3.1 The mRNA expression levels of E2Fs in CRC
To compare E2F expression in tumor and normal tissues, we extracted from the Oncomine, TIMER, and GEPIA databases the E2F mRNA levels in multiple cancer types. In one or more of the datasets, the mRNA expression levels of E2F1, E2F3, E2F4, E2F5, E2F6, E2F7, and E2F8 were significantly upregulated in CRC patients. The mRNA expression levels of E2F2 were significantly downregulated in CRC patients based on the Oncomine and TIMER datasets. See summary in Figure 1, Figure 2A, and Figure 2B.
3.2 The IHC results of E2Fs in CRC
We obtained the IHC results of E2Fs in human colon tissues. In parallel with the increased mRNA expression levels as revealed in the Oncomine, TIMER, and GEPIA datasets, the IHC expression levels of E2F1, 3, 4, 5, 6, 7 and 8 were significantly higher in carcinoma tissues (Figure 3).
Positive correlations between E2F expression levels were the following (Figure 4):
E2F1 with E2F2-E2F8
E2F2 with E2F3, E2F4, E2F7, and E2F8
E2F3 with E2F4, E2F5, E2F6, E2F7, and E2F8
E2F4 with E2F5, E2F6, E2F7, and E2F8
E2F5 with E2F6, E2F7, and E2F8
E2F6 with E2F7 and E2F8
E2F7 with E2F8.
E2F2 had a significant negative correlation with E2F5.
3.3 Prognosis values of E2Fs in CRC
The Kaplan-Meier curve and log rank test GEIPA analyses of E2Fs in CRC revealed that increased expression of E2F3 and E2F4 was associated with poor overall survival (OS) (p < 0.05) [Figure 5]. The prognosis values of E2Fs in CRC determined by PrognoScan revealed that increased expression of E2F1, E2F2, and E2F7 was associated with improved Disease free survival (DFS), OS, and Disease specific survival (DSS) (p < 0.05) [Table Ⅰ].
Table 1 The relationships between E2Fs expression and prognosis of CRC in PrognoScan
GENE
|
DATASET
|
ENDPOINT
|
N
|
P-VALUE
|
HR [95% CI]
|
E2F1
|
GSE17536
|
Disease Free Survival
|
145
|
0.012882
|
0.05 [0.01 - 0.49]
|
|
GSE17536
|
Overall Survival
|
177
|
0.014295
|
0.12 [0.02 – 0.66]
|
|
GSE17536
|
Disease Specific Survival
|
177
|
0.024171
|
0.24 [0.07 – 0.83]
|
E2F2
|
GSE17536
|
Disease Free Survival
|
145
|
0.030286
|
0.23 [0.06 – 0.87]
|
|
GSE17537
|
Disease Free Survival
|
55
|
0.00569
|
0.00 [0.00 – 0.08]
|
|
GSE17537
|
Overall Survival
|
55
|
0.011883
|
0.02 [0.00 – 0.43]
|
|
GSE17537
|
Disease Specific Survival
|
49
|
0.039668
|
0.02 [0.00 – 0.83]
|
E2F7
|
GSE14333
|
Disease Free Survival
|
226
|
0.038041
|
0.67 [0.45 – 0.98]
|
|
GSE17536
|
Overall Survival
|
177
|
0.044223
|
0.29 [0.08 – 0.97]
|
|
GSE17537
|
Disease Specific Survival
|
49
|
0.000754
|
0.00 [0.00 – 0.03]
|
|
GSE17537
|
Disease Free Survival
|
55
|
0.015361
|
0.01 [0.00 – 0.42]
|
|
GSE17537
|
Overall Survival
|
55
|
0.021599
|
0.02 [0.00 – 0.55]
|
3.4 The correlations of the E2Fs expression and clinicopathological parameter
The correlations of E2Fs with tumor stage indicated that expression of the E2F1, E2F3, and E2F5 group varied as a function of tumor stage; however, expression of the E2F2, E2F4, E2F6, E2F7, and E2F8 groups did not vary with tumor stage [Figure 6 A]. Further, we did not find any significant differences in expression of the E2Fs between the non-metastasis and metastasis groups [Figure 6 B]. E2F4 expression was higher in the male group compared with the female group [Figure 6 C]. E2F8 expression was higher in the mucinous adenocarcinoma group compared with the adenocarcinoma group [Figure 6 D].
