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Introduction

The E2F family plays a crucial role in the control of the cell cycle and action of tumor suppressor proteins (1). E2F1 belongs to the E2F family of transcription factors, coordinating the expression of key genes involved in cell cycle regulation and progression (2). The E2F1 gene is located on chromosome 20 q, spanning approximately 10.71 kb, and it contains 7 exons (3). Due to its pivotal and multifunctional role in cell cycle control, E2F1 is expected to be a significant player in carcinogenesis (4). Excess of E2F1 may promote proliferation, but at the same time it may also enhance apoptosis, and there are examples where overexpression or lack of E2F1 has both positive and negative effects on tumorigenesis (5). The delicate balance between growth and death appears to depend on the level of E2F1 deregulation, but also on the cell context background (2).

It is well known that genetic variants in microRNA (miRNA) binding regions can result in altered gene functions. miRNAs are capable of regulating the E2F activity, and miRNA dysregulation has been implicated in malignancy. For instance, E2F1 directly binds to the promoter of the miR-17∼92 cluster, activating its transcription, which in turn negatively modulates translation of E2F1 mRNAs by binding sites in their 3’UTR (6,7). In addition, certain studies have shown that E2F1 and E2F2 variants may play important roles in carcinogenesis (8,9). Considering that E2F1 is crucial for E2F family-dependent apoptosis (10), and the role of variants in miRNA binding sites of E2F1 remains unknown, in the present study, we tested our hypothesis that E2F1 3’UTR variants are associated with its mRNA expression by performing a bioinformatic analysis and genotype-phenotype association analysis based on the HapMap database.

The study was approved by the ethics committee of Nanjing Jinling Hospital.

Materials and methods

Bioinformatic analysis and selection of polymorphisms

The single nucleotide polymorphisms (SNPs) were identified both in the E2F1 gene region and in the coding region by the online database (http://www.ncbi.nlm.nih.gov/SNP/). The distribution of all E2F1 genotypes among the different populations was calculated. We also predicted the potential miRNA binding sites using the bioinformatic tool SNP Function Prediction (FuncPred; http://snpinfo.niehs.nih.gov/cgi-bin/snpinfo/snpfunc.cgi). Additionally, we calculated pairwise linkage disequilibrium (LD) values of all SNPs in the same gene, then selected the SNPs that were not in LD (r2<0.8), and plotted LD maps of those SNPs in E2F1 genes with the online program (http://snpinfo.niehs.nih.gov/cgi-bin/snpinfo/snpfunc.cgi).

Genotype and mRNA expression data of lymphoblastoid cell lines from HapMap database

We used additional data on E2F1 genotypes and mRNA levels available online (http://app3.titan.uio.no/biotools/help.php?app=snpexp) for the genotypephenotype association analysis (11). For the analysis of gene expression variation, we used genome-wide expression arrays (47,294 transcripts) from Epstein-Barr virus-transformed lymphoblastoid cell lines from the same 270 HapMap individuals (142 males and 128 females) (12). The genotyping data were from the HapMap phase II release 23 data set consisting of 3.96 million SNP genotypes from 270 individuals from 4 populations (13). SNPexp v1.2 was used for calculating and visualizing correlations between HapMap genotypes and gene expression levels (Norwegian PSC Research Center, Clinic for Specialized Surgery and Medicine, Oslo University Hospital Rikshospitalet, Norway). We searched for probes representing the gene E2F1 in the file ‘illumina_Human_WG-6_array_content.csv’, and identified one probe, GI_12669910-S. This was analyzed separately and stored as a separate output file with the probe name added to the filename. Running correlation analysis between SNP genotypes and expression levels for probes: GI_12669910-S (additive model assumed).

Statistical methods

We performed genotype and phenotype correlation analysis using the Chi-square test. All statistics tests were two-sided and P<0.05 was considered to indicate a statistically significance result.

Results

E2F1 3’UTR selected variants and their putative function

In total, 183 SNPs were identified in the E2F1 gene region, and 60 in the coding region (http://www.ncbi.nlm.nih.gov/SNP/). Of these, 21 SNPs were reported in the 3’ UTR, of which only five SNPs (rs3213180, rs3213182, rs3213183, rs3213177, rs117423075) had available minor allele frequency (MAF) values, as shown in Table I. Computer prediction analysis revealed the putative miRNA binding sites in the selected 5 SNPs in E2F1 3’UTR (Table I). The most extensively studied SNP of these is the C-to-G transition which includes hsa-miR-1182, hsa-miR-1183, hsa-miR-1231, hsa-miR-140-3p, hsa-miR-220c, hsa-miR-509-3-5p, hsa-miR-638, hsa-miR-760 and hsa-miR-769-3p putative binding sites (http://snpinfo.niehs.nih.gov/cgi-bin/snpinfo/snpfunc.cgi). Combined with other SNPs in the 3’UTR or promoter region, the variant rs3213180 is jointly involved in cancer susceptibility (8,9). Secondly, we used the bioinformatic tool FuncPred (http://snpinfo.niehs.nih.gov/snpfunc.htm) to identify their potential functional relevance. We calculated pairwise LD values of all SNPs in the same gene, then selected the SNPs that were not in LD (r2<0.8), and plotted LD maps of those SNPs in E2F1 genes using FuncPred. Each square number represents the pairwise r2 correlations between the relevant two SNPs. The color of each SNP spot reflects its D’ value, which changes from red to white as the D’ value decreases. The haplotype blocks were estimated with the FuncPred program. The minor allele frequency of all the above alleles was greater than 0.01. rs3213180 was the predicted tag SNP in our study, and rs3213182 and rs3213183 in E2F1 were not included in the LD plot (Fig. 1).

