Comprehensive Analysis of the Expression and Prognosis Value of Chromobox Family Members in Clear Cell Renal Cell Carcinoma

Abstract

Clear cell renal cell carcinoma (ccRCC) accounts for 80% of all renal cancers and has a poor prognosis. Chromobox (CBX) family protein expression has been reported in a variety of human malignancies, but the roles of CBXs in ccRCC remain unclear. In this study, by using ONCOMINE, UALCAN, GEPIA, Kaplan-Meier Plotter, cBioPortal, and TIMER, we found the transcriptional levels of CBX3 and CBX4 in ccRCC tissues were significantly higher than those in normal kidney tissues, whereas the transcriptional levels of CBX1, CBX5, CBX6, and CBX7 were significantly reduced in ccRCC tissues. The promoters of CBX2, CBX3, CBX4, CBX5, CBX6, CBX7, and CBX8 were hypermethylated, whereas the CBX1 promoter was hypomethylated in ccRCC. The expression of CBX1, CBX3, CBX4, CBX5, CBX6, and CBX7 was significantly associated with clinicopathological parameters in ccRCC patients. ccRCC patients with high expression levels of CBX3, CBX4, and CBX8 and low expression levels of CBX1, CBX5, CBX6, and CBX7 showed a strong association with poor overall survival. Genetic alterations in CBXs were correlated with poor overall survival and disease-free survival in patients with ccRCC. Moreover, we found significant associations between the expression of CBXs and infiltration of immune cells (B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells). Our results provide novel insights into the development of CBX-based biomarkers and therapeutic targets for ccRCC.

Keywords: bioinformatics analysis; biomarker; chromobox; clear cell renal cell carcinoma; prognosis.

Figure 1

The transcription levels of CBXs in ccRCC (UALCAN). The transcription levels of CBX3 and CBX4 in ccRCC tissues were significantly elevated compared with normal kidney tissues (C, D), while the transcriptional levels of CBX1, CBX5, CBX6, CBX7 were significantly reduced in ccRCC tissues (A, E–G). The CBX2 and CBX8 were expressed at similar levels in ccRCC tissues and normal kidney tissues (B, H). (*p<0.05, ***p<0.001, ns: no significance).

Figure 2

The mRNA levels of CBXs in normal kidney cell and ccRCC cells (qRT-PCR). The mRNA levels of CBX3 and CBX4 in ccRCC cells were significantly elevated compared with normal kidney cells (B, C), while the mRNA levels of CBX1, CBX5, CBX6, CBX7 were significantly reduced in ccRCC cells (A, D–F). (*p<0.05, **p<0.01).

Figure 3

The protein expression levels of CBX3, CBX6 and CBX7 in normal kidney cell and ccRCC cells (Western blotting). CBX3 protein was much higher in ccRCC cell lines than that in HK2 cells, while CBX6 and CBX7 were significantly reduced in ccRCC cells. The representative western blotting image of CBX3, CBX6 and CBX7 in ccRCC cells and normal kidney cells (A). The statistical results of western blotting of CBX3, CBX6 and CBX7 in ccRCC cells and normal kidney cells (B–D). (*P<0.05).

Figure 4

The protein expression of CBXs in normal kidney tissues and ccRCC tissues (Human Protein Atlas). The protein expression of CBX3 and CBX4 in ccRCC tissues were significantly elevated compared with normal kidney tissues (C, D), while the protein expression of CBX1,CBX5, CBX6, CBX7 were significantly reduced in ccRCC tissues (A, E–G). The CBX2 and CBX8 were expressed at similar levels in ccRCC tissues and normal kidney tissues (B, H).

Figure 5

The protein expression levels of CBX3, CBX6 and CBX7 in ccRCC tissues and paired adjacent normal kidney tissues (Immunohistochemistry). CBX3 was overexpressed in ccRCC tissues compared to normal kidney tissues, while expression levels of CBX6 and CBX7 were reduced in ccRCC tissues. The representative immunohistochemistry image of CBX3, CBX6 and CBX7 in ccRCC tissues and normal kidney tissues (A, C, E). The scatter plot of immunohistochemistry score (B, D, F). (*P<0.05, **P<0.01, ***P<0.001).

Figure 6

The relative expression levels of CBXs in ccRCC (GEPIA).

Figure 7

Promoter methylation status of CBXs in ccRCC (UALCAN). The promoters of CBX2, CBX3, CBX4, CBX5, CBX6, CBX7 and CBX8 were hypermethylated in ccRCC tissues (B–H), while CBX1 promoter was hypomethylated in ccRCC tissues than that in normal kidney tissues (A). (***P<0.001).

Figure 8

Association of mRNA expression of CBXs with tumor grades of ccRCC patients (UALCAN). ccRCC patients who were in more advanced tumor grades tended to express higher mRNA expression of CBX3 and CBX4 (C, D), while lower mRNA expression of CBX1, CBX5, CBX6 and CBX7 (A, E–G). CBX2 and CBX8 did not significantly differ in tumor grades (B, H). (*P<0.05, **P<0.01, ***P<0.001, ns: no significance).

Figure 9

Correlation between CBXs expression and tumor stages in ccRCC patients (UALCAN). Low expression of CBX1, CBX5, CBX6, CBX7 (A, E–G), and high expression of CBX3, CBX4 (C, D) were significantly correlated with advanced cancer stages. CBX2 and CBX8 did not significantly differ in tumor stages (B, H). (*P<0.05, **P<0.01, ***P<0.001, ns, no significance).

Figure 10

The prognostic value of CBXs in ccRCC patients in the OS curve (Kaplan-Meier Plotter). ccRCC patients with high expression levels of CBX3, CBX4, CBX8 (C, D, H), and low expression levels of CBX1, CBX5, CBX6, CBX7 (A, E–G) were strongly associated with poor overall survival. However, CBX2 expression showed no correlation with overall survival in ccRCC patients (B).

Figure 11

The prognostic value of different expressed CBXs in ccRCC patients in the DFS curve (GEPIA). Low expression levels of CBX1 and CBX7 were significantly correlated with poor disease-free survival in ccRCC patients (A, G). CBX2, CBX3, CBX4, CBX5, CBX6 and CBX8 expression showed no correlation with disease free survival in ccRCC patients (B–F, H).

Figure 12

Genetic alteration in CBXs and their relationship with OS and DFS of ccRCC patients (cBioPortal). The genetic alterations rates of CBX1, CBX2, CBX3, CBX4, CBX5, CBX6, CBX7 and CBX8 (A, B). Correlation of CBXs gene alterations with OS and DFS in patients with ccRCC (C, D).

Figure 13

The correlation between differentially expressed CBXs and immune cells infiltration (TIMER). CBX1, CBX2, CBX4, CBX5, CBX6 and CBX7 expression was positively correlated with the infiltration of B cell, CD8+ T cells, CD4+ T cells, macrophage, neutrophils and dendritic cells (A, B, D–G). And there was a positive correlation between CBX3 expression and the infiltration of B cell, CD8+ T cells, macrophage, neutrophils and dendritic cells (C). The expression of CBX8 was positively associated with the infiltration of CD4+ T cells, and negatively associated with the infiltration of CD8+ T cells (H).