3.5 Alterations of E2Fs in CRC
E2Fs expression levels were varied in 175 of 524 patients with CRC (33 %), and the alterations of E2Fs in mucinous adenocarcinoma were more frequent than alterations in the adenocarcinomas. In addition, E2F1 had the highest mutation frequency with amplification [Figure 7]. The network constructed by E2Fs and the 69 most frequently altered co-expressed genes showed that cell cycle-related genes were closely associated with E2Fs alterations [Figure 8].
3.6 GO functional annotation and KEGG pathway enrichment analyses
GO analysis of the functions of E2Fs and co-expressed genes showed that the major E2F functions were regulation of cell cycle, cell division, cell proliferation, DNA replication, and DNA repair [Tables Ⅱ, Ⅲ, and Ⅳ]. KEGG pathway enrichment analysis showed Cell Cycle to be the most enriched pathway [Figure 9].
Table 2 The GO function annotation (BP) of E2Fs and genes significantly associated with E2Fs alterations of CRC analyzed by DAVID
ID
|
DESCRIPTION
|
GENES
|
P-VALUE
|
GO:0051726
|
regulation of cell cycle
|
CCL7, CCL8, CXCL11
|
1.18E-05
|
GO:0006260
|
DNA replication
|
E2F2, E2F5, DTL, FOXM1, CCNF, PKMYT1, MYBL2
|
4.17E-05
|
GO:0051301
|
cell division
|
RECQL4, DTL, RRM1, BRIP1, MCM10, ORC1, CDK2
|
9.08E-05
|
GO:0006977
|
DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest
|
KIF2C, CDCA8, NCAPH, SGO1, CCNF, KATNB1, BUB1B, UBE2C, CDK2
|
1.22E-04
|
GO:0007067
|
mitotic nuclear division
|
KIF2C, SGO1, CCNF, KIF15, PKMYT1, BUB1B, CDK2
|
5.38E-04
|
GO:0007062
|
sister chromatid cohesion
|
KIF2C, CDCA8, SGO1, KIF18A, BUB1B
|
8.50E-04
|
GO:0060707
|
trophoblast giant cell differentiation
|
PLK4, E2F7, E2F8
|
0.001069
|
GO:0000086
|
G2/M transition of mitotic cell cycle
|
PLK4, FOXM1, PKMYT1, MELK, CDK2
|
0.00243234563684612
|
GO:0008283
|
cell proliferation
|
XRCC5, KIF2C, E2F8, KIF15, BUB1B, MCM10, MELK
|
0.00388806554484318
|
GO:0000083
|
regulation of transcription involved in G1/S transition of mitotic cell cycle
|
E2F4, E2F6, ORC1
|
0.0039817619547411
|
GO:0007018
|
microtubule-based movement
|
KIF23, KIF2C, KIF15, KIF18A
|
0.004506
|
GO:0006890
|
retrograde vesicle-mediated transport, Golgi to ER
|
KIF23, KIF2C, KIF15, KIF18A
|
0.004664
|
GO:0019886
|
antigen processing and presentation of exogenous peptide antigen via MHC class II
|
KIF23, KIF2C, KIF15, KIF18A
|
0.006424
|
GO:0032877
|
positive regulation of DNA endoreduplication
|
E2F7, E2F8
|
0.008202
|
GO:0000082
|
G1/S transition of mitotic cell cycle
|
PKMYT1, MCM10, ORC1, CDK2
|
0.00853075482116703
|
GO:0001890
|
placenta development
|
E2F7, E2F8, CCNF
|
0.009072
|
GO:0007080
|
mitotic metaphase plate congression
|
KIF2C, CDCA8, KIF18A
|
0.010101
|
GO:0007049
|
cell cycle
|
E2F2, E2F3, FOXM1, SUV39H1, CCAR1
|
0.0121896039954393
|
GO:0071930
|
negative regulation of transcription involved in G1/S transition of mitotic cell cycle
|
