Identification of the Expression and Clinical Significance of E2F Family in Clear Cell Renal Cell Carcinoma

Abstract

Background: Multiple studies have identified that E2F transcriptions act as important regulators for the tumorigenesis and progression of several human cancers. However, little is known about the function of E2Fs in clear cell renal cell carcinoma (ccRCC).

Methods: We firstly investigated the expression levels, genetic alteration, and biological function of E2Fs in patients with ccRCC and the connections between the immune cell infiltration and the overall survivals of ccRCC patients with the E2Fs expression levels based on UALCAN, The Cancer Genome Atlas database, Gene Expression Profiling Interactive Analysis, TIMER, STRING, GSCALite and cBioPortal databases.

Results: Results revealed that the expression levels of E2F1/2/3/4/6/7/8 were markedly upregulated in patients with ccRCC, while the expression of E2F5 displayed an opposite trend. We also experimentally validated the overexpression of E2F3/4/7 in human ccRCC tissues and ccRCC cell lines. Furthermore, the high E2F1/2/3/4/7/8 expression levels were clearly associated with worse pathological characteristics of ccRCC, including high pathological stage, poor molecular subtypes and high tumor grade. Meanwhile, high expression levels of E2F1/2/4/7/8 were evidently associated with worse overall survivals (OSs) and progression-free survivals (PFSs) of patients harboring ccRCC. Univariate and multivariate analyses illustrated that the expressions of E2F4/5/7 were independent factors associated with the OSs and PFSs of patients with ccRCC. Meanwhile, the mutations in E2Fs were also significantly related to poor OSs and PFSs of patients with ccRCC. Mechanically, the E2Fs genes synergistically promoted the progression of ccRCC by accelerating the cell cycle and inhibiting DNA damage response and apoptosis after performing the protein structure, functional enrichment, and PPI network analyses. In addition, E2Fs genes were also significantly associated with tumor immune cells infiltration and the drug sensitivity in ccRCC.

Conclusion: As a result, E2F4/7 were highly expressed in ccRCC and significantly associated with worse pathological characteristics of ccRCC, including high pathological stage, poor molecular subtypes and high tumor grade, tumor immune cell infiltration, and drug sensitivity, consequently translating into poor OSs and PFSs of patients with ccRCC. Our results indicated that E2F4/7 could be potential biomarkers and therapeutic targets of ccRCC patients.

Keywords: E2Fs; GEPIA; UALCAN; clear cell renal cell carcinoma; prognosis.

Figure 1

Transcriptional expression of E2Fs in 72 pairs of kidney cancer tissues and adjacent normal kidney tissues (TCGA and GTEx database) (A). mRNA expression of distinct E2Fs members in ccRCC tissues and normal kidney tissues (UALCAN) (B–I). ns indicates not significant; *p < 0.05; **p < 0.01; ***p <0.001.

Figure 2

qRT-PCR were used to detect the mRNA levels of E2F1 (A), E2F2 (B), E2F3 (C), E2F4 (D), E2F5 (E), E2F6 (F), E2F7 (G), E2F8 (H) in ccRCC tissues and paired-adjacent normal kidney tissues. Comparison of the mRNA levels of E2Fs in ccRCC cell lines between normal cell lines. The mRNA levels of E2F1 (I), E2F3 (J), E2F4 (K), E2F5 (L), and E2F7 (M) in kidney cancer and normal cell lines were estimated with qRT-PCR. ns indicates not significant; *p < 0.05; **p < 0.01; *** p < 0.001.

Figure 3

Correlations between E2Fs’ mRNA level and clinical stages in ccRCC patients by GEPIA database were described with violin plots. The mRNA expressions of E2F1/2/7 were significantly related to patients’ pathological stages (A–G), while mRNA expressions of E2F3/4/5/6/8 were not associated with patients’ pathological stages (C–H).

Figure 4

The mRNA levels of E2F1/2/3/4/5/6/7/8 in ccA and ccB subtypes of KIRC (A–H). ns indicates not significant; **p < 0.01; ***p < 0.001.

Figure 5

Associations of E2F1/2/3/4/5/6/7/8 expressions with tumor grades of ccRCC were presented in (A–H), respectively. ns indicates not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 6

The overall survivals and progression-free survivals of ccRCC patients with high or low E2F1/2/3/4/5/6/7/8 expression were presented in (A and B), respectively.

Figure 7

The genetic alteration of E2Fs in ccRCC (A) Alteration frequency of E2Fs based on the cBioProtal database (B) Kaplan–Meier plots revealed the overall survivals and progression-free survivals of ccRCC patients with or without E2Fs alterations (C and D). The network among E2Fs and miRNAs (E).

Figure 8

A network (A) and enrichment analysis (B) of E2Fs with the 50 neighboring genes related to the mutations of E2Fs in ccRCC.

Figure 9

Association between E2Fs and crucial immune checkpoint genes (A) and the comparison of tumor infiltration levels in ccRCC with different somatic copy number alterations for E2Fs (B) ns indicates not significant; p ≥ 0.05; *p < 0.05; **p<0.01; and ***p <0.001.

Figure 10

Drug resistance analysis of the E2Fs genes were completed using GSCALite.

Figure 11

The pathway enrichment analysis of eight E2Fs genes (GSEALite).