Screening And Expression Verification Of Cancer-Testis Antigen In Glioma Based On Bioinformatics

Objective: Previous studies have found that cancer-testis antigen(CTA)OY-TES-1 is highly expressed in glioma and is related to the prognosis.Because of the expression heterogeneity,the application of OY-TES-1 as a tumor vaccine alone is limited.It has been reported that multiple clusters of CTAs were co-expressed in tumors,which provides the possibility for the development of multivalent tumor vaccine based on the combination of CTAs.In this study,CTAs with high expression in glioma were screened using bioinformatics methods,which were related to tumor purity and prognosis.Experimental verification and co-expression analysis of candidate CTAs were performed,so as to find potential targets for glioma immunotherapy.Methods:(1)The RNA-seq and clinical data of glioma patients were obtained from TCGA database.CTAs in CT antigen library were used to screen.GEPIA database was used to identify differential expressed genes(DEGs)and data were processed by R language.Linked Omics online website was used to investigate the correlation between candidate CTAs and tumor purity.Kaplan-Meier survival curve,univariate and multivariate Cox regression analysis were also performed to evaluate the prognostic implications.(2)The protein-protein interaction network of candidate CTAs was analyzed by STRING software.Functional annotation of CTAs co-expressed genes was performed by GO and KEGG analysis.(3)The mRNA expression of candidate CTAs and OY-TES-1 in 53 glioma tissues and 3 normal brain tissues was detected by RT-PCR.The co-expression of CTAs,their prognosis and clinical significance were also explored.Results:(1)The RNA-seq of 681 glioma tissues and clinical information of 675 glioma patients were obtained from the TCGA database.The DEGs analysis in245 CTAs showed that 12 CTAs(PBK,IL13RA2,KIF2 C,NUF2,CEP55,OIP5,TTK,DCAF12,CASC5,ATAD2,RQCD1 and IGSF11)were significantly up-regulated in glioma.The tumor purity analysis showed that 7 CTAs(PBK,KIF2 C,NUF2,OIP5,TTK,CASC5 and ATAD2)specifically expressed in tumor cells.Prognostic analysis showed that 7 CTAs mRNA were associated with the survival prognosis of glioma patients.The overall survival of patients with high expression of those CTAs was significantly lower than patients with low CTAs expression(P=0e+00).(2)PPI,GO and KEGG analysis showed that the 7 candidate CTAs were mainly involved in cell cycle regulation,DNA replication initiation,chromosome separation and other biological functions.(3)The results of RT-PCR showed that the expression of OY-TES-1,PBK,ATAD2,KIF2 C,TTK,NUF2,OIP5 and CASC5 mRNA in glioma tissues were84.9%(45/53),75.5%(40/53),71.7%(38/53),67.9%(36/53),66.0%(35/53),64.2%(34/53),56.6%(30/53)and 45.3%(24/53),respectively.They are not expressed in normal brain tissues.Among the glioma specimens examined,100%(53/53)of the CTA transcripts were expressed at least 3 CTAs,88.7%(47/53),69.8%(37/53),43.4%(23/53),18.9%(10/53)and 11.3%(6/53)were co-expressed in 4,5,6,7 and 8 CTAs,respectively.Statistical analysis showed that the expression of OY-TES-1 mRNA was related to the WHO grade of glioma(P=0.020).The expression of 7 CTA mRNA has not been found to be related to the prognosis of glioma patients.Conclusion: Based on bioinformatics high-throughput data analysis,the candidate CTAs with high expression in glioma and related to tumor purity and prognosis were identified.OY-TES-1,PBK,ATAD2,KIF2 C,TTK,NUF2,OIP5 and CASC5 mRNA were all highly expressed in glioma with high frequency of co-expression,suggesting that their combined application may be a potential immunotherapy target for glioma.