E2F1, E2F7
|
0.012277
|
GO:0000398
|
mRNA splicing, via spliceosome
|
RALY, PRPF4B, RNPS1, CPSF3, CCAR1
|
0.013159
|
GO:0032508
|
DNA duplex unwinding
|
XRCC5, RECQL4, BRIP1
|
0.014085
|
GO:0006281
|
DNA repair
|
RECQL4, FANCI, FOXM1, RAD54L, CDK2
|
0.015907
|
GO:1990086
|
lens fiber cell apoptotic process
|
E2F1, E2F2
|
0.016337
|
GO:0070345
|
negative regulation of fat cell proliferation
|
E2F1, E2F3
|
0.02038
|
GO:0060718
|
chorionic trophoblast cell differentiation
|
E2F7, E2F8
|
0.02038
|
GO:0006351
|
transcription, DNA-templated
|
E2F1, XRCC5, RALY, UTP4, E2F3, E2F4, E2F5, E2F6, FOXM1, E2F7, E2F8, SUV39H1, RNPS1, ZBTB24, CCAR1
|
0.026128
|
GO:0032466
|
negative regulation of cytokinesis
|
E2F7, E2F8
|
0.028417
|
GO:0006302
|
double-strand break repair
|
XRCC5, RECQL4, BRIP1
|
0.030147
|
GO:0051436
|
negative regulation of ubiquitin-protein ligase activity involved in mitotic cell cycle
|
BUB1B, UBE2C, CDK2
|
0.034473
|
GO:0000733
|
DNA strand renaturation
|
RECQL4, RAD54L
|
0.036388
|
GO:0007019
|
microtubule depolymerization
|
KIF2C, KIF18A
|
0.04035
|
GO:0070365
|
hepatocyte differentiation
|
E2F7, E2F8
|
0.04035
|
GO:0006310
|
DNA recombination
|
XRCC5, RECQL4, RAD54L
|
0.045755
|
GO:0006396
|
RNA processing
|
PNPT1, U2SURP, SSB
|
0.06037
|
GO:0006406
|
mRNA export from nucleus
|
NUP153, RNPS1, CPSF3
|
0.063686
|
GO:0007099
|
centriole replication
|
PLK4, CDK2
|
0.063785
|
GO:0000122
|
negative regulation of transcription from RNA polymerase II promoter
|
E2F1, E2F6, E2F7, FOXM1, E2F8, SUV39H1, DNAJA3
|
0.074986
|
GO:0051276
|
chromosome organization
|
CDCA8, RAD54L
|
0.075291
|
GO:0016925
|
protein sumoylation
|
NUP153, CDCA8, SENP6
|
0.083562
|
GO:0051439
|
regulation of ubiquitin-protein ligase activity involved in mitotic cell cycle
|
UBE2C, CDK2
|
0.090414
|
GO:0071398
|
cellular response to fatty acid
|
XRCC5, E2F1
|
0.090414
|
GO:0007126
|
meiotic nuclear division
|
RAD54L, CDK2
|
0.094157
|
GO:0002040
|
sprouting angiogenesis
|
E2F7, E2F8
|
0.097885
|
Table 3 The GO function annotation (CC) of E2Fs and genes significantly associated with E2Fs alterations of CRC analyzed by DAVID
ID
|
DESCRIPTION
|
GENES
|
P-VALUE
|
GO:0005654
|
nucleoplasm
|
E2F1, XRCC5, KIF23, GPN1, E2F2, UTP4, E2F3, E2F4, PRPF4B, E2F5, E2F7, FOXM1, U2SURP, PKMYT1, COPS8, MYBL2, MCM10, GTSE1, SENP6, CDCA8, FANCI, EIF3E, ORC1, CCAR1, NUP153, DTL, SUV39H1, BRIP1, RNPS1, UBE2C, RAD54L, CDK2, ADRM1, SGO1, RRM1, NUTF2, CPSF3, WDR43
|
4.96E-13
|
GO:0005634
|
nucleus
|
E2F1, RALY, XRCC5, KIF23, E2F3, E2F4, E2F5, E2F6, E2F7, FOXM1, E2F8, U2SURP, KATNB1, COPS8, MCM10, KIF2C, CDCA8, NCAPH, NUDCD1, EIF3E, ORC1, DNAJA3, RECQL4, SPATA33, DTL, CCNF, SUV39H1, KIF18A, BRIP1, SSB, RNPS1, RAD54L, CDK2, ZBTB24, ARMC1, EMC8, ADRM1, SGO1, CPNE3, MELK, ATAD2B
|
2.61E-06
|
GO:0005667
|
transcription factor complex
|
E2F2, E2F3, E2F4, E2F5, E2F6, E2F7, E2F8, CDK2
|
1.01E-05
|
