Prognostic Significance Of ARL9 And Its Methylation In Low-grade Glioma

Objectives:Low-grade glioma(LGG)represents the most common primary malignancy occurred in the brain,and LGG exhibits great intrinsic heterogeneity with regard to tumor biological behavior.Despite comprehensive therapy for LGG,including neurosurgical resection,chemotherapy and radiotherapy,therapeutic resistance and tumor recurrence appear to be inevitable.Some patients with LGG may progress to high grade glioblastoma,which usually represents an unfavorable outcome.Hence finding new target gene for therapy and novel biomarker for predicting prognosis is urgently needed.ADP ribosylation factor(ARF)small GTPase are a family of low molecular weight GTP-hydrolyzing enzymes,which belongs to the Ras superfamily of small GTPases.ARFs proteins have been identified in diverse kinds of eukaryotes and the structure are highly conserved through evolution.Its binding of GTP activates itself,however,it becomes inactive soon after the GTP hydrolyzed.ARF family proteins are regulated by Guanine nucleotide Exchange Factors(GEF)which facilitate activation and GTPase-Activating Proteins(GAP)which cause inactivation.Once activated,the GTP-bound active form binds effectors and mediates downstream cascade reaction.ARF family have been implicated in many cellular activities,from membrane trafficking,ciliogenesis,lipid metabolism through energy use and cell motility,differentiation,apoptosis to transcriptional regulation.Researches have showed that cells of several cancer types can subvert the membrane trafficking regulators to enhance their migration and invasion.As a part of ARF family,ADP ribosylation factor-like proteins(ARL)are also reported to play significant roles in the tumorigenesis and tumor progression.However,the clinical and prognostic significance of ARL9 in glioma is still unknown,and its function role in LGG has never been documented.In this study,we first analyzed the RNA-seq data retrieved from TCGA to compare the m RNA level between LGG and normal tissue,moreover,methylation data were download from TCGA to analyzed the correlation between ARL9 expression and ARL 9 DNA methylation,and clinical data were used to assess the prognostic significance of ARL9 expression and its methylation.To validate the prognostic role of ARL9,we further analyzed the relevant data in CGGA database,Gravendeel and Rembrandt datasets one by one.Then we conducted a meta-analysis on those four independent LGG datasets to assess the combined effects of ARL9 on prognosis.We also use the deconvolution methods to evaluate the potential correlation of ARL9 with immune infiltration levels in LGG.Then,we examined the biological processes which ARL9 is involved through gene ontology enrichment analysis and KEGG analysis,to study the functional mechanism of ARL9 in LGG.Lastly,we detect the expression level of ARL9 m RNA and proteins in LGG samples or normal brain tumor by real time quantification PCR and western blot,respectively.Materials and methods:Bioinformatic analysis on four independent datasetsLGG transcriptional,methylation and clinical data were downloaded from TCGA database.We first compare the m RNA level of ARL9 between LGG and normal tissue,then the association between ARL9 expression and ARL DNA methylation was analyzed.We also analyzed the association between ARL9 expression and clinical characteristics,including age,gender,histological type,WHO grade,1p19 q codeletion and IDH1-mutation status.Moreover,survival analyses were utilized to assess the association between ARL9 expression as well as ARL9 methylation and prognosis of patients with LGG.To consolidate the aforementioned analyses,we retrieved the data from CGGA database and similar analyses were conducted again to verify the conclusions.In addition,we conducted survival analyses on another two independent datasets(Gravendeel and Rembrandt datasets)to validate the prognostic significance of ARL9 on LGG patients.Meta-analysis of ARL9 expression on LGG patients' prognosisWe searched the Pub Med,Web of Science,and Embase for published studies on ARL9 and glioma.The strategy we used were “(‘ARL9' or ‘ADP-ribosylation factor-like 9 protein')and(‘glioma' or ‘astrocytoma' or ‘ependymoma' or ‘oligodendroglioma' or ‘glioblastoma')”.As no previous studies have reported an association between ARL9 expression and OS among patients with LGG,we only included the results generated from four independent datasets in meta-analysis.Combined HR and 95% CI were calculated to evaluate the correlation of ARL9 expression with the prognosis of LGG patients.The heterogeneity across four datasets was assessed by the Q test(I2 statistics).A fixed-effects model would be selected for combination if no obvious heterogeneity(I2 < 50%).Otherwise,a random effects model would be applied.Influence of ARL9 expression on immune infiltration and exploration of its underlying mechanismDifferent deconvolution methods were used for the calculation of immune infiltrate levels of each sample through the transcriptional data.We assessed the difference of each immune cells between patients with ARL9 high expression and patients with low expression.Moreover,we analyzed the differential expressed genes between the two group,then