Effect Of PD-L1 On Glycolysis Of Acute Myeloid Leukemia And Its Mechanism

BackgroundAcute myeloid leukemia(AML)is a highly invasive and highly heterogeneous hematological malignancy,and one of the most common leukemias in adults.It appears that the current complete remission(CR)rate of induction chemotherapy(chemotherapy)can reach 70%-80%,but there are still about 10%-40%of patients who have failed induction therapy.Patients with regular consolidation therapy still have a relapse rate of up to 50%.Patients who failed induction therapy and relapsed eventually developed refractory and relapsed leukemia.At present,the mechanism of the occurrence and development of AML has not been fully elucidated.Its pathogenesis involves multiple factors.Among them,cytogenetic abnormalities play an important role in the pathogenesis of AML.An important area affected by genetics is the glucose metabolism pathway.Glycolysis is the main energy metabolism process of tumor cells.In the 1920s and 1930s,Otto Warburg et al.found that,under the condition of sufficient oxygen,tumor cells can convert most of the glucose into lactic acid in the form of glycolysis.This type of metabolism characterized by high glucose consumption,aerobic glycolysis and lactic acid production is called the "Warburg effect".A series of key enzymes in glycolysis play an important role in proliferation,infiltration,metastasis and drug resistance of solid tumors and acute leukemias.Glycolysis not only provides energy supply for rapid proliferation of tumor cells,but also provides them with a large number of synthetic raw materials necessary for proliferation.By inhibiting glycolysis,tumor cell proliferation inhibition,increased apoptosis,and reversal of drug resistance can occur to achieve anti-tumor purposes.The AKT/m-TOR signaling pathway is one of the regulatory pathways most closely related to tumor cell glycolysis,and also involved in the expression and regulation of the transcription factor HIF-lα.PD-L1(programmed death ligand 1),as a member of the B7 superfamily,is often expressed on the surface of antigen-presenting cells and tumor cells,and participates in the immune escape process of tumor cells through interaction with its receptor PD-1(programmed death 1).PD-L1 can be detected on the surface of a variety of tumor cells and on the surface of immune cells in the tumor microenvironment.PD-L1 is not only involved in tumor immune escape but also related to the malignancy and poor prognosis of tumor.Several studies have confirmed that PD-L1 is closely related to the occurrence and development of malignant tumors.Other studies have shown that PD-L1 on the surface of tumor cells can directly activate the intracellular AKT/m-TOR signaling pathway,leading to upregulation of glycolytic gene expression and enhanced glycolytic metabolism.There are few reports on the research of PD-L1 in AML,and the current research mainly focuses on the combination of PD-L1 and PD-1 to affect antitumor immunity.The lack of PD-L1 on the occurrence and development of AML and the specific mechanism Research.Among them,it has not been reported whether PD-L1 can directly participate in regulating the biological behavior of AML cells,and whether abnormally expressed PD-L1 in AML is related to the high glycolytic phenotype of AML.To solve these problems,this study first explored the relationship between the expression of PD-L1 in primary AML and its clinical characteristics and prognosis,and then the effect and mechanism of PD-L1 on the biological behavior and glycolysis of AML cells.To study the expression of PD-L1 in bone marrow mononuclear cells in newly treated AML patients,bone marrow specimens of newly treated non-M3 AML patients admitted to the First Affiliated Hospital of Zhengzhou University between 2015 and 2017 were collected.After the follow-up and the compilation of case data,there were 90 cases.After collecting fresh specimens,they were centrifuged with lymphocyte separation solution,the mononuclear cells were extracted after centrifugation,and then mRNA were extracted and detected by fluorescent real-time quantitative PCR method to detect mRNA