The Effect Of Mitochondrial Autophagy Regulated By Acylglycerol Kinase On Aerobic Glycolysis And Immunity In Human Glioblastoma Cell

BackgroundGlioma is the most common primary tumor in the central nervous system,accounting for 80% of all malignant brain tumors.Glioblastoma multiforme(GBM)is the most malignant glioma,accounting for about half of all gliomas.It has the characteristics of high recurrence,poor prognosis and high mortality.At present,the treatment of glioma is mainly surgical resection,and adjuvant therapy is radiotherapy,chemotherapy,immunotherapy,targeted therapy and tumor electric field therapy.Although the modern active comprehensive treatment methods are constantly improving,the treatment effect of glioma is still very poor.How to improve the therapeutic effect of glioblastoma is still an urgent problem in neurosurgery.Aerobic glycolysis is one of the important characteristics of GBM.GBM needs a lot of energy and nutrition in the process of its rapid proliferation.Aerobic glycolysis can produce a lot of metabolic intermediates while providing energy,which provides raw materials for the synthesis of substances needed for cell proliferation.Studies have shown that the gene expression of key enzymes of aerobic glycolysis in GBM is increased.Inhibition of GBM aerobic glycolysis can inhibit the proliferation,invasion and migration of GBM,but the molecular mechanism of regulating GBM aerobic glycolysis is still unclear.Acylglycerol kinase(AGK),as a lipid metabolism kinase,is up-regulated in GBM and is associated with the proliferation,invasion and migration of GBM,but the specific mechanism is still unclear.Previous studies have shown that AGK can regulate PI3 K /AKT/ NFκB signal and affect the proliferation,apoptosis,invasion and migration of GBM.At the same time,AGK is located in the inner membrane of mitochondria,which plays an important role in maintaining the stability of mitochondrial membrane structure.The depolarization of mitochondrial membrane is one of the inducements of mitochondrial autophagy,and mitochondria is the site of aerobic glycolysis.Mitochondrial autophagy can affect the level of aerobic glycolysis.Previous studies found that the level of mitochondrial autophagy and aerobic glycolysis water of GBM were changed by changing the expression of AGK.At the same time,bioinformatics analysis also found that AGK was related to GBM immunity.However,the molecular mechanism of how AGK regulates mitochondrial autophagy and affects GBM aerobic glycolysis and immunity is still unclear.Therefore,we propose that AGK affects the proliferation,apoptosis,invasion and migration of GBM by regulating PI3 K / AKT /NFκB signal,and affects the aerobic glycolysis and immunity of GBM by regulating mitochondrial autophagy.In order to verify the above hypothesis,this paper will be divided into four parts:(1)the expression of AGK in human glioma and its impact on the prognosis of patients;(2)the effect and mechanism of AGK on GBM proliferation,apoptosis,invasion and migration;(3)the effect and mechanism of AGK on GBM mitochondrial autophagy and aerobic glycolysis;(4)the effect of AGK on GBM tumor microenvironment.Part Ⅰ Expression of acylglycerol kinase in human glioma and analysis of its clinical prognosisObjective: To analyze and detect the expression of AGK in human glioma,and analyze the relationship between the expression of AGK and clinical prognosis of human glioma.Methods: TCGA,CGGA and GTEX databases were used to download the gene expression data and clinical characteristics of glioma and normal brain tissues.R language was used to sort out the data.All gene expression data were converted into TPM.The relationship between AGK expression and tumor grade and clinical characteristics was analyzed by one-way ANOVA.At the same time,glioma specimens and normal brain tissues were collected from the Department of Neurosurgery,RENMIN Hospital of Wuhan University.According to the diagnosis of Pathology,the expression level of AGK in each specimen was detected by real-time fluorescent quantitative PCR and Western blot.The expression level of AGK in 71 patients with glioma was analyzed retrospectively by immunohistochemistry.According to the median immune score,the expression level of AGK was lower than the median 40 patients were divided into high expression group and 31 patients were divided into high expression group.Wilcox test and Kaplan Meier test were used to analyze the effect of high and low expression of AGK on survival time.The localization of AGK in glioblastoma was analyzed by immunofluorescence staining.The difference between the two groups was statistically significant(P < 0.05).Results: The expression of AGK in gliomas was higher than