| Objective.Colon cancer is one of the most common and major cause of death and malignant tumor in the world.N6-methylation of adenosine(m6A)is a common reversible posttranslational modification of mRNA in eukaryotic cell,and it plays an important role in various biological functions in human body.Dysregulated expression and genetic changes of m6A regulators have been correlated with tumorigenesis,cancer cell proliferation,tumor microenvironment,and prognosis in cancers.This study used colon cancer clinical data and RNA-seq data from public database to explore the relationship between colon cancer and N6-methylation.Method.Public gene expression data and completed clinical annotation information from Gene-Expression Omnibus(GEO)and The Cancer Genome Atlas(TCGA)databases were analyzed using various R packages.Quantitative real-time PCR and Western blot were used for detecting the expression level of m6A regulators in different colon cancer cell lines.The effect of CBLL1-knockdown on the migration ability of HT26 cells was detected by scratch test.Based on the expression level of 7 m6A regulators,Consensus Clusterplus package was used for classifying patients.The CIBERSORT was used for quantifying the proportion of immune cells,and the patients were grouped as m6A.Cluster1-3 by R package Limma.Clusterprofiler R package conducted gene annotation enrichment analysis on m6A-related genes.,and samples from the training and the test sets were grouped according to the median of m6A risk score in the training set,and then Kaplan-Meier survival curve of Log-rank test was ploted to construct the prognosis model.Result.The mRNA expression levels of 21 m6A regulatorys in normal and colon cancer samples in the TCGA database were studied.It can be found that there are differences in the expression of these 21 m6A regulatorys in colon cancer samples and normal samples.Through the univariate Cox regression model,we revealed the prognostic value of 21 m6A regulatorys in colon cancer patients,of which CBLL1,ELAVL1,LRPPRC,RBM15 B,YTHDF1,YTHDF2 and ZC3H13 are related to the prognosis.Based on the expression of seven m6A regulatorys related to the prognosis,patients were divided into three molecular subtypes of colon cancer,m6A.cluster1-3.Analysis of the three defined m6A modified subtypes showed that m6A.cluster1/2 has a particularly significant survival advantage compared to m6A.cluster3.By comparing the composition of immune cells and the expression differences of immune checkpoint genes in the three subtypes,there are fewer CD4+T cells and NK cells,more M2 cells,and the expression of immune checkpoint genes is highest in these three subtypes,suggesting that m6A.cluster3 patients are more likely to have immune escape during immunotherapy.Furthermore,299 differentially expressed genes related to the m6A phenotype were used to classify patients into different genome subtypes,named m6 Gene cluster1-3.In m6Agene cluster2,there are fewer CD4+T cells,plasma cells,and NK cells,and more M0/M2 macrophages,indicating that these patients have a poor anti-tumor environment.And most of the checkpoint genes are up-regulated in m6 Gene.cluster2.Finally,using 299 m6A-related genes and 5 clinical information,we constructed a prognostic model and validated the model,proving that the model can be used clinically for the prognosis of patients.Conclusion.Seven m6A regulatory factors(CBLL1,ELAVL1,LRPPRC,RBM15 B,YTHDF1,YTHDF2 and ZC3H13)are related to tumorigenesis,tumor microenvironment and tumor prognosis.Based on the 299 m6A phenotype-related genes,three subtypes were studied.One of the subtypes is immunosuppressive tumor.Patients with this subtype may be more suitable for immunotherapy than other subtypes.Finally,using m6A-related genes and clinical information,a prognostic model was constructed and tested to prove that the prognostic model can be used for the prognosis of clinical treatment of CRC patients. |
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