expression level.AML cell lines with stable overexpression and low expression of PD-L1 were further constructed,and the effects of PD-L1 expression on the proliferation,apoptosis,cycle,and glycolytic function of AML cells were studied in vitro and in vivo.RNA-Seq technology was used to analyze the gene changes of PD-L1 gene overexpression group and control group leukemia cell lines at the transcriptome level.Bioinformatics technology was used to analyze the pathway and functional enrichment of the genes it affected,and the possible mechanism of PD-L1 in AML was analyzed.Finally,the effects of PD-L1 expression on glycolysis-related proteins and key proteins in AKT/mTOR signaling pathway were detected by Western blot technology,and the specific mechanism of action was analyzed.This research is divided into three parts:Part 1.Expression of PD-L1 in AML and its effect on clinical prognosisObjectiveQ-PCR method was used to detect the mRNA level of PD-L1 in monocytes of patients with AML,to explore the relationship between changes in PD-L1 expression levels and clinical characteristics and prognosis,and to analyze the PD-L1 gene and glycolytic genes and Correlation between HIF-la genes.Methods1.Using the published tumor sequencing results data provided by GEPIA website,analyze the expression of PD-L1 gene in AML patients and normal controls,and analyze the relationship between the level of PD-L1 gene expression and survival.Finally,the correlation between PD-L1 gene and glycolysis-related genes and HIF-la gene were analyzed.2.Detecting the expression levels of PD-L1,glycolysis-related genes and HIF-la gene in bone marrow mononuclear cells of 90 newly treated AML patients and 18 bone marrow transplant donor mononuclear cells using Q-PCR.Correlation analysis were performed between genes.3.Collect clinical data of 90 patients with primary AML,then divide them into continuous complete remission group and refractory relapse group according to the efficacy and follow-up results of induction therapy and subsequent consolidation therapy.Analyze AML patients and normal controls and continuous complete remission group and difficulty.The difference in PD-L1 gene expression between the relapsed groups was analyzed,and the difference in glycolysis-related gene expression between AML patients and the normal control group was analyzed.4.Chi-square test was used to analyze the relationship between PD-L1 expression and clinical characteristics of AML patients.Kaplan-Meier method was used to analyze the survival rate and draw the survival curve.COX regression model was used for univariate and multivariate analysis.Results1.By searching on the GEPIA website,it was found that the expression of PD-L1 gene was increased in AML patients compared with normal controls,but the difference was not statistically significant.Survival search revealed that the survival time of some patients with AML whose PD-L1 expression was up-regulated was significantly lower than that of PD-L1 expression down-regulation(P=0.046).Analysis of the correlation between PD-L1 gene expression and glycolysis-related genes(PFKM,PDK1,ENO2,GLUT1)and HIF-1α gene expression(all P<0.05)were confirmed.2.Q-PCR detection of mRNA expression levels of PD-Ll,glycolysis-related genes(PFKM2,ALDOA,PGK1,PDK1,LDHA,HK2)and HIF-lα showed that PDL1 expression was higher in newly treated AML patients than in normal controls.(P<0.05),the expression level of refractory relapse group was higher than that of continuous complete remission group(P<0.05),and glycolysis-related genes and HIF-la expression levels were higher in newly treated AML patients than in normal controls(both P<0.01).There was a positive correlation between the expression of PD-L1 and the expression of glycolysis-related genes and HIF-la in newly treated AML patients(all P<0.01).3.Chi-square test was used to analyze the relationship between the expression of PD-L1 gene and clinical characteristics.The results showed that the expression level of PD-L1 gene was positively correlated with the clinical characteristics of whether AML patients had a course of treatment.4.The results of survival analysis