Expression And Clinical Significance Of CGN In Renal Cell Carcinoma

Part Ⅰ Bioinformatics method to establish key genes related to the diagnosis and prognosis of renal cell carcinomaObjectiveRecently,with the development of bioinformatics,weighted gene co-expression networks and differential gene analysis have become important methods for screening tumor diagnostic markers.This study used the gene expression profiles of TCGA-KIRC,GSE46699,GSE36895 and GSE16449,combined with the results of weighted gene co-expression network and differential gene analysis,explored differentially co-expressed genes in renal cell carcinoma.At the same time,based on survival analysis,prognostic genes associated with renal cell carcinoma survival were identified.MethodsFirst,genes were divided into tumor groups and normal groups according to tissue characteristics.Genes in core modules were obtained through the weighted gene co-expression network.Then,the intersection of the core module and the differential genes obtained by the differential expression analysis is called differentially co-expressed genes.The differentially co-expressed genes were included in univariate and multivariate Cox survival analysis to obtain independent prognostic key genes associated with overall survival in renal cell carcinoma.Finally,Kaplan-Meier survival analysis was performed on the independent prognostic key genes,and the key gene most related to the overall survival of renal cell carcinoma were selected according to the statistical results.In order to verify the reliability of the results,Pearson correlation analysis was used to explore the correlation between the key gene most associated with overall survival and the classic biomarkers of renal cell carcinoma.ResultFinally,124 differentially co-expressed genes were screened out by weighted gene co-expression network and differential gene expression analysis.According to univariate and multivariate Cox survival analysis,CGN,FECH,UCHL1 and WT1 were independently associated with overall survival in renal cell carcinoma patients.After Kaplan-Meier survival analysis of the above genes,it revealed that CGN was the key gene with the strongest correlation with the prognosis of renal cell carcinoma patients.Pearson correlation analysis showed that CGN was highly correlated with BAP1,SETD2,BIRC5 and CXCR4.ConclusionsCGN,FECH,UCHL1 and WT1 were independent prognostic key genes associated with overall survival in renal cell carcinoma,and CGN was the most independent prognostic key gene associated with renal cell carcinoma survival.This study provided a new strategy for screening the diagnostic and prognostic biomarkers of renal cell carcinoma,and provides a new research filed of understanding the mechanism of occurrence and development of renal cell carcinoma.Part Ⅱ Expression of CGN in renal cell carcinoma and its correlation with clinical featuresObjectiveAs one of the tight junction proteins,CGN has been confirmed as a key factor in the occurrence and development of various tumors,but there is no study exploring the expression of CGN in renal cell carcinoma.In this study,we conducted experiments to investigate the expression of CGN in human renal cell carcinoma and its correlation with the clinical characteristics of renal cell carcinoma by collecting renal cell carcinoma tumor tissue specimens and normal tissue specimens in clinical practice.MethodsTumor tissue of renal cell carcinoma and normal tissue specimens were extracted from the database of renal tumors in Beijing Hospital.According to the inclusion and exclusion criteria,the included tissue samples were subjected to immunohistochemical staining,quantitative Real-time Polymerase Chain Reaction(RT-qPCR)and western blotting(WB)to evaluate the expression of CGN in tumor tissue and normal tissue.Then,the clinicopathological information of the above patients and the TCGA-KIRC database were collected,the correlation between CGN expression and the clinicopathological characteristics of renal cell carcinoma was compared by Wilcox rank sum test.ResultFresh tumor tissue and normal tissue from a total of 59 renal cell carcinoma patients were included in this study,of which 30 were subjected to RT-qPCR experiments and 29 were subjected to WB as verification.In addition,this study collected a total of 20 cases of renal cell carcinoma patients with paraffin sections in the pathology department for immunohistochemical study.In the RT-qPCR experiment,the results showed that the expression of CGN in tumor tissue was significantly lower than that in normal tissue,and the difference was statistically significant(P<0.05).Meanwhile,at the protein level,both WB and immunohistochemical staining experiments confirmed that CGN was lowly expressed in tumor tissues compared with normal tissues.In clinical samples,according to the results of RT-qPCR experiments,there was no correlation between CGN expression and age and gender(P>0.05),while low CGN expression were highly correlated with higher T stage and Fuhrman grade,and the difference was statistically significant(P<0.05).The results of gray value analysis of WB test results showed that there was no correlation between CGN expression and age and gender(P>0.05).But CGN expression was negatively correlated with T stage and Fuhrman grade(P<0.05).Due to the sample size selected in the specimen database of our center is small,data from the TCGA-KIRC database were used to further validate the correlation between CGN expression and clinicopathological features of renal cell carcinoma.We found that there was no statistical correlation between CGN and gender(P>0.05),the expression of CGN decreased with age,and was negatively correlated with higher T,N,M stages,tumor stages and tumor grades,the difference was statistically significant(P<0.05).ConclusionsThis study confirmed the low expression of CGN in tumor tissue compared with normal tissue samples through the tissue samples of Beijing hospital.At the same time,a high correlation between CGN and the clinicopathological features of renal cell carcinoma has also been demonstrated.A novel diagnostic biomarker for renal cell carcinoma was confirmed and provide a new strategy for precise diagnosis and treatment of renal cell carcinoma.Part Ⅲ Correlation between CGN and tumor microenvironment in renal cell carcinomaObjectiveThe tumor microenvironment is closely related to the occurrence and development of renal cell carcinoma.Meanwhile,low expression of CGN in renal cell carcinoma.Therefore,the correlation between CGN and the tumor microenvironment of renal cell carcinoma was further explored in this study.MethodsRNA-sequencing and tumor mutational burden(TMB)data of TCGA-KIRC renal cell carcinoma tissues were obtained in this study,and used the "CIBERSORT" R package to analyze the components of tumor-infiltrating immune subsets in each sample.The linear relationship between 22 immune cells and CGN was discussed using Pearson correlation analysis,and the correlation between CGN and TMB was explored by Pearson correlation analysis and Wilcox rank sum test analysis.Meanwhile,Kaplan-Meier survival analysis showed survival curves of CGN and TMB stratification.Finally,CGN gene expression was divided into two groups according to the median value,and the Wilcoxon rank sum test was used to analyze the relationship between the two groups of CGN high and low expression and PD-L1 in TCGA-KIRC,GSE16449 and GSE22541 data sets.ResultAfter obtaining the proportion of tumor-infiltrating immune subsets using the"CIBERSORT" algorithm,the results showed that activated NK cells,activated dendritic cells and resting mast cells were positively correlated with the expression of CGN,while CD4+memory activated T cells,regulatory T cells and M0 macrophages were negatively correlated with the expression of CGN(P<0.05).According to the median value of CGN expression,they were divided into high and low group.The Wilcox rank sum test showed that the CGN high expression group showed lower TMB(P<0.05).Pearson correlation analysis also found that the expression of CGN was negatively correlated with TMB.When the Kaplan-Meier method was used to analyze the survival curves of CGN stratification and TMB stratification,the group with high TMB and low CGN expression had the worst prognosis.The results of Wilcoxon rank sum test showed that the expression levels of CGN and PD-L1 in TCGA-KIRC,GSE16449 and GSE22541 datasets were closely related(P<0.05).ConclusionsIn this study,through the TCGA database,it was found that CGN gene expression was closely related to the tumor microenvironment of renal cell carcinoma.The results help to deeply understand the role of CGN in the occurrence and development of renal cell carcinoma,and provide new ideas for precise immunotherapy of renal cell carcinoma.