IDH1 Mutation Promotes Glucose Metabolism And Growth Capacity In Glioma Through The Promoter Region Methylation Of MiR-34a

Background and objective:Recently,the metabolism of cancer has become the hot spot in tumor research field.The changes of energy metabolism and genomic instability are known as the hallmarks of cancer.Normal differentiated cells intake glucose and use it through mitochondrial oxidative phosphorylation to generate the energy needed for cellular processes in aerobic condition;metabolize glucose to lactate through glycolysis in hypoxic conditions.While,cancer cells instead relying on glycolysis to extract energy even in aerobic condition.This phenomenon is termed as the Warburg effect,also known as aerobic glycolysis.Aerobic glycolysis is an inefficient way to generate ATP,however,it can provide materials for cancer cells to synthesis the biological macromolecular,such as nucleotides,proteins and lips,which are needed to produce a new cell.However,the interest on metabolism has been recently renewed by the discovery of recurrent mutation of isocitrate dehydrogenase 1(IDH1)gene in cancer.IDH1 gene encodes the cytoplasmic NADP+ dependent IDH1 that catalyzes the oxidative decarboxylation of isocitrate into α-ketoglutarate(α-KG),with simultaneous production of NADPH.IDH1 mutation was frequently found in low gliomas and some secondary glioblastoma multiforme(GBM).IDH1 mutation results in a new enzymatic activity transform α-KG into 2-hydroxyglutarate(2-HG),with the decrease of NADPH.The oncometabolite 2-HG accumulates in the cancer cell and it acts as a competitive inhibitor of many α-KG dependent enzymes about methylation,such as TET2 and JHDMs.When TET2-mediated DNA demethylation is inhibited by 2-HG,thus it will lead to anti-oncogene silence because of DNA hypermethylation.Micro RNAs(miRNAs)are the class of short,endogenous,non-coding RNA molecules that regulate the gene expression by binding with target m RNA incomplete post-transcriptionally.Interestingly,miRNAs are abnormally expressedin a wide variety of human cancers including glioma,playing the critical role in tumorigenesis.Recent studies have demonstrated that epigenetic mechanisms,including DNA methylation and histone modification,not only regulate the expression of protein-encoding genes,but also miRNAs.miR-34 a,as a key regulator of tumor suppression,is decrease expression in many cancers,including glioma.It controls the necessary processes for basic cancer cell viability including cell cycle,differentiation,apoptosis and metabolism.It is known to us that the promoter regions of miR-34 a loci contain P53-binding sites,and is regulated by the P53 signal.Interestingly,the host gene of miR-34 a is associated with a Cp G island surrounding its transcriptional start site,which is frequently methylated in various malignancies.In this study,we explore the effect of methylation of IDH1 mutation,which lead to miR-34 a promoter region methylation and promote the glucose metabolism and growth of glioma via silenting miR-34 a.Methods: 1.miRNAs expression data for glioma was downloaded from Chinese Glioma Genome Atlas(CGGA)data portal and The Cancer Genome Atlas(TCGA)data portal.We validate the IDH1 mutation specific miRNA signature of glioblastoma in CGGA and TCGA.2.The relative expression of miR-34 a in human glioma cells were determined by real-time quantification RT-PCR,after the the IDH1 mutation lentivirus were transfected into glioma cells U87 and U251.miR-34 a is associated with a Cp G island surrounding its transcriptional start site and bisulfate-treated DNA sequencing(BSP)was used to analyze the methylation of miR-34 a.In addition,to test the effects of IDH1 mutation on glioma,we employed U87 glioma cell xenograft model.3.5-Aza-2′-Deoxycytidine(5-Aza)was used to treat the mutant IDH1 glioma cells,and then the relative expression of miR-34 a was detected by real-time quantification RT-PCR.4.miR-34 a mimics、HK2 depletion(via HK2-sh RNA)and 3-bromopyruvic acid(3-Br PA),a HK2 inhibitor,were used to processglioma cells.5.XF glycilysis stress test kit,CCK8 and colony formation assay were used to detect the glycolysis ability and growth capacity of glioma cells.6.Bioinformatics,luciferase reporter assay,real time RT-PCR and western blot assay were used to analysis whether glycolytic enzymes(HK1、HK2、GPI、LDHA)are the target of miR-34 a.Results: 1.miR-34 a expression was decline in mutant IDH1 glioma tissues compared towild IDH1 glioma tissues in CGGA and TCGA.2.The relative expression of miR-34 a was declined,in glioma cells with IDH1 mutation.BSP assay was proved that the transcriptional start site of miR-34 a showed hypermethylation pattern in IDH1 mutant glioma cells.Moreover,the expression of miR-34 a was ascend,after 5-Aza process the IDH1 mutant glioma cells.3.IDH1 mutation promotes glioma growth in vitro and in vivo,and it also enhancesthe glycolysis ability of glioma cells.While,when we use 5-Aza to treat mutant IDH1 glioma cells,the glycolysis ability and growth capacity of glioma cells were decreased.4.Overexpression of miR-34 a decreases the glycolysis ability and growth capacity of glioma cells.Down-regulation of HK2 or use 3-Br PA(HK2 inhibitor)impairs the glycolysis ability and growth capacity of glioma cells.5.Bioinformatics,RT-PCR,western blot and luciferase reporter assays showed that miR-34 a modulated glycolytic enzymes(HK1、HK2、GPI、LDHA)expression by directly targeting the binding site within the 3′ UTR.Conclusions: 1.The discovery of mutant IDH1/miR-34a/ glycolytic enzymes signaling have profound effects on cellular metabolism,which could renewed interest in cancer metabolism and renewed hope of taking therapeutic advantage of cancer metabolism.2.IDH1 mutation through the promoter region hypermethylation leading to miR-34 a silencing in glioma cells.Eepigenetic mechanism is an important way to regulate the expression of miR-34 a at transcription level,and not rely on P53 signal.Moreover,5-Aza and 3-Br PA have shed light on how small-molecule inhibitor hamper the progression of tumor.