| Background:clear cell renal cell carcinoma(ccRCC)is the most common pathological subtype of renal cell carcinoma,which seriously endangers human health.Although patients with early ccRCC can be cured by radical surgery,the mortality rate of patients with advanced CCRCC,especially those with metastasis,is still high.The insensitivity of ccRCC to radiotherapy and chemotherapy leads to the limitation of its treatment methods.Therefore,the research on the pathogenesis and target genes of renal cell carcinoma has important theoretical and clinical value.The research on biomarkers based on target genes can also promote the individualized and accurate prognosis evaluation of patients with renal cell carcinoma,and is of great significance for guiding postoperative review and clinical medication.Gap junction proteins(GJP)are a family of channel proteins in organisms,and the stability of their functions is crucial for maintaining the normal growth and development of the body.Recent studies have confirmed that GJPs have tumor suppressive effects.However,no studies of GJPs in ccRCC have been reported.Therefore,the present study aimed to systematically screen GJPs significantly associated with ccRCC prognosis and to preliminarily explore the biological functions of GJA5 and GJB1 in renal cancer cells.Method:Using high-throughput sequencing data of ccRCC in the Gene Expression Omnibus(GEO)database and the Cancer Genome Atlas(TCGA)database,LASSO regression,univariate,and multivariate Cox regression were used to screen for GJPs that are independently associated with the prognosis of ccRCC patients,and the candidate genes were analyzed by pan-cancer analysis and Kaplan-Meier(KM)survival analysis.The risk score(RS)of each patient with ccRCC was calculated based on the coefficient value of multivariate Cox regression and the expression of GJPs.The patients were divided into high-RS and low-RS groups,taking the median of RS as the cutoff value,and a RS prognostic model was constructed.Then,the KM survival analyses were performed to examine the prognosis of the two RS groups.A nomogram that incorporating the RS grouping and clinicopathological parameters was constructed.Time-dependent ROC curve analysis was performed to evaluate the specificity and sensitivity of the RS prognostic model and the nomogram.We performed a differential analysis of immune cell infiltration,tumor microenvironment related molecular abundance,tumor mutation load,and drug sensitivity between the RS risk groups,and explored the active pathways enriched in high-RS groups by GO and KEGG pathway analysis.Finally,qRT-PCR and Western blot were performed to detect the expression of mRNAs and proteins.EDU experiments were performed to detect cell.Transwell assay and cell scratch assay were performed to examine cell migration.Result:1.LASSO regression and Cox regression analysis were performed to screen the GJPs with prognostic values in TCGA ccRCC RNA-seq data,and GJA5 and GJB1 turned out to be the top candidates.GJA5 and GJB1 are lowly expressed in pan-cancer analysis,and GJA5 and GJB1 low-expression is associated with poor prognosis in patients with ccRCC(p<0.001).2.The KM survival curve analysis demonstrated that high-RS group was significantly associated with bad prognosis in terms of overall survival(OS)and progression-free survival time(PFS)(p<0.001),and the risk of death in patients with high-RS was 1.695 times higher than that in patients with low-RS(HR=1.695,95%CI:1.439～1.996,p<0.001).3.Time-dependent ROC curves showed that the Area Under Curve(AUC)of RS prognostic models in predicting 1-year,3-year,and 5-year survival rates was 0.740,0.781,and 0.771,while the AUC of nomograms in predicting 1-year,3-year,and 5-year survival rates was 0.863,0.848,and 0.817.4.Differential analysis showed that the high-RS group and the low-RS group have different immune status,TMB,and targeted drug sensitivity.5.qRT-PCR and Western blot showed significantly lower expression of GJA5 and GJB1 in ccRCC cell lines compared to the normal renal epithelial cells(p<0.001).EDU assay showed that GJA5 knockdown and GJB1 knockdown significantly promoted the proliferation of ccRCC cells(p<0.001);Transwell assay and cell scratch assay demonstrated that GJA5 knockdown and GJB1 knockdown significantly promoted the migration ability of ccRCC cells;Western blot analysis showed that the expression of E-cad and Bcl-2 was downregulated,while the expression of N-cad,VIM and Bax were upregulated in the GJA5 and GJB1 knockdown group.Conclusion:Gap junction proteins GJA5 and GJB1 are promising prognostic biomarkers for ccRCC patients,and could serve as potential drug targets. |