showed that the expression level of PD-L1 gene was correlated with the prognosis of patients with AML.AML patients with high expression of PD-L1 gene had worse overall survival compared with patients with low expression(P=0.041).Multivariate analysis of the prognosis of AML patients,WBC number(P=0.007),prognostic stratification(P=0.011),whether a course of remission(P=0.04)and PD-L1 gene(P=0.02)are independent prognosis of AML factor.Conclusion1.Compared with normal control,the expression of PD-L1 gene was significantly increased in patients with newly treated AML.The expression of refractory relapse group was higher than that of continuous complete remission group,and its expression was related to the prognosis of patients.2.Compared with normal controls,the expression of glycolysis-related genes(PFKM2,ALDOA,PGK1,PDK1,LDHA,HK2)and HIF-la gene were significantly increased in newly treated patients with AML.3.There is a positive correlation between the expression of PD-L1 and the expression of glycolysis-related genes and HIF-la in newly treated AML patients,suggesting that PD-L1 may regulate the glycolytic process of AML through HIF-1α.Part 2.Effects of PD-L1 on the biological function of AML cells in vitro and in vivoObjectiveThe effects of PD-L1 expression on the proliferation,apoptosis and cycle of AML cells were studied by in vitro cell function experiments.To study the role of PD-L1 in the occurrence and development of AML,a nude mice subcutaneously transplanted tumor model was constructed,and the effect of PD-L1 expression on tumor volume and weight was examined.Methods1.Culture 5 AML cells:HL-60,Molm-13,K562,KG-la and THP-1 cells,and apply magnetic bead sorting technology to isolate CD34+cells from the peripheral blood of patients with normal bone marrow transplantation as normal controls.Promote cell proteins and RNA during logarithmic growth phase.2.Q-PCR method was used to detect the expression level of PD-L1 gene in the above five AML cell lines and CD34+ cells;flow cytometry detection and Western blot were used to detect the cell PD-L1 protein expression.3.Use PD-L1 overexpression and shRNA interference plasmid to package lentivirus,transfect PD-L1 overexpression plasmid into Molm-13 cells,transfect shRNA plasmid into THP-1 cells,use puromycin to screen for stability.The expression strains were finally detected by fluorescence microscopy,Q-PCR,Western blot,and flow cytometry for the screened cell lines to detect the fluorescence expression,PD-L1 gene and protein expression.4.CCK-8 kit was used to detect cell proliferation,apoptosis kit was used to detect cell apoptosis,cell cycle kit was used to detect cell cycle distribution changes,and the effect of PD-L1 expression changes on the biological behavior of AML cells was observed.5.Construct a subcutaneous xenograft model by inoculating Mol-13-vec and Mol-13-PD-L1 cells subcutaneously in nude mice,and observe the growth rate,volume,and final weight changes of the two groups.Finally,the expression of Ki67 in transplanted tumors was detected by immunohistochemistry.Results1.According to the expression of PD-L1 in 5 AML cell lines,finally selected the cell line Molm-13 that lowly expressed the PD-L1 gene and protein and the cell line THP-1 that highly expressed the PD-L1 gene and protein.2.The use of Q-PCR and western blot to detect stable cell lines showed that PD-L1 gene and protein were increased in Molm-13-PD-L1 compared to Mol-13-vec and Mol-13-blank,and THP1-sh1 and THP1-sh2 are lower than in THP1-NC.3.PD-L1 overexpression can increase the proliferation of Molm-13 cells and suppress apoptosis while increasing the proportion of S-phase cells.Decreased PD-L1 can cause suppression of THP-1 cell proliferation and increase apoptosis while increasing G0/G1 Proportion of cells in the phase,reducing the proportion of cells in S phase.4.Results of subcutaneous xenograft tumors in nude mice show that PD-L1 can significantly increase the expression of proliferation-related protein Ki67 in AML xenografts and promote the proliferation of AML xenografts.ConclusionIn vitro and in vivo experiments have confirmed that changes in