GO:0000776
|
kinetochore
|
KIF2C, SGO1, KIF18A, BUB1B, WDR43
|
2.81E-04
|
GO:0005813
|
centrosome
|
KIF23, PLK4, DTL, SGO1, KIF15, KATNB1, WDR43, CDK2
|
0.001332
|
GO:0000775
|
chromosome, centromeric region
|
KIF2C, CDCA8, SGO1, SUV39H1
|
0.001426
|
GO:0005730
|
nucleolus
|
XRCC5, UTP23, UTP4, NUP153, PLK4, CDCA8, E2F5, DTL, MCM10, WDR43, ORC1
|
0.001716
|
GO:0005829
|
cytosol
|
SPATA33, KIF23, XRCC5, KIF15, KIF18A, PKMYT1, RNPS1, UBE2C, GTSE1, CDK2, KIF2C, NCAPH, PLK4, CDCA8, SGO1, EIF3E, RRM1, CTU2, BUB1B, CPNE3, NUTF2, ARHGAP11A, ORC1, DNAJA3
|
0.002805
|
GO:0005737
|
cytoplasm
|
GPN1, E2F3, E2F5, FOXM1, PNPT1, KATNB1, COPS8, MCM10, ACP1, SENP6, NUDCD1, FANCI, EIF3E, CTU2, ORC1, DNAJA3, RECQL4, NUP153, DTL, KIF18A, BRIP1, RNPS1, UBE2C, CDK2, ARMC1, EMC8, ADRM1, SGO1, RRM1, BUB1B, CPNE3, SHCBP1
|
0.004871
|
GO:0005874
|
microtubule
|
KIF23, KIF2C, KIF15, KIF18A, KATNB1, WDR43
|
0.007286
|
GO:0005694
|
chromosome
|
RECQL4, UTP4, PRPF4B, DTL
|
0.007999
|
GO:0030496
|
midbody
|
KIF23, CDCA8, KATNB1, SHCBP1
|
0.013959
|
GO:0005871
|
kinesin complex
|
KIF23, KIF2C, KIF18A
|
0.018145
|
GO:0001650
|
fibrillar center
|
UTP4, WDR43
|
0.023152
|
GO:0016020
|
membrane
|
XRCC5, RECQL4, PNPT1, KIF15, KATNB1, PKMYT1, GTSE1, ADRM1, KIF2C, EMC8, NCAPH, FANCI, EIF3E, MELK, TMEM30A
|
0.04265
|
GO:0000777
|
condensed chromosome kinetochore
|
KIF2C, SGO1, BUB1B
|
0.045277
|
GO:0044613
|
nuclear pore central transport channel
|
NUP153, NUTF2
|
0.049496
|
GO:0031965
|
nuclear membrane
|
NUP153, DTL, BRIP1, NUTF2
|
0.060074
|
GO:0051233
|
spindle midzone
|
CDCA8, BUB1B
|
0.071518
|
GO:0005739
|
mitochondrion
|
E2F1, GPN1, EMC8, SLC25A32, BRI3BP, PNPT1, RPUSD1, CTU2, DNAJA3, ARMC1
|
0.072749
|
GO:0005819
|
spindle
|
KIF23, KIF15, SHCBP1
|
0.080833
|
GO:0005680
|
anaphase-promoting complex
|
BUB1B, UBE2C
|
0.085919
|
GO:0000784
|
nuclear chromosome, telomeric region
|
XRCC5, SSB, ORC1
|
0.091333
|
GO:0015630
|
microtubule cytoskeleton
|
KIF2C, KIF18A, KATNB1
|
0.099761
|
Table 4 The GO function annotation (MF) of E2Fs and genes significantly associated with E2Fs alterations of CRC analyzed by DAVID
ID
|
DESCRIPTION
|
GENES
|
P-VALUE
|
GO:0005515
|
protein binding
|
RALY, KIF23, XRCC5, E2F1, E2F2, E2F3, E2F4, PRPF4B, E2F5, E2F6, E2F7, E2F8, U2SURP, PKMYT1, COPS8, MCM10, UHRF1BP1, GTSE1, SENP6, KIF2C, CDCA8, NUDCD1, FANCI, CTU2, ORC1, CCAR1, DTL, KIF15, CCNF, UBE2C, CDK2, ZBTB24, EMC8, ADRM1, SGO1, RRM1, BUB1B, CPSF3, WDR43, MELK, SHCBP1, GPN1, UTP4, FOXM1, PNPT1, MYBL2, ACP1, NCAPH, EIF3E, DNAJA3, TMEM30A, RECQL4, UTP23, NUP153, VAC14, SUV39H1, KIF18A, BRIP1, SSB, RNPS1, RAD54L, ARMC1, PLK4, NUTF2, CPNE3
|
2.49E-11
|
GO:0001047
|
core promoter binding
|
E2F1, E2F2, E2F3, E2F7, E2F8, CCAR1
|
7.69E-06
|
GO:0005524
|
ATP binding
|
RECQL4, KIF23, XRCC5, PRPF4B, KIF15, KIF18A, PKMYT1, BRIP1, UBE2C, RAD54L, CDK2, KIF2C, PLK4, RRM1, BUB1B, ORC1, MELK, DNAJA3, ATAD2B
|
