Regulation Of Glutamine Metabolism And Mitochondrial Damage By PKM2 And The Intervention Effect Of Resveratrol

In order to adapt to the tumor microenvironment of hypoxia and hypo-nutrient,tumor cells always undergo altered metabolism,which is called “metabolic reprogramming”,showing in two aspects mainly: Abnormal glucose metabolism and addiction of glutamine.Tumor cells provide a large number of intermediate metabolites through metabolic remodeling,which indicates that metabolic reprogramming plays an important role in the process of malignant transformation.The glucose and glutamine metabolism of tumor cells are regulated by complex signal transduction pathways.Therefore,to explore the relationship between these two metabolic pathways and their causes will provide a theoretical basis for tumor therapy that the tumor metabolism is as the target.Along with the further research on the energy metabolism of tumor cells in recent years,M2 pyruvate kinase(PKM2),which dephosphorylates phosphoenol pyruvate(PEP)into pyruvate,the last step of glycolysis,shows an important role in the process of glycolysis.It's well known that PKM2 plays a pivotal role in sustaining aerobic glycolysis,pentose phosphate pathway and serine synthesis process.However,the participation of PKM2 in the regulation of glutamine metabolism and mitochondrial function remains unclear.This study aims to explore the relationship between PKM2 and glutamine metabolism and mitochondrial function,as well as the molecular mechanism;explore the intervention effect of resveratrol on PKM2 and the underlying molecular mechanism,providing an effective therapeutic target and a novel function of resveratrol in apoptosis induction.The main results obtained are as follows:(1)The exploration of the relationship between PKM2 and glutamine metabolism and the underlying molecular mechanism.The results showed that stable PKM2 knockdown increased the expression of glutaminase Gls1 and Gls2,glutamine transport receptor SLC1A5 and upstream transcription factor ?-catenin /c-Myc;PKM2 overexpression inhibited the expression of the genes above,indicating that the ?-catenin/c-Myc signaling pathway mediated the regulation of glutamine metabolism by PKM2.In addition,PKM2 up-regulated ?-catenin mRNA expression at the transcriptional level.The detection of ?-catenin expression in PKM2 mutant cell lines showed that R399 E mutant cells could significantly inhibit the expression of ?-catenin,that was,the dimeric PKM2 played a key role in the regulating ?-catenin.PKM2 overexpression up-regulated the expression of miR-200 a,miR-200 a suppressed ?-catenin expression through binding to the 3'UTR of ?-catenin.(2)Analysis of the relationship between PKM2 and mitochondria.After the mitochondria in PKM2 overexpression and knockdown cells were labeled with mitochondrial probes,the changes of mitochondria were observed using Delta Vision.The results showed that the mitochondria in PKM2 overexpression cells were distributed around the nucleus,the proportion of the fused mitochondria increased,and the number of mitochondria decreased.Instead,PKM2 knockdown cells exhibited smaller mitochondria in size and increased mitochondrial number in quantity.Furthermore,the overexpression of PKM2 resulted in decreased expression of mitochondrial fission protein Drp1,increased expression of mitochondrial fusion protein Mfn2;while PKM2 knockdown exhibited increased expression of Drp1 and decreased expression of Mfn2.The detection of mitochondrial function implicated that PKM2 overexpression correlated with increased mitochondrial DNA copy number,decreased expression of electron transport chain complex and ATP production.These results show the relationship between PKM2 and mitochondrial function.(3)Investigation into the molecular mechanism underling PKM2 regulating mitochondrial dysfunction through p53/Drp1 axis.Detection of Drp1 transcription factor p53 using qPCR and western blot showed that in protein level PKM2 overexpression reduced the expression of p53,and PKM2 knockdown promoted the expression of p53,but in m RNA level no effect existed.The study showed that Drp1 was decreased along with the decrease of p53 expression by p53 siRNA treatment,suggesting that PKM2 suppressed Drp1 via p53.Protein stability experiment indicated that PKM2 overexpression promoted p53 degradation through proteasome pathway,while PKM2 knockdown increased the stability of p53 protein,and which was mediated by MDM2.Immunoprecipitation and GST pull-down experiments showed that PKM2 directly bound with both p53 and MDM2 forming a ternary complex to promote p53 ubiquitination,thereby promoting its degradation.Immunohistochemical results showed that the expression of PKM2 was negatively correlated with Drp1,which was identical with the results of cell lines.(4)Reveal of the interdependency of mitochondrial dysfunction with the high or excessive expression PKM2 and Mfn2.Quantitative and morphological analyses showed that the expression of miR-106 b was decreased in the PKM2 overexpressed cells,the reduction of Mfn2 expression and the recovery of mitochondrial functions were induced by the treatment of miR-106 b mimic,demonstrating that miR-106 b played important roles in the down-regulation of Mfn2 expression and the PKM2 mediation of mitochondrial fusion.3'UTR of Mfn2 was cloned into the luciferase reporter vector psicheck2.0.Co-transfection of the luciferase reporter gene vector and miR-106 b mimic showed decreased luciferase activity,and that was increased in co-transfection of the luciferase reporter gene vector and miR-106 b inhibitor,indicating miR-106 b directly targeted the Mfn2 3'UTR.Clinical investigation was performed and results showed that the higher expression levels of PKM2 corresponded with the higher expression levels of Mfn2 in breast cancer tissues by comparison of their expression in adjacent normal tissues,which was identical with the results of cell lines.(5)Elucidation of the intervention effect of resveratrol on PKM2 and its underlying molecular mechanism.Resveratrol significantly inhibited the expression of PKM2.After cancer cells were treated with resveratrol,the mitochondria divided into smaller and more units,the expression of mitochondrial fission protein Drp1 and Fis was significantly increased,the expression of mitochondrial fusion protein Mfn2 was decreased;In addtion,the expression of endoplasmic reticulum(ER)stress markers GRP78,CHOP and Caspase12 was significantly elevated.Moreover,PKM2 overexpression reversed the phenomenon.The results of PKM2 knockdown were the same as that of resveratrol treatment.These results indicated that PKM2-mediated ER stress and mitochondrial dysfunction were involved in resveratrol-induced apoptosis.In order to explore the molecular mechanism of resveratrol in inhibiting PKM2,the candidated microRNAs targeting PKM2 were analyzed by bioinformatics and further verified by experiments,the results showed that resveratrol inhibited the expression of PKM2 through upregulation of miR-326,and miR-326 could directly bind to 3'UTR of PKM2.In conclusion,PKM2 depletion induces the compensation of glutaminolysis through ?-catenin/c-Myc pathway in tumor cells.PKM2 induces excessive fusion of mitochondria through both the p53/Drp1 axis and the mi R-106b/Mfn2 axis,ultimately leading to the mitochondrial dysfunction.Resveratrol inhibits the expression of PKM2 by upregulating miR-326,which thereby causes the ER stress and mitochondrial dysfunction,eventually inducing the apoptosis of the cells.In view of the important role of PKM2 in tumor metabolism and growth,PKM2 is expected to be an effective target for tumor therapy,and resveratrol displays the potential inhibition on PKM2 expression.