Identifying Molecular Biomarkers Of Renal Cell Carcinoma Associated With Clinicopathological Features By Weighted Gene Co-expression Network Analysis

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Identifying molecular biomarkers of papillary renal cell carcinoma associated with pathological stage by weighted gene co-expression network analysisObjective To investigate the correlation between gene expression data and clinicopathological features of PRCC patients with a system-level WGCNA and other methods.Methods The RNA sequencing data sets and corresponding clinical information of kidney PRCC patients were downloaded from the TCGA repository website.Two R packages,DEseq2 and limma were utilized to screen differently expressed genes between normal and cancer samples.Scale-free gene co-expression networks were constructed and gene modules were identified by the WGCNA package.Then the highest correlation in the Module-feautre relationship was found between corresponding module and clinical feature.Based on gene significance and module membership,the function "networkScreening" in the WGCNA package was applied to screen hub genes in module of interest.Correlation between hub genes and corresponding clinical feature was tested with one-way ANOVA and an independent sample t-test.The stability of target module,and the correlation between hub genes and the clinical feature were confirmed by an validation cohort.A good molecular biomarker for pathological stage of RCC must be a prognostic gene in clinical practice.Survival analysis was adopted to reversely validate if hub genes are associated with pathological stage.Results The mRNA sequencing data of PRCC and adjacent normal tissue in The Cancer Genome Atlas was analyzed to identify 1148 differentially expressed genes,on which weighted gene co-expression network analysis was performed.Eleven co-expressed gene modules were identified.The highest association was found between blue module and pathological stage(r = 0.45)by Pearson's correlation analysis.All Forty hub genes in blue module can distinguish localized(pathological stage ?,?)from non-localized(pathological stage ?,?)PRCC(P<0.01).Survival analysis unveiled that all hub genes were associated with patient prognosis(P<0.01).The validation cohort GSE2748 verified that blue module belonged to most stable modules,30 hub genes could differentiate localized from non-localized PRCC(P<0.01),and 18 hub genes are prognosis-associated(P<0.01).ROC curve indicated that 17 hub genes exhibited excellent specificity and sensitivity on the diagnostic efficiency for localized and non-localized PRCC(AUC>0.7).Conclusion The 17 key genes may serve as molecular biomarkers and help distinguish different pathological stages for PRCC patients.Part ? Identifying molecular biomarkers of clear cell renal cell carcinoma associated with tumor histological grade by weighted gene co-expression network analysisObjective To investigate the correlation between gene expression data and clinicopathological features of CCRCC patients with a system-level WGCNA and other methods.Methods The RNA sequencing data sets and clinical information of kidney CCRCC patients were downloaded from the TCGA repository website.DEseq2 and limma were utilized to screen differentially expressed genes between normal and cancer samples.Scale-free gene co-expression networks were constructed and gene modules were identified by the WGCNA package.Then the highest correlation in the Module-feautre relationship was found between corresponding module and clinical feature.The hub genes were screened by directly setting the threshold for gene significance and module membership.Correlation between hub genes and corresponding clinical feature was tested with one-way ANOVA and an independent sample t-test.The stability of target module,and the correlation between hub genes and the clinical feature were confirmed by an independent validatioin cohort.Results The mRNA sequencing data of CCRCC and adjacent normal tissue in The Cancer Genome Atlas was analyzed to identify 1532 differentially expressed genes,on which weighted gene co-expression network analysis was performed.Twelve co-expressed gene modules were identified.The highest association was found between yellow module and histological grade(r = 0.3)by Pearson's correlation analysis.Twenty-three out of forty-one hub genes in yellow module can distinguish the different histological grades(Fuhrman ?,Fuhrman ? and Fuhrman ?/?)of CCRCC(P<0.01).GSE781,GSE19949,GSE47032,GSE76207 and GSE89563 based on microarray mRNA expression data of CCRCC patients were combined as an independent validation cohort,which confirmed that yellow module belonged to most stable modules,also confirmed that BUB1 and NCAPG could identify different histological of CCRCC(P<0.01).Conclusion Two key genes BUB1 and NCAPG could be selected as molceular biomarkers and help differentiate distinct histological grades of CCRCC patients.Part ? Study on the correlation between BUB1 protein expression and Furhman grades of clear cell renal cell carcinoma and mechanism explorationObjective To investigate the correlation between BUB1 protein expression and histological grads of clear cell renal cell carcinoma(CCRCC)by immunohisto-chemistry,and to preliminarily explore the potential mechanism of BUB 1 affecting histological grades of CCRCC.Methods Immunohistochemical analysis was carried out to observe the correlation of BUB1 protein expression and clinicopathological features of 52 patients with CCRCC in our hospital,the expression difference between normal kidney tissue and CCRCC.Through transient transfection on human CCRCC cell line 786-0 with BUB1 shRNA plasmid,different cell lines,including 786-0-NC,786-0-shNC and 786-0-shBUB1 were constructed.The ability of proliferation,invasion and migration in different cell lines and protein expression level of Ki67 were detected.Results Immunohistochemical analysis showed that the expression levels of BUB 1 protein in CCRCC was positively correlated with different histological grades of CCRCC.The expression level of BUB1 protein in normal kidney tissue was higher than that of Fuhrman ?/? CCRCC,which was similar to the expression level of Fuhrman ?/? CCRCC.The CCRCC cell line 786-0 of low BUB1 expression was successfully constructed by sh-RNA technique.Proliferation,invasion and migration ability of 786-0 cell line,as well as protein expression of Ki67 were significantly decreased when BUB1 expression was reduced.Conclusion At the protein level,BUB1 was confirmed to be a molecular biomarker of different histological grades of CCRCC.BUB1 can regulate the proliferation,migration and invasion of CCRCC cell,thus affecting the malignancy of CCRCC.