| Part I Bioinformatics analysis and verification of gene targets for clear cell renal cell carcinoma(ccRCC)Objective:To study the clinical pathogenesis of ccRCC.Methods:Two expression profiling datasets(GSE68417,GSE71963)were downloaded from the GEO database.Differentially expressed genes(DEGs)between ccRCC and normal tissue samples were identified by GEO2 R.Functional enrichment analysis was made by the DAVID tool.Protein-protein interaction(PPI)network was constructed.The hub genes were excavated.The clustering analysis of expression level of hub genes was performed by UCSC(University of California Santa Cruz)Xena database.The hub gene on overall survival rate(OS)in patients with ccRCC was performed by Kaplan-Meier Plotter.Finally,we used the ccRCC renal tissue samples to verify the hub genes.Results: 1.1182 common DEGs between the two datasets were identified.The results of GO and KEGG analysis revealed that variations in were predominantly enriched in intracellular signaling cascade,oxidation reduction,intrinsic to membrane,integral to membrane,nucleoside binding,purine nucleoside binding,pathways in cancer,focal adhesion,cell adhesion molecules.2.10 hub genes ITGAX,CD86,LY86,TLR2,TYROBP,FCGR2 A,FCGR2B,PTPRC,ITGB2,ITGAM were identified.FCGR2 B and TYROBP were negatively correlated with the overall survival rate in patients with ccRCC(P<0.05).3.qRT-PCR analysis showed that the relative expression levels of CD86,FCGR2 A,FCGR2B,TYROBP,LY86,and TLR2 were significantly higher in ccRCC samples,compared with the adjacent renal tissue groups.Part II Higher FCGR2 A as a predictive biomarker for poor prognosis of clear cell renal cell carcinomaObjective:ccRCC is the main type of RCC and the most aggressive histological type.However,the molecular mechanism of FCGR2 A in the ccRCC was not clear.Methods:A total of 151 patients with clear cell renal cell carcinoma(ccRCC)were recruited.Pearson’s chi-squared and spearman test were performed to analyze the correlation between FCGR2 A and relative parameters.Univariate and multivariate Cox proportional hazards regression analysis were also performed.HE staining was performed to observe the pathological morphologic changes of ccRCC.the immunohistochemical and immunofluorescence assay were made to detect the protein expression of FCGR2 A.Strong correlation between the expression of FCGR2 A and the survival time of ccRCC patients was analyzed by the BP neural network and support vector machine(SVM).Results: 1.TNM(P<0.001),family history of ccRCC(P=0.01)and Fuhrman stage(P<0.001)were markedly related to the FCGR2 A.FCGR2A were significantly correlated with the TNM(ρ = 0.406,P<0.001),family history of ccRCC(ρ = 1.000,P<0.01)and Fuhrman stage(ρ =0.577,P<0.001).2.Patients who had high FCGR2 A,had obviously lower OS than patients who had low FCGR2 A level,and the HR is 66.901(95% CI,28.251-159.428,P<0.001).Through the ROC result,FCGR2 A had more advantage as a biomarker for ccRCC.3.By the immunohistochemical assay,protein expression of FCGR2 A in the ccRCC was higher than the control tissues(P<0.05).Part III Higher TYROBP as a predictive biomarker for poor prognosis of clear cell renal cell carcinomaObjective:To study the molecular mechanism of TYROBP in ccRCC.Methods:A total of 151 patients with clear cell renal cell carcinoma(ccRCC)were recruited.Pearson’s chi-squared and spearman test were performed to analyze the correlation between TYROBP and relative parameters.Univariate and multivariate Cox proportional hazards regression analysis were also performed.HE staining was performed to observe the pathology result of ccRCC.Immunohistochemistry and Immunofluorescence assay were made to detecting the protein expression of TYROBP.Total RNA was extracted using the TRIzol to exam the m RNA expression of TYROBP via the qRT-PCR.Strong correlation between the expression of TYROBP and the survival time of ccRCC patients was performed by the BP neural network and support vector machine(SVM).Results: 1.Compared with the Ⅰ Fuhrman Staging,the expression of TYROBP was higher in the Ⅲ and Ⅳ Fuhrman Staging(P<0.05).Among the individuals,TNM(P<0.001),and Fuhrman stage(P<0.001)were markedly related to the TYROBP.2.Furthermore,TYROBP(HR = 7.838,95% CI: 4.834-12.711,P <0.001)were significantly associated with overall survival.And the area under the curve(AUC)of TYROBP was used as the degree of confidence in judging each factor: survival time(AUC= 0.85748,P= 0.03116).Conclusions : In conclusion,FCGR2 A and TYROBP are highly expressed in ccRCC,indicating that FCGR2 A and TYROBP may be potential targets for the diagnosis and treatment of ccRCC. |
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