normal brain tissues.As the tumor grade changes,the expression of AGK is different.For example,the expression of AGK in glioblastomas was higher than that in low grade gliomas;the expression of AGK in gliomas with IDH mutation,MEMT promoter methylation and1 p / 19 q co deletion was lower than that in control group.The overall survival,diseasefree survival and progression free interval in the low expression group were higher than the high expression group,but there was no significant difference between the two groups in disease-free intervals.The results of real-time quantitative PCR showed that the expression of AGK was increased in gliomas and positively correlated with tumor grade.Immunohistochemical results showed that the expression level of AGK was the highest in glioblastoma and the lowest in grade I gliomas.There was no significant difference between grade Ⅰ and grade Ⅲ gliomas.The survival time was longer in the low expression group.Univariate analysis of variance showed that high expression of AGK was a risk factor for poor prognosis.Immunofluorescence staining showed that AGK mainly existed in cytoplasm.Mitotracker staining showed that AGK mainly expressed in mitochondria.Conclusion: AGK is expressed in normal brain tissue and glioma tissue,but the highest expression is in glioblastoma.AGK is mainly located in mitochondria in glioblastoma.The expression of AGK was positively correlated with the grade of gliomas,and negatively correlated with IDH mutation,MEMT promoter methylation and 1p / 19 q co deletion.AGK is mainly expressed in mitochondria.Part Ⅱ The effect of AGK on the proliferation,apoptosis,invasion and migration of GBM and its mechanismObjective: To explore the effect of AGK on GBM proliferation,apoptosis,invasion and migration and its molecular mechanism.Methods: Four groups of GBM cell lines were constructed by using AGK overexpression and knockdown plasmids and glioblastoma cell lines U87,which were labeled as pcDNA group,AGK group,shCtl group and shAGK group respectively.The effect of AGK knockdown on the size and survival of intracranial tumor in nude mice was observed.Results mann-Whitney’s test and Kaplan Meier test were used to analyze whether there was significant difference between the two groups.The cell proliferation was detected by CCK-8 and EdU.The cell cycle and apoptosis were analyzed by flow cytometry.The cell invasion and migration were detected by Transwell assay,Western blotting was used to analyze the levels of AGK,Cyclin D1,BCL2,Bax,MMP2,MMP9,PI3 K,p-PI3 K,AKT,p-AKT and NF κ B.Results: the survival time of nude mice in the shAGK group was significantly longer than that in the shCtl group(P<0.05),and the size of intracranial tumor in the shAGK group was significantly smaller than that in the shCtl group(P<0.05).CCK-8 results showed the cell viability in AGK group was significantly higher than pcDNA group after the second day(P<0.05),while the cell viability in shAGK group was significantly lower than shCtl group after the second day(P<0.05),and the difference was more obvious on the third and fourth day.Edu results showed the positive rate of EdU in AGK group was significantly higher than pcDNA group(P<0.05),while the positive rate of EdU in shAGK group was significantly lower than shCtl group(P<0.05).Flow cytometry analysis showed that compared with pcDNA group,the proportion of G1 phase increased,the proportion of S phase decreased,and the proportion of G2 phase had no significant difference in AGK group.Compared with shCtl group,the proportion of G1 phase decreased,the proportion of S phase increased,and the proportion of G2 phase decreased in shAGK group(P<0.05).Flow cytometry analysis showed the apoptosis rate of AGK group was lower than pcDNA group,while apoptosis rate of shAGK group was higher than shCtl group(P<0.05).Transwell migration assay showed that compared with pcDNA group,the proportion of cell migration increased in AGK group,but decreased in shAGK group(P< 0.05).Transwell invasion assay showed that the proportion of cell migration increased in AGK group compared with pcDNA group,but decreased in shAGK group compared with shCtl group(P<0.05). Western blot analysis showed that the expression levels of AGK,Cyclin D1,BCL2,MMP2,MMP9,p-PI3 K,p-AKT and NFκB in AGK group were higher than those in pcDNA group,but the expression levels of Bax were lower than those in pcDNA group.Compared with shCtl group,the expression levels of AGK,Cyclin D1,BCL2,MMP2,MMP9,p-PI3 K,p-AKT and NFκB in shAGK group were lower,but the expression levels of PI3 K and AKT were not significantly different.The expression level of Bax increased than those in shCtl group.