| Objective:Clear cell renal cell carcinoma(cc RCC)is the most common pathological type of renal cancer and the most common cause of death in patients with urogenital tumors.There was no suitable biomarkers applicated to guide the clinical diagnosis,treatment and prognosis of cc RCC.Emerging evidences have indicated that interferon regulatory factor 6(IRF6),as a member of the Interferon regulatory factors(IRFs)family,plays important roles in a variety of tumors.However,the expression status of IRF6 and its prognostic value in cc RCC remain unclear.The purpose of this study was to investigate the expression pattern and prognostic value of IRF6 in cc RCC,and initially to explore the possible molecular mechanism.Methods:(1)Based on the online geo and TCGA databases,we explored the expression pattern and prognostic value of IRF6 in cc RCC.(2)The expression pattern and prognostic value of IRF6 in cc RCC were verified by using cc RCC clinical samples and clinical data,combined with immunohistochemistry(IHC)experiment performed on clinical samples.(3)Western blot(WB)was used to detect the expression of IRF6 in cc RCC cell lines;Methylation specific PCR(MSP)was used to detect the DNA methylation level of IRF6 in cc RCC cell lines.In addition,demethylating agents was used to analyze the effect of DNA methylation level on IRF6 expression.(4)The correlation between IRF6 expression and cc RCC tumor immune cells infiltration was studied by using online TCGA and TIP databases.Results:(1)The results of comprehensive analysis of GEO and TCGA databases data showed that the expression of IRF6 was significantly down regulated in cc RCC compared with normal renal tissues(P <0.0001),besides,decreased expression of IRF6 was associated with adverse clinicalpathological characters(P < 0.0001);In addition,in the TCGA cohort,decreased IRF6 expression was associated with poor prognosis of patients with cc RCC(P < 0.0001).Multivariate Cox regression analysis showed that decreased IRF6 expression was an independent risk factor for predicting shorter overall survival(HR:0.8524,95% CI: 0.7614-0.9543,P = 0.0056)and shorter disease-free survival(HR: 0.7024,95% CI: 0.6087-0.8104,P < 0.0001).(2)IHC results showed that IRF6 was significantly down regulated in clinical samples of cc RCC compared with adjacent normal renal tissues(P < 0.0001);The results of K-M survival curve analysis showed that patients of low IRF6 expression group had poor prognosis.(3)WB experiment results showed that the expression level of IRF6 was significantly lower in cc RCC cell lines compared with normal renal tubular epithelial cells;In addition,MSP experiment result showed that compared with normal renal tubular epithelial cells,IRF6 DNA methylation level was significantly decreased in cc RCC cells,while the use of demethylating agents could significantly increase the expression level of IRF6 in cc RCC cells.(4)The data analysis based on TCGA and TIP databases showed that the decreased expression of IRF6 was related to the change of infiltration level of various immune cells in cc RCC.Conclusion:IRF6 expression was significantly down-regulated in cc RCC,which was related to DNA hypermethylation,and the reduced expression of IRF6 was associated with poor clinicopathological features and predicted an unfavorable prognosis of cc RCC patients.These findings provide new ideas and scientific basis for developing new effective biomarkers for cc RCC and exploring important therapeutic targets in the future. |
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