those immune related differential genes were further used as input for Gene ontology and KEGG analysis.Lastly,we choose hub genes to construct a formula to calculate immune scores of each sample,and more importantly,construct a survival model based on risk score to predict the survival time of each sample.Detecting the expression level of ARL9 in glioma samplesThe PCR method was used to detect m RNA levels in 12 cases of glioma(8 cases of grade II glioma,4 cases of grade III glioma)and 12 cases of normal brain tissues(5 cases of peritumoral tissues and 7 cases of normal brain tissues collected from epilepsy surgery).At the same time,the Western Blot method was used to detect the protein levels in these tissues,then the differences in m RNA and protein expression between the glioma tissues and the normal brain tissues were compared.Statistical analysisThe data were analyzed using R(version 3.6.3)or Graph Pad Prism(version 6.0).The meta-analysis was completed using STATA 15.1 software.Low and high ARL9 expression groups were established based on the median ARL9 m RNA expression value in the separate datasets.Similarly,ARL9 hypomethylation and hypermethylation groups were established according to the median value of ARL9 DNA methylation in the TCGA-LGG dataset.The relationships between ARL9 expression or its DNA methylation and a series of categorical variables were analyzed by chi-square or Fisher exact-tests.Continuous indexes with normal distribution between two groups was determined by Student's t-test and continuous indexes with skew distribution were inspected by a nonparametric test.The correlation of ARL9 expression with ARL9 DNA methylation level was measured by Pearson correlation coefficient.Moreover,we employed univariate along with multivariate Cox regression models to probe whether ARL9 expression was an independent prognostic index in patients with LGG.Kaplan-Meier curves were utilized to evaluate the prognostic significance of ARL9 expression along with ARL9 DNA methylation.Time dependent-receiver operating characteristic(td-ROC)analyses were utilized to assess the predictive performance of ARL9 in predicting OS.P-values less than 0.05 on both sides were statistically significant.Results:Lower ARL9 expression predicts better prognosis of patients with LGG,and ARL9 expression is negatively correlated with its DNA methylation,moreover,significant association is observed between ARL9 expression and tumor recurrence,1p19 q codeletion and IDH1 mutation status.After analyzing the transcriptional data of LGG patients in TCGA,we found that the m RNA level of ARL9 is downregulated in LGG tissue,and upregulated in normal tissue.In addition,the methylation data revealed that ARL9 expression is negatively regulated by its methylation.Moreover,we also found that ARL9 expression is of important clinical relevance.That is,lower ARL9 expression and hypermethylation predicts better prognosis in patients with LGG,and the expression and methylation level of ARL9 are significantly related with tumor recurrence,1p19 q codeletion and IDH1 mutation status.Later,the aforementioned results are validated in the transcriptional and clinical data of CGGA database,more importantly,the prognostic significance of ARL9 expression on LGG patients was further consolidated in another two independent datasets.Finally,we conducted a metaanalysis to combine the results from those four independent datasets,the pooled HR and the pooled HR along with 95% CI for the association between low ARL9 expression and OS was0.57(0.49–0.67),and no significant heterogeneity among the 4 datasets was observed(I2 =22.2%,P = 0.278).The ROC curves showed ARL9 expression could predict 1-year,3-year,and 5-year OS with good predictability.Relationships of ARL9 with immune cells and exploration its functionWe found a consistent result among CIBERSORT,EPIC,quan TIseq and TIMER calculations in three different datasets,i.e.,the amount of myeloid dendric cells,Tregs,MDSCs and M2 phenotype of macrophages were elevated in patients with ARL9 high expression,while more CD4+ T cells,CD8+ T cells,NK cells and M1 phenotype of macrophages were presented in patients with ARL9 low expression.In the correlation analysis of ARL9 expression and biomarkers of 28 immune cells,we found ARL9 was positively correlated with biomarkers of MDSCs and Tregs,but negatively related with CD4+T cell and CD8+ T cell landmarks.GO and KEGG analysis revealed that ARL9 was mainly enriched on T cell related biological functions and pathways.ARL9 is downregulated in glioma tissue than in normal tissue,and the expression of ARL9 is positively associated with the WHO grade of LGG.Real time quantification PCR was used to detect the m RNA level of ARL9 and Western blot was used to determine the protein level of ARL9 both in tumor and normal tissue.The m RNA and protein level are downregulated in LGG tissue.Conclusions:ARL9 is downregulated in patients with LGG,and negatively regulated by its methylation.Lower ARL9 expression predicts better OS,thus ARL9 could act as a promising biomarker in LGG patientsARL9 potentially contributes to the regulation of CD4+ T cells,CD8+ T cells,microglial cells and MDSCs,thus playing an important role in the immunosuppression of LGG.