A Novel Six-methylated Pseudogenes Signature Predicts Prognosis Of Glioma Patients

Objective Glioma is the most common primary intracranial tumor,accounting for 81%of malignant brain tumors.Despite advances in medical and surgical treatment,glioma remains challenging to treat,with poor treatment success and overall survival.Abnormal pseudogene expression is significantly associated with the prognosis of glioma patients.Nevertheless,the roles of pseudogene abnormal methylation in glioma prognosis have not been investigated.Therefore,we speculate that the detection of pseudogene methylation is crucial for predicting the prognosis of glioma patients.The purpose of the study was to develop a novel 6-methylated pseudogene signature to predict the prognosis of glioma patients.Methods The clinical information of glioma patients and 450 K methylation data were downloaded from TCGA,and 649 glioma samples were included after the incomplete cases were removed.Clinical information for the data included survival status,survival time,tumor grade,clinicopathologic features,and age.The selected samples were then randomly divided into a training cohort(n = 325)and a verification cohort(n = 324).A total of 365,687 Cp G sites were extracted from the methylation chip data downloaded from TCGA,and 1324 Cp G sites of pseudogenes were obtained,corresponding to 203 Cp G promoter regions of pseudogenes(multiple Cp G sites correspond to one pseudogene,and their average values were taken).Six prognostic methylated pseudogenes were identified by univariate Cox regression analysis.Regression coefficients of six methylated pseudogenes were obtained based on the multivariate Cox proportional hazard regression model.Next,linear combination methods were used to treat methylation expression levels and promoter region risk factors for each pseudogene.According to the median risk score,the clinical samples were divided into high-risk and low-risk groups.Kaplan-Meier survival analysis was used to determine the difference of two groups of patients survival rate,whether univariate and multivariate risk factors could be used as independent prognostic factors,and the AUC curve was performed to analyze the specificity and sensitivity of risk factors as prognostic factors.GO and KEGG performed functional analysis of related genes to predict the function and pathway of related genes.Finally,two pseudogenes,SBF1P1 and SUMO1P1,which have not been studied in glioma and have survival significance in glioma patients,were selected for the study.siRNA was used to construct silencing models of pseudogenes SBF1P1 and SUMO1P1.Single-cell cloning assay and MTT assay were used to detect the proliferation of glioma cells after the knockdown of pseudogene SBF1P1 and SUMO1P1.Finally,the Transwell migration and invasion assay was performed to detect the effect of silencing pseudogenes SBF1P1 and SUMO1P1 on the migration and invasion of glioma cells of T98 G and U251.Result(1)Analysis of pseudogene methylation levels in high and low-grade glioma samples.(2)Six methylated pseudogenes associated with survival were obtained by univariate analysis.(3)Six gene models composed of methylated pseudogenes were identified and validated and showed good prognostic ability in the training dataset,validation dataset,and the whole dataset.(4)Pseudogene methylation clustering showed that gliomas were divided into two subtypes,and the prognosis of low pseudogene methylation samples was worse than that of high pseudogene methylation samples.(5)Cellular immune infiltration in different risk groups: the proportion of B cells and CD4+T cells was significantly increased in the high-risk group,while the proportion of Mono cells was lower.(6)The biological functions associated with glioma in high-risk groups were correlated with malignant phenotypes.(7)Silencing the expression of pseudogenes SBF1P1 and SUMO1P1 can inhibit the proliferation,migration,and invasion of glioma.Conclusion The 6 methylated pseudogene signature may be a novel predictor for the prognosis assessment of glioma patients,which can accurately predict the prognosis of patients.In addition,the knockdown of pseudogenes SBF1P1 and SUMO1P1 inhibited cell proliferation,migration,and invasion.These results suggest that pseudogenes take a vital part in glioma.