The Correlation Analysis Of N6-methyladenosine Regulators And The Immune Infiltration In Clear Cell Renal Cell Carcinoma Based On Bioinformatics

Background:The tumor immune microenvironment(TIME)was related to the progression and prognosis of cancer.Moreover,the role of RNAN6-methyladenosine(m6A)in tumors has been investigated in many studies.Objective:This work aims to analyze the correlation between m6A regulators and TIME.Methods:The transcriptome data,tumor mutation burden(TMB)data,copy number variation(CNV)data,and immunohistochemistry data of ccRCC were downloaded from the public database.Based on the previous literature on m6A studies,23 m6A RNA regulators were enrolled in this study.With the k-means clustering algorithm in the R package "ConsensusClusterPlus",the ccRCC samples were subgrouped according to the expression of m6A regulators.Based on the principal component analysis(PCA)algorithm,the R package "factoextra" was utilized to screen m6A regulators that play an important role in the subgroup.R packages "survminer" and "survivor" were utilized to analyze the relationship between the critical m6A regulators and the prognosis of patients with ccRCC.R package "WGCNA" was performed to conduct weighted gene co-expression network analysis(WGCNA),obtaining the module related to the subgroups.Then gene ontology(GO)enrichment analysis and gene set enrichment analysis(GSEA)was conducted to explore the potential biological functions associated with the subgroup.The single sample gene set enrichment analysis(ssGSEA)algorithm in the R package"GSVA" was used to calculate the immune cell infiltration score of ccRCC samples.Next,we identify the immune infiltrating cells associated with the grade and stage of ccRCC.Correlations between hub genes and immune cells in subgroups were analyzed using the Spearman correlation analysis algorithm.Further,we utilized correlation analysis to screen the genes associated with hub m6A regulators and observed the immunohistochemistry of hub genes in the HPA database.Results:This study downloaded 607 ccRCC samples from TCGA database,including 73 adjacent normal tissue samples and 534 tumor samples,and downloaded two ccRCC datasets,including GSE53757 and GSE36895,from GEO database.We found that some m6A regulators differently expressed in several clinical character groups,such as grade and stage.Based on the result of the clustering analysis,the ccRCC samples were divided into 3 groups,and the overall survival(OS)of cluster 2 was less than the other two subgroups.The expression of IGF2PB2 and IGF2PB3 is higher expressed than cluster 1/3 significantly,meanwhile,the TMB and CNV in cluster 2 are different from that in cluster 1/3.Then the result of PCA indicated that IGF2BP2 and IGF2PB3 were the essential components in the cluster 2 subgroup.The result of WGCNA showed that the brown gene module was correlated with cluster 2 significantly,further,the result of GO enrichment analysis and GSEA demonstrated that the genes in the brown module were related to the T cell subtypes,which implied that there were some correlations between the module related to cluster 2 and the immune cell infiltration.Next,we calculated the immune infiltration score of ccRCC samples via ssGSEA algorithm.Then we conducted the K-M survival analysis and univariate Cox analysis for immune cells in ccRCC,which indicated that the T cell subtypes were associated with the prognosis of the patients with ccRCC.Furthermore,the immune cell infiltration score,immune regulators,and immunomodulator agonists were differently expressed in subgroups significantly.Meanwhile,we found that IGF2BP2 and IGF2BP3 had a strong correlation with T cell subtypes in cluster 2 via Spearman correlation analysis.Then we found that there was a strong positive correlation between HMGA2 and IGF2PB2,and the immunohistochemistry indicated that HMGA2 and IGF2PB2 were highly expressed in ccRCC.Conclusion:The m6A regulators may affect ccRCC immune cell infiltration,where IGF2PB2 and IGF2PB3 play an important role in regulating immune cell infiltration.Moreover,HMAGA2 may affect the expression of IGF2BP2 and regulate ccRCC immune cell infiltration via the HMGA2-IGF2BP2 axis.