Decreased interferon regulatory factor 6 expression due to DNA hypermethylation predicts an unfavorable prognosis in clear cell renal cell carcinoma

Abstract

Background: Emerging evidences have indicated that 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 clear cell renal cell carcinoma (ccRCC) remain unclear. Methods: In this study, we used TCGA-KIRC, GEO and TIP databases and immunohistochemistry staining to determine the expression profile, clinico-pathological features and prognostic value of IRF6 in ccRCC. MSP and demethylation analysis were utilized to verify the regulatory effect of DNA methylation on IRF6 expression. Results: Our results found that IRF6 expression was downregulated in ccRCC tissues and cell lines, and decreased IRF6 expression was associated with worse clinicopathological features and poorer prognosis. Besides, the results of multivariate Cox regression analysis also confirmed that decreased IRF6 expression was an independently risk factor predictor of shorter Overall Survival (OS) (HR: 0.8524, 95%CI: 0.7614-0.9543, P=0.0056) and Disease Free Survival (DFS) (HR: 0.7024, 95%CI: 0.6087-0.8104, P<0.0001) in ccRCC patients. Moreover, the results of MSP and demethylation analysis validated that decreased IRF6 expression was caused by DNA hypermethylation. Furthermore, our results showed that IRF6 expression was associated with the infiltration levels of multiple immune cells in ccRCC. Conclusions: These findings demonstrated that IRF6 expression was significantly reduced in ccRCC and DNA hypermethylation played an important role in decreased IRF6 expression. In addition, the decrease of IRF6 was related to the unfavorable prognosis of ccRCC patients and the alterations of tumor immune cells infiltration.

Keywords: DNA hypermethylation; IRF6; ccRCC; immune cells infiltration; prognosis.

Figure 1

IRF6 expression was downregulated in ccRCC based on GEO datasets. (A) IRF6 expression was significantly decreased in ccRCC than that in adjacent normal renal tissue. (B-E) Decreased IRF6 expression was associated with higher histological grade, advanced tumor stage, higher pathological stage and distant metastasis. ****p < 0.0001.

Figure 2

Decreased IRF6 expression was associated with worse pathological features and poor prognosis based on TCGA-KIRC data. (A) IRF6 expression was significantly decreased in ccRCC. (B) Decreased IRF6 expression was associated with higher histological grade, advanced tumor stage, higher pathological stage, lymph node invasion and distant metastasis. (C) IRF6 low expression group have shorter OS and DFS compared to IRF6 high expression group. Patients were separated into two groups according to the median cutoff of IRF6 expression. ****p < 0.0001.

Figure 3

Validation of the expression pattern and prognostic value of IRF6 in ccRCC. (A) Comparison of IRF6 protein expression in 50 ccRCC tissues and 20 matched adjacent normal renal tissue using immunohistochemistry staining. (B) Decreased IRF6 expression was associated with higher histological grade, advanced tumor stage, lymph node invasion and distant metastasis. (C) IRF6 low expression group have shorter OS and DFS compared to IRF6 high expression group. Patients were separated into two groups according to the median cutoff of IRF6 expression. *p <0.05, **p <0.01, ****p < 0.0001.

Figure 4

Comparison of IRF6 DNA methylation status in ccRCC and adjacent normal renal tissues. (A) Heatmap and (B) statistical comparison of the difference in methylation levels of 16 CpG sites of IRF6 DNA. ***p<0.001, ****p < 0.0001.

Figure 5

The prognostic role of the CpG sites of IRF6 DNA in ccRCC. (A) High methylation level groups of cg12034118 and cg16030177 had shorter OS than their related low methylation level groups. (B) High methylation level groups of cg00989853, cg10074409, cg16030177, cg21951975 and cg23283495 had shorter DFS than their related low methylation level groups. Patients were separated into two groups according to the median cutoff of the methylation levels of these CpG sites.

Figure 6

IRF6 expression was regulated by DNA methylation in ccRCC. (A) IRF6 expression was negatively correlated with the methylation levels of cg12034118, cg16030177, cg00989853, cg10074409, cg21951975 and cg23283495. (B) IRF6 expression was decreased in ccRCC cell lines. (C) IRF6 is hypermethylated in ccRCC cell lines compared to HK2 cell line. (D) DNA methylation level of IRF6 in OSRC2 and Caki-1 cells were significantly decreased after exposed to demethylating agents. (E) IRF6 expression in OSRC2 and Caki-1 cells was remarkably increased after exposed to demethylating agents. M = Methylated, U = Unmethylated.

Figure 7

IRF6 expression was associated with immune cells infiltration in ccRCC. (A) IRF6 expression was significantly positively correlated with the infiltration levels of CD4 Naïve, Th cell and pDC, and negatively correlated with the infiltration levels of CD4 Memory, CD8 Effector, DC and NK cells. (B) IRF6 expression was negatively correlated with CD4, CD45, ITGAX, CD40, CD80 and CD86 expression, and positively correlated with CD29 expression.

Figure 8

The prognostic value of IRF6-related immune cells in ccRCC. (A) Lower CD4 Naïve infiltration level and higher CD4 Memory and DC infiltration levels were related to shorter OS. (B) Lower CD4 Naïve and pDC infiltration levels and higher CD4 Memory and DC infiltration levels were related to shorter DFS.

Figure 9

MAPK, ERBB, ERK and CREB pathways were significantly activated in the IRF6 high expression group.