(P< 0.05).Conclusion: AGK can promote the proliferation,migration and invasion of GBM,and inhibit the apoptosis of GBM.The molecular mechanism may be related to the regulation of PI3 K / AKT / NFκB signal by AGK.Part Ⅲ The effect of AGK on mitophagy and aerobic glycolysis of GBM and its mechanismObjective: To study the effect of AGK on mitochondrial autophagy and aerobic glycolysis in GBM and its mechanism.Methods: The levels of LPA and lactate in pcDNA,AGK,shCtl and shAGK groups were detected by LPA and lactate detection kits.The levels of glycolysis were analyzed by seahorse metabolic analyzer.JC-1 was used to detect the changes of mitochondrial membrane potential and ROS was used to detect the level of oxidative stress.The expression levels of HK2,LDHA,PFK1 and PKM2 were detected by realtime PCR.LC3 and Mitotracker immunofluorescence staining were used to analyze the level of mitochondrial autophagy.The morphological changes of mitochondria in shCtl group and shAGK group were observed by electron microscope.The expression levels of PGAM5,BNIP3,NIX and LC3 were detected by real-time PCR.GBM cells were transfected with AGK overexpression plasmid and treated with DMSO and mitochondrial autophagy activator FCCP respectively.The cell viability on the 1st,2nd and 3rd day was detected by CCK8 method.At the same time,GBM cells were transfected with shAGK plasmid and treated with DMSO and mitochondrial autophagy inhibitor cyclosporin A respectively.The cell viability on the 1st,2nd and 3rd day was detected by CCK8 method.CCK8 method was used to detect the cell viability of pcDNA group and AGK group after PGAM5 overexpression(AGK + PGAM5 group)and control pcDNA.The protein expression of AGK,PGAM5,BNIP3,NIX and LC3 in pcDNA group,AGK group,shCtl group,shAGK group and AGK + PGAM5 group,shAGK + shPGAM5 group were detected by Western blotting.Results: compared with pcDNA group,the concentration of LPA in the supernatant of AGK group increased,while that in the supernatant of shAGK group decreased(P< 0.05).Compared with pcDNA group,the lactate concentration in the cell supernatant of AGK group was higher,while the lactate concentration in the cell supernatant of shAGK group was lower than that of shCtl group(P < 0.05).Seahorse analysis showed that the glycolysis ability of AGK group was higher than that of pcDNA group,while the glycolysis ability of shAGK group was lower than that of shctl group(P < 0.05).JC-1 results showed the level of mitochondrial membrane potential in AGK group was higher than that in pcDNA group,while that in shAGK group was lower than that in shCtl group(P < 0.05).Compared with pcDNA group,ROS level of AGK group decreased,while that of shAGK group increased(P < 0.05).Real time PCR results showed that the mRNA levels of HK2,LDHA,PFK1 and PKM2 in AGK group were higher than those in pcDNA group,while the mRNA levels of HK2,LDHA,PKF1 and PKM2 in shAGK group were lower than those in shCtl group(P < 0.05).Immunofluorescence results showed that the fluorescence intensity and number of LC3 overlapped with Mitotracker increased in AGK group compared with pcDNA group,while the fluorescence intensity and number of LC3 overlapped with Mitotracker decreased in shAGK group compared with shCtl group(P < 0.05).Real time PCR results showed the mRNA levels of PGAM5,BNIP3,NIX and LC3 in AGK group were lower than those in pcDNA group,while the mRNA levels of PGAM5,BNIP3,NIX and LC3 in shAGK group were higher than those in shctl group(P < 0.05).The results of CCK-8 showed that compared with AGK + FCCP group,pcDNA + DMSO group had no significant difference in cell viability on day 1,2 and 3(P > 0.05).Similarly,compared with shAGK + CSA group,shCtl + DMSO group had no significant difference in cell viability on day 1,2 and 3(P > 0.05).Compared with AGK + PGAM5 group,pcDNA + DMSO group had no significant difference in cell viability on day 1,2 and 3(P > 0.05).Similarly,compared with shAGK + PGAM5 group,shCtl + DMSO group had no significant difference in cell viability on day 1,2 and 3(P > 0.05).Western blotting showed that the expression of AGK protein in AGK group was higher than that in pcDNA group,while the expression of PGAM5,BNIP3,NIX and LC3 protein in AGK group was lower than that in pcDNA group.Compared with shCtl group,the expression level of AGK protein was decreased in shAGK group,while the expression levels of PGAM5,BNIP3,NIX and LC3 were increased.There was no significant difference in the protein expression levels of PGAM5,BNIP3,NIX and LC3 between pcDNA group and AGK + PGAM5 group,as well as between shCtl group and shAGK + shPGAM5 group.Conclusion: AGK can regulate the aerobic glycolysis and mitochondrial autophagy of GBM,and its molecular mechanism is that AGK can inhibit the mitochondrial autophagy induced by