L-Arg/P4HA1 Pathway Mediates Epithelial-Mesenchymal Transition And Promotes Glioblastoma Multiforme Invasion Under Hypoxic Microenvironment

OBJECTIVE:Glioblastoma multiforme(GBM)is the most malignant and invasive intracranial tumor.Hypoxia microenvironment plays an important role in the invasion of GBM.This study is based on metabonomics and proteomics to explore the mechanism of hypoxia’s effect on GBM invasion and construct a GBM hypoxia prognosis model,which provides a theoretical basis for exploring the pathogenesis and risk assessment of GBM.METHODS:LN18 cells were treated with hypoxia and normoxia for 12 h,24 h,48 h and 72 h,separately.Wound-healing and transwell assays were used to detect the migration and invasion of LN18 cells.Metabonomics and proteomics of LN18 cells cultured in hypoxia and normoxia for 24 h were detected by liquid chromatograph-mass spectrometer and labelfree methods.Expression of different proteins in LN18 and T98G cells was verified by MRM assay.Differential metabolites and proteins were analysed by Pearson correlation.Biological function experiments demonstrated a negative correlation between the L-Arg and the P4HA1 on the invasiveness of GBM.The differential protein-converting genes identified by proteomics were crossed with TCGA and CGGA databases to obtain genes for further analysis.Single variable Cox regression and Lasso regression were used to construct the risk model of glioblastoma multiforme hypoxia prognosis.RESULTS:We showed that the migration and invasiveness of LN18 cells was significantly enhanced after 24 h of hypoxia treatment.The metabolomic and proteomic profiling were conducted in LN18 cells cultured under hypoxia condition:1 160 and 1 958 significant differential anions and cations were identified respectively;there were 62 differentially expressed proteins,of which 28 were up-regulated and 34 were down-regulated.7 proteins and 10 metabolites were revealed in significant differences common pathway by correlation analysis,of which metabolite L-Arg was negatively correlated with P4HA1 protein;MRM further showed P4HA1 protein was highly expressed in two glioblastoma cell lines(LN18 and T98G)in hypoxic environment.Meanwhile,the expression of hypoxia inducible factor-1α(HIF1α),neuronal nitric oxide synthase(nNOS)and P4HA1 was up-regulated,and the concentration of L-Arg and NO was decreased and increased respectively.Knockdown of HIF1α reduced the expression of nNOS and P4HA1,the concentration of NO and the invasiveness of cells,while increased the concentration of L-Arg.Similar changes on P4HA1 expression,the concentration of L-Arg and NO were observed when the expression of nNOS was disrupted.Lastly,knockdown of P4HA1 impaired the invasion of LN18 and T98G cells,probably through regulating the expression of Vimentin,MMP2,MMP9,Snail and E-cadherin.Consistent trends on both the overexpression of these relevant genes,as well as the concentration of L-Arg and NO were also observed in all our overexpression experiments.426 differential proteins screened by proteomics were transformed by UniProt database,and 262 proteins had corresponding gene names,among which 212 had annotation genes in TCGA database.The 212 genes were analyzed by single factor Cox analysis and 19 differentially expressed genes were identified.Lasso regression analysis identified seven key genes.Roc curve analysis and survival analysis showed that the clinical risk model was sensitive and effective in distinguishing high-risk from low-risk groups.Meanwhile,the survival curve and differential expression of these 7 key genes showed significant differences.CONCLUSION:Hypoxia promotes the migration and invasion of glioblastoma multiforme via the L-Arg-P4HA1 axis mediated epithelial-mesenchymal transition(EMT).Meanwhile,FKBP2,GLO1,IGFBP5,NSUN5,RBMX,TAGLN2 and UBE2V2 were used to construct the model of hypoxia related prognosis,which accurately evaluated the risk of GBM.