PD-L1 expression can affect the proliferation,apoptosis and cycle of AML cells.Part 3.Effect of PD-L1 on Glycolysis of AML Cells and Its MechanismObjectiveThe transcriptome sequencing of Molm-13-vec and Mollm-13-PD-L1 cells,functional analysis of the sequencing results,and pathway enrichment analysis were performed to predict the mechanism of PD-L1 in AML.The effect of PD-L1 expression on glycolysis of AML cells was examined and the specific signal pathways of PD-L1 on glycolysis were explored.Methods1.The stable-transformed cell lines Molm-13-PD-L1 and Molm-13-vec were cultured,collected,and washed,and an appropriate amount of TRizol was transported on dry ice to BGI Shenzhen Co.,Ltd.for transcriptome sequencing.Differential genes were analyzed by KEGG function and pathway enrichment to predict the role of PD-L1 in AML and its mechanism.2.Use Q-PCR and western blot to detect the effects of PD-L1 expression on glycolysis-related genes and key proteins,and use glucose and lactate detection kits to detect changes in PD-L1 expression on glucose uptake and lactic acid production.The effects of changes in PD-L1 expression on extracellular acidification rate(ECAR)were examined using Seahorse experiments.3.Western blot was used to detect the key proteins in the enriched pathway obtained by sequencing,then pathway inhibitors were used to detect changes in key proteins of the pathway and to detect the effects of pathway inhibitors on glycolysis of AML cells.4.Double-luciferase expression experiments were used to verify the targeted regulation of HIF-1α on key glycolytic genes HK2 and LDHA.Results1.Differential gene KEGG functional enrichment analysis PD-L1 can regulate AML carbohydrate metabolism;KEGG pathway enrichment analysis shows that the effect of PD-L1 on AML cell metabolism may be achieved through the PI3K/AKT signal transduction pathway.2.Overexpression of PDL-1 gene can lead to upregulation of AML cell glycolysis-related genes(ALDOA,PGK1,LDHA,HK2)and glycolysis key proteins HK2 and LDHA,and interference with PD-L1 expression in AML cells can lead to glycolysis.The expression of unrelated genes and key glycolytic proteins HK2 and LDHA were down-regulated.3.Overexpression of PDL-1 gene can lead to increased glucose uptake,increased ECAR and increased lactic acid production in AML cells.Interfering with the expression of PD-L1 can lead to decreased glucose uptake,reduced ECAR,and reduced lactic acid production in AML cells.4.Detection of AKT/mTOR/HIF-1α pathway key proteins in AML cell lines showed that P-AKT,P-S6,and HIF-1α proteins were highly expressed in PD-L1 compared with PD-L1.Increased expression in line THP1.Compared with the blank cell and empty plasmid cell group,the expression of P-AKT,P-S6 and HIF-1 prion protein increased in AML cells overexpressing PD-L1.Compared with control group,the expression of P-AKT,P-S6 and HIF-1 prion protein decreased in AML cells that interfered with PD-L1 expression.5.The application of AKT inhibitors in AML cells overexpressing PD-L1 can lead to a decrease in the expression of P-AKT,P-S6,HIF-1α,HK2 and LDHA proteins,and at the same time lead to decreased glucose uptake,reduced ECAR in AML cells and reduced lactic acid production.6.The application of mTOR inhibitor rapamycin in AML cells over-expressing PD-L1 can lead to a decrease in the expression of P-S6,HIF-la,HK2 and LDHA proteins,as well as a decrease in glucose uptake,ECAR and Reduced lactic acid production.7.Using si technology to interfere with the expression of HIF-1α gene in AML cells over-expressing PD-L1 can cause the expression of HIF-1α,HK2 and LDHA proteins to decrease,while the expression of P-AKT and P-S6 proteins is not affected.At the same time,it results in reduced glucose uptake,reduced ECAR and reduced lactic acid production in AML cells.8.Application of double luciferase experiments to verify that HIF-la can target HK2 and LDHA genes.Conclusion1.PD-L1 expression changes can affect the glycolysis process of AML cells.2.PD-L1 can increase the expression levels of HK2 and LDHA proteins by activating the AKT/mTOR/HIF-1α signaling pathway and promote glycolysis in AML cells.