Methylation-mediated Down-regulation Of MiR-155 Contributes To The Less Aggressive Phenotypes Of IDH Mutant Gliomas

Background:Recent years,the discovery of mutations in Isocitrate Dehydrogenases-1/2(IDH1/2)has provided tremendous help for undetstanding the underlying molecular mechanisms of gliomas and evaluating the prognosis of glioma patients.Firstly,IDH1/2 mutations are thought to be the earliest mutations and play key roles in gliomagenesis.Secondly,IDH1/2 mutant glioma(IDHmt)patients bear favorable prognosis than their IDH1/2 wildtype(IDHwt)counterparts.After adjustment for grade,age,genomic profile,and treatment,multivariate analysis confirmed that IDH mutations was an independent favorable prognostic marker.This indicates that IDH1/2 mutations play key roles in affecting brain glioma patients’ prognosis and long-term survival,and IDH1/2 mutations itself may cause a less aggressive glioma phenotype.However,the underlying mechanisms of IDH1/2 mutation to a less aggressive glioma phenotype remains unknown.On the other hand,a great numer of studies have demonstrated the vital role of microRNAs(miRNAs)in gliomagenesis and progression,but the potential role of miRNAs in IDHmt gliomas remain largely unknown.Objectives:This study aimed to analyze the differentially expressed miRNAs(DE-miR)between IDHwt and IDHmt gliomas,and to explore the role of DE-miRs in contributing to the less aggressive phenotype of IDHmt gliomas and the potential underlying molecular mechanisms,so as to shed new light on deeply understanding the biology features of IDHmt gliomas.Methods:1.To identify the DE-miRs between IDHmt and IDHwt gliomas via microarray analysis,bioinformatic analysis and quantitative PCR(qPCR)detection of clinical glioma samples;2.To explore the role of DE-miRs in glioma patients’ survival and prognosis via Survival analysis and Multivariate COX regression model.3.To explore the differentially expressed mechanisms of miRNAs via BSP(Bisulfite Sequencing PCR),Pearson correlation analysis,Reporter gene assay,lentivirus transfection for establishment of IDH R132 H overexpressed glioma cells and demethylation drug 5-azacitidine treatment,etc.4.To explore the role of DE-miRs in glioma proliferation,invasion and migration via transfection of miRNA mimics and inhibitors,CCK-8 and Transwell assays.5.To explore the downstream targets of DE-miRs via Online website(TargetScan and miRanda)prediction,Pearson correlation analysis and Reporter gene assay,etc.6.To explore the molecular mechanisms of DE-miRs downstream targets in leading to the phenotype changes via Tissue chip,Immunohistochemistry(IHC),Gene Enrichment analysis and Western blot assay,etc.Results:1.MiR-155,which is an independent risk factor affecting glioma patients’ long-term survival,is significantly down-regulated in IDHmt gliomas.2.MiR-155 is down-regulated via IDH1 mutation-mediated promoter CpG island methylation.3.Human cancer/testic antigen FAM133 A is a direct downstream target of miR-155.4.FAM133 A predicts a favorable prognosis for glioma patients.5.Down-regulation of FAM133 A promotes glioma invasion and migration via up-regulating MMP14 expression.Conclusions:Methylation-regulated miR-155-FAM133 A axis may contributes to the attenuated invasion and migration of IDHmt gliomas by targeting MMP14.Our findings can help better understand the biology of IDHmt gliomas and provide basis for potential therapeutic intervention strategies.