BNIP3/NIX by inhibiting PGAM5,thereby increasing the aerobic glycolysis ability of GBM.Part Ⅳ The effect of AGK on immune microenvironment of GBMObjective: To explore the effect of AGK on the immune microenvironment of GBM and its mechanism.Methods: The gene expression data of gliomas and normal brain tissues were downloaded from Xena and CGGA databases,including 200 normal brain tissues,523 low-grade gliomas and 166 glioblastomas.According to the expression of AGK,all gliomas were divided into high expression group and low expression group.The immune score and matrix score were evaluated by estimate package.The differences between the two groups were analyzed,and GO and KEGG were analyzed.The relationship between AGK and various subtypes of immune cells was analyzed online on TIMER2.0.The data of TAMs in glioma were downloaded from GEO database,including 20 glioma samples and 15 control samples in 4 data sets.The expression of AGK in TAMs and its relationship with CTLA4,PD1,PDL1,LAG3,TIM3,TIGIT and LPAR1,LPAR2,LPAR3,LPAR4,LPAR 5,LPAR6 were analyzed.At the same time,three GEO datasets GSE80338,GSE115397 and GSE135437 were used to analyze the differentially expressed genes,and the intersection was selected.KEGG and GO were used to analyze the differentially expressed genes.The single cell data set GSE84465 was used to analyze the expression of AGK and LPA receptor family genes in various cell types of glioblastoma samples.Results: There were 436 differentially expressed genes between AGK high expression group and AGK local expression group,including 369 up-regulated genes and 67 down regulated genes.Go and KEGG analysis showed that the expression of AGK was related to T cell activation,granulocyte activation,granulocyte degranulation and lymphocyte differentiation.The mechanism score and immune score of AGK high expression group were higher than those of AGK low expression group.The results of GSE80338 data set analysis on TIMER2.0 website showed that the expression of AGK in TAMs was lower than that in normal control group,the expression of PD1 in TAMs was significantly increased,while the expression of PDL1 in TAMs was significantly decreased,while the expression of CTLA4,LAG3,TIM3 and TIGIT in TAMs was not significantly different from that in normal control group.The expression of LPAR1,LPAR2,LPAR5 and LPAR6 in TAMs was lower than that in normal control group,while the expression of LPAR3 and LPAR4 in TAMs was not significantly different from that in normal control group.According to the difference analysis of three TAMs related data sets GSE80338,GSE115397 and GSE135437,a total of 64 differential genes were obtained,including 43 up-regulated genes and 21 down regulated genes.The down regulated genes include LPAR5 and LPAR6.Go and KEGG analysis showed that the differentially expressed genes were related to HIF-1 signaling pathway,cell adhesion molecule,signal transduction,cell adhesion and angiogenesis.Single cell data set GSE84465 analysis showed that AGK was expressed in astrocytes,tumor cells,oligodendrocytes,vascular endothelial cells,immune cells,neurons and oligodendrocyte precursor cells of GBM,and the main expression was concentrated in tumor cells.The expression of LPAR1 in GBM neurons was very low.LPAR2 is mainly expressed in immune cells.LPAR3 was only expressed in astrocytes and immunocytes.LPAR4 was almost not expressed in neurons and oligodendrocytes,but less expressed in other types of cells.LPAR5 was mainly expressed in immune cells,a small amount in tumor cells,and almost no expression in other types of cells.LPAR6 expression is very low in neurons,but mainly in immune cells and tumor cells.The results showed that the expression level of AGK was positively correlated with the infiltration level of CD4 + T cells,monocytes,myeloid dendritic cells and NK cells in GBM,and negatively correlated with the infiltration level of macrophages.Conclusion: AGK is related to the immunity of GBM,and LPAR5 and LPAR6.And they may play a role in the immune microenvironment of GBM together with AGK.Summary(1)AGK was highly expressed in GBM and correlated with tumor grade.The increase of AGK expression was also correlated with the decrease of survival time and adverse prognostic factors.(2)AGK could promote the proliferation,invasion and migration of GBM,and inhibit the apoptosis of GBM.(3)AGK could regulate PI3 K / AKT / NFκB signaling pathway.(4)AGK could promote the aerobic glycolysis of GBM.(5)AGK could negatively regulate GBM mitochondrial autophagy.(6)AGK affected GBM aerobic glycolysis by regulating mitochondrial autophagy.(7)AGK is mainly distributed in tumor cells and immune cells of GBM,and is associated with a variety of immune cell infiltration.