The Metabolism And Prognostic Significance Of GLUD1 In Acute Myeloid Leukemia

Acute myeloid leukemi(AML)is a kind of hematological malignancies with high heterogeneous and incidence.Recent years,with the development of genetics,molecular biology,second-generation sequencing,and other technologies have significantly improved the further understanding of the etiology and pathology of AML,but the specific mechanisms involved in are still not yet been clearly defined.With the further popularization of molecular targeted drugs and chimeric antigen receptor T-cell immunotherapy(CAR-T)and hematopoietic stem cell transplantation,the treatment of AML has entered a new field.But compared with the traditional chemotherapy,In addition to the high cost of new therapy,long-term efficacy remains to be further confirmed.Metabolomics is an emerging branch of system biology following genomics and proteomics.It aims to reveal abnormal metabolism associated with the development of tumors by analyzing the metabolic spectrum of tumors,thus providing a new indicator for stratified treatment and prognosis assessment of AML.And provide new potential targets for tumor therapy in metabolic pathways.At present,metabolomics studies of tumor metabolism reprogramming are mainly focused on abnormal metabolism in glucose and glutamine.The uptake,utilization,and conversion of glucose and glutamine by tumor cells are significantly different from those of normal ones.Glutamine has two metabolic pathways in cells.Including glutaminase(GLS),which converted glutamine to glutamate,and then catalyzed by glutamate dehydrogenase(GLUD)to Ketoglutaric acid(?-KG).and transaminases to generate another non-essential amino acid and ?-KG.Both pathways generate ?-KG that can enter the tricarboxylic acid cycle to provide energy for cell survival.GLUD has two forms including GLUD1/2 in the human body and is mainly in GLUD1 form.GLUD1 catalyzes the conversion of glutamate to a-KG and provides raw materials for the synthesis of proteins,nucleic acids,and fatty acids.It is one of the key enzymes for glutamine metabolism.Studies in prostate cancer and glioma shown that GLUD1 is closely related to the occurrence,development and metastasis of tumors and has a significant impact on the prognosis of patients.In this study,we will focus on the effect of GLUD1 on the prognostic and metabolism significance in AML.Object:By comparing the expression of GLUD1 in bone marrow/peripheral blood of AML patients and healthy people,the relationship between the different expression of GLUD1 and the prognosis of AML patients was analyzed,and the regulation mechanism of GLUD 1 expression was explored.Investigate the effect of GLUD1 overexpression/knockout on the biological behavior in AML cell line and its effect on the tumor burden and survival of Xenografts models.miRNA sequencing was carried out to investigate the regulation mechanism of GLUD1,quantitative metabolomics method based on chromatography-mass spectrometry was used to study the metabolic profile of AML cell line after knockout of GLUD1,GLUD1 inhibitor R-162 was administrated to study its effect on AML cell.Method:(1)The expression of GLUD 1 in 320 cases of AML and 19 cases of normal controls was detected by real-time PCR,and the relationship between GLUD1 expression and the prognosis of AML patients was analyzed;(2)Ovexpression and knockout GLUD1,CCK8,clone formation test,drug sensitive test,flow test cell apoptosis and cell cycle,Western-blot were used to detect related proteins to further validate GLUD1 biofunction in AML;(3)GLUDl overexpression/knockout and control AML cell lines were injected into the tail vein of mice to establish Xenografts models.The mice were mainly observed for tumor burden and survival;(4)Using the miRNA sequencing technology to detect different miRNA profile in AML patients,and through bioinformatics website forecast miRNA which may regulate GLUD1,investigate the molecular mechanism of GLUD1 regulation;(5)Chromatography-mass spectrometry for quantitative metabolomics study of the metabolic profile of AML cell lines after knockout GLUD1,analyzing the role of GLUD1 in AML metabolic disorders.Result:(1)The expression of GLUD1 was significantly up-regulated in part of AML patients compared with normal controls,and the overall survival of patients with high expression was poor,and high expression of GLUD1 was an independent prognostic factor for AML patients;(2)GLUD1 overexpression promoted the AML cell proliferation;Improved the clone formation;Reduced the sensitivity to chemotherapy drugs.GLUD1 knockout inhibited AML cell proliferation;Reduced the clone forming;induced cell apoptosis;Enhanced the sensitivity to chemotherapy drugs;(3)Overexpression of GLUD1 increased tumor burden in mice;shortened survival time.GLUDl knockout reduced the tumor burden and prolonged the survival time.(4)AML patients with GLUD1 high expression exerted lower level of miR-493.Bioinformatics predicted miR-493 was the upstream regulatory factor of GLUD1,using fluorescence quantitative PCR,Western blot and luciferase double report system further confirmed that GLUD1 was the direct target of miR-493;(5)GLUD1 knockout induced glutamine catabolism,inhibited and a-KG levels.Transaminase is the main route to produce a-KG after GLUD1 knockout,the glycolytic pathway was inhibited and the AML cells gained energy mainly through the tricarboxylic acid cycle.(6)R-162 inhibited AML cell proliferation.Conclusion:(1)GLUD1 was highly expressed in part of AML,and it was an independent risk factor for poor prognosis in AML patients;(2)GLUD1 functions as an oncogene in AML;(3)miR-493 was low expressed in GLUD1 high-expressed AML patients,and GLUD1 was the direct target of miR-493;(4)GLUD1 knockout inhibited glutamine metabolism in AML,inhibited anaerobic glycolysis of glucose and reductive carboxylation of ?-KG,enhanced the tricarboxylic acid cycle.(5)R-162 inhibit AML cell proliferation.