4.06E-05
|
GO:0046983
|
protein dimerization activity
|
E2F1, E2F2, E2F3, E2F4, E2F5, E2F6
|
4.49E-04
|
GO:0044822
|
poly(A) RNA binding
|
XRCC5, RALY, UTP23, UTP4, PRPF4B, PNPT1, U2SURP, SSB, RNPS1, EIF3E, CPNE3, WDR43, CCAR1
|
0.002773
|
GO:0003677
|
DNA binding
|
XRCC5, E2F1, E2F2, NUP153, E2F3, E2F4, E2F5, E2F6, FOXM1, E2F7, KIF15, BRIP1, RAD54L, ZBTB24, FANCI, ORC1
|
0.004163
|
GO:0003777
|
microtubule motor activity
|
KIF23, KIF2C, KIF15, KIF18A
|
0.004833
|
GO:0004674
|
protein serine/threonine kinase activity
|
PLK4, PRPF4B, PKMYT1, BUB1B, CPNE3, MELK, CDK2
|
0.005327
|
GO:0003723
|
RNA binding
|
RALY, PNPT1, RPUSD1, U2SURP, SSB, RNPS1, THUMPD2, CPSF3
|
0.008637
|
GO:0008017
|
microtubule binding
|
KIF23, KIF15, KIF18A, KATNB1, WDR43
|
0.012002
|
GO:0000049
|
tRNA binding
|
CTU2, SSB, THUMPD2
|
0.019992
|
GO:0003700
|
transcription factor activity, sequence-specific DNA binding
|
E2F1, E2F2, E2F3, E2F4, E2F5, E2F6, E2F7, FOXM1, E2F8, MYBL2
|
0.020575
|
GO:0008134
|
transcription factor binding
|
E2F1, E2F2, E2F4, E2F5, DNAJA3
|
0.033089
|
GO:0036310
|
annealing helicase activity
|
RECQL4, RAD54L
|
0.033623
|
GO:0016887
|
ATPase activity
|
KIF23, KIF2C, KIF15, ATAD2B
|
0.043371
|
GO:0051010
|
microtubule plus-end binding
|
KIF2C, KIF18A
|
0.045942
|
GO:0003714
|
transcription corepressor activity
|
E2F6, E2F7, E2F8, CCAR1
|
0.05588
|
GO:0004672
|
protein kinase activity
|
PRPF4B, PKMYT1, BUB1B, MELK, CDK2
|
0.06716
|
GO:0045502
|
dynein binding
|
KATNB1, WDR43
|
0.070118
|
3.7 The mRNA‑target of E2F2, E2F3, and E2F4
The E2F2, E2F3, and E2F4 mRNAs were identified as‑targets of miRNAs (Figure 10) according to the standards described in Methods (Figure 9 and 10).
3.8 Differential miRNA expression between CRC tissues and corresponding normal tissues
To assess the potential targeting of miRNAs to E2F2, E2F3, and E2F4, we compared the miRNAs between CRC tissues and corresponding normal tissues in the GEO database (GSE 115513). We obtained 148 miRNAs, 90 upregulated and 58 downregulated, between CRC tissues and corresponding normal tissues. The miRNAs to potentially target E2F2 included hsa-miR-17-5p, hsa-miR-93-5p, hsa-miR-20b-5p, hsa-miR-106b-5p, and hsa-miR-20a-5p. The results also showed that E2F3 was a target of hsa-miR-10b-5p and hsa-miR-497-5p. There were no miRNAs to target E2F4 [Table Ⅴ].
Table 5 The miRNA-target expression of E2F2/E2F3 between CRC tissues and corresponding normal tissues in GSE115513
GENE
|
miRNA
|
adj. P-VALUE
|
logFC
|
CANCER
|
NORMAL
|
E2F2
|
has-miR-17-5p
|
6.92E-148
|
1.547963
|
750
|
654
|
|
hsa-miR-93-5p
|
2.02E-101
|
1.169552
|
750
|
654
|
|
hsa-miR-20b-5p
|
1.32E-93
|
1.465394
|
750
|
654
|
|
hsa-miR-106b-5p
|
8.20E-58
|
1.024036
|
750
|
654
|
|
hsa-miR-20a-5p
|
4.93E-131
|
1.605937
|
750
|
654
|
E2F3
|
hsa-miR-10b-5p
|
2.19E-20
|
-0.59542
|
750
|
654
|
|
hsa-miR-497-5p
|
1.30E-26
|
-0.84106
|
750
|
654
|