| Objective: The potential mechanism of sunitinib resistance in Renal cell carcinoma(RCC)remains unclear.In this study,bioinformatics was used to explore the genes associated with sunitinib resistance in renal cell carcinoma.To explore the relationship between target genes and prognosis of patients with renal cell carcinoma,and to further explore the influence of target genes on the invasion ability of renal cell carcinoma and sunitinib resistance of renal cell carcinoma.Finally,the signal transduction pathways of target genes were analyzed.Methods and materials: In this study,we collected and analyzed two microarray transcriptome data from sunitini-resistant renal cell carcinoma(RCC)tissue samples from Gene Expression Omnibus(GEO)database,and screened out Differentially expressed genes(DEG).The online HPA database was used to analyze the expression of candidate genes in renal cancer and finally select the target genes.The protein-protein interaction(PPI)network of the target gene was constructed to identify the core genes closely related to the target gene,and GO analysis and KEGG pathway analysis were performed.Clinical information of TCGA patients with renal cell carcinoma was downloaded and analyzed.Univariate,multivariate and survival analysis of prognosis of RCC patients were further performed to explore the relationship between target genes and prognosis of RCC patients.Clinical renal cancer tissue samples and adjacent normal kidney tissues were collected,and qRT-PCR and Western Blot were used to analyze the expression of target genes in clinical tissue samples.Lentivirus transfection technique was used to establish 786-0 cell lines with low expression of target genes,and the m RNA and protein expression levels of target genes were detected by qRT-PCR and Western Blot to verify the success of modeling.Transwell assay was used to detect the influence of target genes on the invasion ability of renal cancer cell line 786-0.CCK8 assay was used to detect the proliferation activity of the target gene on the renal cancer cell line 786-0,and the effect of the target gene on the sunitinib resistance of the renal cancer cell line 786-0 was further analyzed.Finally,the transcriptome data of TCGA,an online database,was analyzed by correlation analysis to explore the possible signal transduction pathways involved in the target genes.Results: Analysis of GEO microarray data indicated that EPSTI1、OAS2、DUSP1、PLAUR、SIK1 and CTGF were highly expressed in Sunitinib-resistant RCC samples compared with those in Sunitinib-sensitive RCC samples.Immunohistochemical profilograms of the online HPA database showed that EPSTI1 was significantly overexpressed in renal carcinoma.This study selected EPSTI1 for target genes,and build EPSTI1 related protein--protein interaction networks,and filter core gene,gene ontology analysis was carried out on the core genes found that EPSTI1 and core gene in molecular function mainly combining,single-stranded RNA and ATP,etc.,of cells mainly in terms of cytoplasm,etc.,biological process level mainly defense responses to the virus,viral genome replication of negative control,etc.KEGG pathways were mainly influenza A and chemokine signaling pathways.Univariate analysis of clinical information in RCC patients with TCGA showed that high expression of EPSTI1 was associated with patients’ AJCC pathological stage(HR,7.379;95%CI,5.281-10.312;P <0.001)and survival status(HR,1.090;95%CI,1.052-1.129;P<0.001),multivariate analysis showed that high expression of EPSTI1 was an independent risk factor for poor prognosis in patients with RCC(HR,1.070;95%CI,1.028-1.114;P<0.001),survival analysis showed that RCC patients with high EPSTI1 expression had a worse survival outcome compared with RCC patients with low EPSTI1 expression(P<0.0001).Lentivirus transfection with si RNA,qRT-PCR and Western Blot results indicated that the renal carcinoma cell line 786-0 with low expression of EPSTI1 was successfully constructed.Further Transwell experiments indicated that knockdown of EPSTI1 expression could reduce the invasion ability of 786-0 cells(P<0.05),CCK8 results suggested that the sensitivity of 786-0 renal carcinoma cells to sunitinib was significantly increased after the expression of EPSTI1 was knockdown(P<0.05),Pearson correlation analysis suggested that EPSTI1 might be related to the JAK-STAT pathway.Conclusion: Bioinformatic analysis suggested that EPSTI1 was one of the major genes involved in sunitinib resistance in RCC.Further analysis showed that EPSTI1 was also highly expressed in RCC tissues and was an independent risk factor for poor prognosis in RCC.It was confirmed that the invasion ability of786-0 cells was weakened and the resistance to sunitinib was significantly alleviated after the expression of EPSTI1 was knocked down.The specific molecular mechanism remains to be further studied,and EPSTI1 may be a new target for the treatment of sunitinib resistance in RCC in the future. |
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