediated leukemia cell apoptosis.Indirect immunofluorescence assay was used to detect the expression of NAT10 and NPM1.Western blot was used to detect the overall expression of NAT10 and NPM1 and their expression in cytoplasm and nucleus.Results: NAT10 m RNA levels were assayed by RT-qPCR in bone marrow mononuclear cells that had been collected from 48 AML patients and 20 normal controls.Because of the good prognosis in patients with acute promyelocytic leukemia,we only analyzed the clinical data of 40 non-M3 AML patients.AML patients had higher NAT10 expression level than the control group(P<0.01).There were no significant differences in NAT10 expression among different age,gender,or FAB classification or among patients with good,medium,or poor prognosis.25 of the 40 patients received standard induction chemotherapy,and 14 achieved CR,and one achieved PR.The overall response(OR)rate was 60%.10 patients got non-remission.Pretreatment-NAT10 expression was significantly lower in the OR than that in NR patients(P<0.05).The median follow-up was 5.5 months.The survival curves indicate that patients with high NAT10 expression had significantly shorter OS and PFS than those with low expression(P<0.05).The results are supportive of NAT10 expression as an AML biomarker with prognostic value.As the expression of NAT10 m RNA was higher in the NB4 and U937 than in the other AML cell lines(HL-60,KG1α,and K562),they were selected for further investigation.Immunofluorescence results showed that NAT10 was expressed in nucleus in both NB4 and U937 cells.Remodelin is a NAT10 inhibitor.NB4 and U937 cells were treated with Remodelin for 24 h at 25,50,100,or 200 μM).Remodelin treatment reduced both NAT10 m RNA and protein expression in a dose-dependent manner,which confirmed its expected activity as an NAT10 inhibitor.NB4 and U937 cell cultures were treated with25,50,100,or 200 μM Remodelin for 12,24,and 48 h,and proliferation was assayed with a CCK8 kit.The anti-proliferative activity of Remodelin in AML cells was both dose-and time-dependent.The effect of Remodelin on apoptosis was assayed by flow cytometry in NB4 and U937 cells incubated with 25,50,100,or 200 μM Remodelin for24 h.Remodelin induced apoptosis in a dose-dependent manner.Next,we used caspase inhibitor Z-VAD-FMK(20 μM)to treat AML cells together with Remodelin.Z-VAD-FMK was added 30 min before Remodelin.The results showed that the apoptosis rates of Remodelin group were higher than those of Remodelin+Z-VAD-FMK group(P<0.05).Western blot assays of AML cell lines cultured under the same conditions found that caspase 3,caspase 7 and caspase 9 expression decreased and the expression of cleaved caspase 3 increased,consistent with Remodelin-induced apoptosis of the AML cells.As the mitochondrial apoptosis pathway is a classical intrinsic apoptosis pathway, Bcl-2,BAX,and BAK expression were assayed.Bcl-2 was downregulated and BAX and BAK were upregulated,indicating that mitochondrial pathway participated in Remodelin-mediated apoptosis in the AML cells.The cell cycle strongly influences proliferation,and to investigate the effect of NAT10 on the cell cycle,NB4 and U937 were incubated 25,50,and 100 μM Remodelin for 24 h.Cell cycle analysis was performed with PI-stained cells by flow cytometry.The proportion of cells in G1 phase was significantly increased by Remodelin,indicating that inhibition of NAT10 caused G1 phase arrest.The mechanism of cell cycle arrest was investigated by assays of the expression of the cell cycle regulators p21,cyclin E,cyclin D1,CDK2,and CDK4 by western blotting.p21 expression increased and cyclin D1,cyclin E,CDK2,and CDK4 expression decreased.The results consistently indicated that downregulation of NAT10 caused G1 phase arrest.3.Rhodamine 123 staining flowcytometry was used to detect MMP and revealed that MMP of NB4 and U937 cells decreased as NAT10 downregulation,further demonstrating the activation of mitochondrial apoptotic pathway.As activation of ERS can induce apoptosis,NB4 and U937 cells were treated with Remodelin and the expression of the ER stress marker proteins GRP78,PERK,e IF2α,caspase 12,cleaved caspase 12,and CHOP were assayed.At Remodelin concentrations of 100 and 200 μM,expression of GRP78,PERK,e IF2α,caspase 12,cleaved caspase 12 and CHOP proteins were all upregulated.The results confirmed that Remodelin evoked ERS.PI3K/Akt is a classical pathway that regulates the cell cycle and apoptosis11,12.PI3 K,Akt,and p-Akt.PI3 K,Akt and p-Akt were all downregulated in Remodelin-treated AML cells.To confirm the effect of Remodelin on PI3K/Akt pathway,PI3 K activator IGF-1 and inhibitor LY294002 were added to NB4 and U937.Following pretreatment IGF-1(200 ng/ml)or LY294002(25 μM)for 30 min,NB4 and U937 cells were incubated in 100 μM Remodelin for 24 h.The apoptosis rate was highest with Remodelin+LY294002,followed by Remodelin alone,and Remodelin+IGF-1(P<0.05).Expression of PI3 K,Akt,and p-Akt was the highest with Remodelin+IGF-1,followed by Remodelin alone,and Remodelin+LY294002(P<0.05).The results consistently indicated that Remodelin induced apoptosis by downregulating the PI3K/Akt pathway.4.Indirect immunofluorescence assay showed that NAT10 and NPM1 co-located in the nucleus.NPM1 was downregulated when expression of NAT10 was inhibited in NB4 and U937 cells.Cytoplasmic protein and nuclear protein were extracted respectively.Western blot was used to detect the expression of NAT10 and NPM1 in the cytoplasm and nucleus after NAT10 was downregulated.The results showed that the expression of NAT10 in the nucleus and cytoplasm were both downregulated while the expression of NPM1 was downregulated in the cytoplasm but upregulated in the nucleus in U937 cells.However,in NB4 cells,both NPM1 in nucleus and cytoplasm were downregulated.The results indicated that downregulation of NAT10 could inhibited the expression of NPM1 and might promote translocation of NPM1 from cytoplasm to nucleus.Conclusion: 1.Acute myeloid leukemia patients are higher NAT10 expression than normal control group.Patients with high NAT10 m RNA expression have lower response rate and poorer survival than patients with low NAT10 m RNA expression.2.The downregulation of NAT10 can inhibit the proliferation,promote caspase-dependent apoptosis and cause G1 phase arrest in acute myeloid leukemia cells.3.Decrease of MMP and promotion of endoplasmic reticulum stress can induce apoptosis in Remodelin-treated AML cells.NAT10-induced apoptosis can be promoted by inhibiting the PI3K/Akt signaling pathway.4.NAT10 and NPM1 co-localized in the nucleus.Downregulation of NAT10 inhibits the expression of NPM1 and promotes the migration of NPM1 from the cytoplasm to the nucleus.