| Background and Aim:Colorectal cancer(CRC)is one of the most common malignant tumors in the world.The incidence of CRC and mortality ranking third and second,respectively.Currently,the pathogenesis of colon cancer has not been well understood.Exploring the molecular mechanisms underlying CRC progress is crucial to find novel therapeutic targets and consequently improve the prognosis of patients.Chaperonin containing TCP1 complex(CCT)has been shown to play an important role in the development of several cancers including breast cancer,lung cancer.CCT is a chaperonin composed of two stacked rings.Each ring consists of eight paralogous subunits.Chaperonin Containing TCP-1 subunit 6A(CCT6A)is one of the CCT subunits.The expression and the roles of CCT6A in tumor remain unknown.In this study,we explored the expression and significance,as well as the underlying molecular mechanisms of CCT6A in colon cancer using bioinformatics analyses.Furthermore,the expression and clinical value of CCT6A was verified in colon cancer samples from our institute.Thus,this study will elucidate the role of CCT6A and its potential molecular mechanism and provide valuable reference for identifying CCT6A as a potential therapeutic target in colon cancer.Method:1.A Cox risk model was established based on the expression level of CCT family genes in TCGA database using multivariate Cox regression.Another dataset(GSE39582)was used as a validation set to evaluate the significance of CCT risk score and its correlation with clinicopathological parameters in colon cancer.A nomogram prognostic model for survival prediction was constructed.2.The expression and survival analyses of nine CCT-related genes in colon cancer were performed using GEPIA database in order to screen the differential expression gene related to overall survival(OS).3.The mRNA expression array and clinical information of thirty-three types of cancer were downloaded from the Cancer Genome Atlas Program(TCGA).The expression level of CCT6A between tumor sample and normal sample was compared among different types of cancer.Survival analyses were performed to explore the prognostic value of CCT6A using GEPIA database.Next,the correlation between the expression level of CCT6A and immune cells infiltration,DNA methyltransferase and mismatch repair(MMR)genes were analyzed in multiple cancers.Three colon cancer datasets were downloaded from GEO database to verify the expression level of CCT6A between colon cancer and normal colon tissues.Univariate and multivariate Cox regression were used to validate risk score as the independent prognosis factor in colon cancer cohort.Then,Cox regression were performed to build a risk models based on CCT family genes.Univariate and multivariate Cox regression analyses were used to validate the independent prognostic factors.4.Kyoto encyclopedia of genes and genomes(KEGG)and Hallmark pathway enrichment analysis were used to analyze target genes of CCT6A.miRDB,Target Scan and DIANA databases were used to predict miRNA which was related with CCT6A.The overlaps of potential targets of CCT6A were conducted by Venn diagrams and the expressions of upstream miRNAs in colon cancer were then analyzed based on the TCGA database.5.The expression of CCT6A was detected by immunohistochemical analysis in 56 pairs of colon cancer and adjacent normal tissue samples.The correlation between CCT6A expression and clinicopathological parameters was further analyzed.Results:1.Corresponding risk scores of colon cancer patients were calculated based on CCT family gene expression matrix using cox risk model and was divided into low-and high-risk subgroups by the median value of risk scores.The CCT risk score was an independent predictor of prognosis in colon cancer patients.High risk group had significant poorer prognosis(P <0.05).A prognostic nomogram model was constructed based on TCGA database,using the following variables including CCT risk score,age,sex,tumor TNM stage,T stage,N stage and M stage.This model exhibited excellent performance and could predict the OS of colon cancer patients well(AUC = 0.719).2.The expression level of CCT6A was significantly higher in tumor samples than normal samples among a series types of cancer.In multiple cancer species,CCT6A expression levels were significantly correlated with immune cell infiltration,immune checkpoint genes,DNA methyltransferases and mismatch repair(MMR)genes.Take colon cancer as an example :2.1 The expression level of CCT6A in colon cancer samples was significantly higher than normal colon samples(P < 0.05).2.2 Univariate Cox analyses showed that CCT6A expression levels were significantly associated with overall survival(OS)(HR=1.343,95%CI:1.092-2.127,P=0.043)in colon cancer patients.Multivariate Cox regression analysis revealed that CCT6A wasn’t an independent prognostic factor for OS.2.3 CCT6A gene expression levels were significantly correlated with five types of immune cell infiltration(T cell regulatory: R =-0.369,P < 0.001;CD8+ T cells:R =-0.174,P < 0.001;resting natural killer cells: R =-0.107,P = 0.022;M1macrophages: R = 0.125,P < 0.01,M2 macrophages: R = 0.131,P < 0.01).In addition,CCT6A expression levels had significant correlation with several immune checkpoints including CTLA4(R =-0.133,P < 0.01),LAG3(R =-0.15,P < 0.01),PDCD1(R =-0.119,P = 0.01)and SIGLEC15(R =-0.142,P < 0.01).However,the correlation was not very strong.2.4 CCT6A expression levels were significantly correlated with the expression levels of DNA mismatch repair genes(MMR),DNA methylase level and m6A regulatory factor expression level(P < 0.05),which may serve as a new potential evaluation indicator for tumorigenesis and development.3.KEGG Pathway enrichment analysis indicated that Pyrimidine metabolism(ES =-0.69,NES =-2.4,FDR = 0.0011),Purine metabolism(ES =-0.58,NES =-2.3,FDR = 6e-04)and cell cycle(ES =-0.73,NES =-2.3,FDR = 6e-04)pathways were significantly enriched in CCT6A high expression group.Hallmark pathway enrichment analysis suggested that the top three Hallmark pathways enriched in CCT6A high expression group were m TORC1 signaling pathway(ES =-0.71,NES=-2.3,FDR = 0),MYC target V1(ES =-0.81,NES =-2.3,FDR = 0)and G2 M checkpoints(ES =-0.81,NES =-2.3,FDR = 0).Furthermore,we found hsa-miR-92b was a potential regulators of CCT6A.4.The results of Immunohistochemistry(IHC)showed that the expression level of CCT6A was significantly higher in colon cancer tissue than adjacent normal colon tissues.The expression level of CCT6A was significantly correlated with TNM stage,T stage,N stage,OS status(P < 0.05).The median OS of CCT6A low expression group was 66.4 months,and was 54.1 months in CCT6A high expression group.Multivariate Cox regression analysis showed that T stage(P = 0.005,HR =5.11,95%CI:3.512-9.95),N stage(P = 0.03,HR:2.56,95%CI:1.882-4.256),M stage(P = 0.035,HR:5.61,95%CI:1.132 ～ 17.865),TNM stage(P = 0.002,HR = 2.35,95%CI:1.956 ～ 7.764)and BRAF mutation(P = 0.001,HR: 4.01,95%CI: 1.508 ～16.753)were independent risk factors in predicting overall survival after surgery.Conclusion:1.CCT high risk score is an independent predictor of poor prognosis in colon cancer.2.A nomogram survival prediction model including CCT risk score exhibited excellent performance in colon cancer patients.3.Although CCT6A isn’t an independent prognostic factor of CRC patient,patients with high CCT6A expression have poorer prognosis.The expression level of CCT6A could be a potential prognostic indicator in colon cancer.4.hsa-miR-92b could be a potential regulatory factor of CCT6A.CCT6A might exert its effect through regulating Pyrimidine metabolism,Purine metabolism and cell cycle pathways.5.CCT6A may participate in initiation and progression of colon cancer and could be a potential therapeutic target of colon cancer. |
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