Figure 1 Linkage disequilibrium plot of the E2F region using SNP Function Prediction (FuncPred). Each square number represents the pairwise r2 correlations between the relevant two SNPs. The color of each SNP spot reflects its D’ value, which changes from red to white as the D’ value decreases. SNP, single nucleotide polymorphism.

Table I Selected SNPs of E2F1 3’UTR and putative miRNA binding sites.

Table I

Selected SNPs of E2F1 3’UTR and putative miRNA binding sites.

Name	Alleles	MAF	Putative miRNA binding sites
rs3213180	C>G	0.1224	hsa-miR-1182, hsa-miR-1183, hsa-miR-1231, hsa-miR-140-3p, hsa-miR-220c, hsa-miR-509-3-5p, hsa-miR-638, hsa-miR-760, hsa-miR-769-3p
rs117423075	C>T	0.0053	NA
rs3213182	T>G	0.067	NA
rs3213183	C>T	0.267	NA
rs3213177	G>T	0.0095	hsa-miR-133a, hsa-miR-133b, hsa-miR-331-3p

[i] SNP, single nucleotide polymorphism; 3’UTR, 3’ untranslated region; MAF, minor allele frequency; NA, not available.

Frequency distribution of selected E2F1 3’UTR variants among different populations

Due to the lack of frequency distribution data available for rs117423075 in the HapMap website, only the frequency distribution data of the other four SNP variants among different populations are summarized in Table II. The frequencies of the CC, CG and GG genotypes of rs3213180 among the African population were 95.0, 5.0 and 0%, respectively, compared with those of the global population of 77.0, 20.7 and 2.3%, respectively. The G alleles of rs3213180 in the European, African and global population were 5.8, 2.5 and 12.6%, respectively. Frequencies of rs3213180 genotypes have not been reported for European and Asian populations (Table II).

Table II Frequency distributions of selected variables in different populations.

Table II

Frequency distributions of selected variables in different populations.

Genotypes	European	Asian	African	Global
rs3213180
CC	-	-	0.095	0.770
CG	-	-	0.050	0.207
GG	-	-	0.000	0.023
G alleles	0.058	-	0.025	0.126
rs3213182
TT	0.841	1.000	0.968	0.920
TG	0.150	0.000	0.032	0.068
GG	0.009	0.000	0.000	0.011
G alleles	0.084	0.000	0.016	0.045
rs3213183
CC	0.478	0.430	0.467	0.511
CT	0.460	0.384	0.417	0.433
TT	0.062	0.186	0.117	0.045
T alleles	0.292	0.378	0.325	0.267
rs3213177
GG	0.929	1.000	0.984	0.841
GT	0.071	0.000	0.016	0.159
TT	0.000	0.000	0.00	0.000
T alleles	0.035	0.000	0.008	0.080

E2F1 mRNA expression by genotype in lymphoblastoid cell lines

For mRNA expression of the prohibitin gene in the lymphoblastoid cell lines, we took advantage of the available HapMap-cDNA expression database for the correlation analysis of prohibitin genotype and mRNA expression in 270 HapMap lymphoblastoid cell lines. With the exception of the one cell line with unavailable values for rs3213183, 122 (45.4%) cell lines with the CC genotype, 116 (43.1%) cell lines with the CT genotype and 31 (11.5%) cell lines with the TT genotype were identified. For rs3213180, 86 (95.6%) cell lines had the GG genotype and 4 (4.4%) had the CG genotype. There were 256 (95.5%) cell lines with TT, 11 (4.0%) with TG, and 1 (0.4%) with GG genotype for rs3213182. Fig. 2 shows the E2F1 mRNA expression levels of cell lines by E2F1 genotype. The rs3213180 GG genotype was found to have significantly lower expression levels than the CG (P=0.045; Fig. 2A), and there was no significant difference in E2F1 mRNA expression level among cell lines carrying rs3213182 (P=0.345) and rs3213183 (P=0.402) genotypes (Fig. 2B and C).

Figure 2 mRNA expression level of the genotype-phenotype association analysis of E2F1 variants (A) rs3213180, (B) rs3213182 and (C) rs3213083, and mRNA expression in Epstein-Barr virus-transformed lymphoblastoid cell lines from the HapMap database.

Discussion

The E2F family consists of transcriptional factors that play pivotal roles in regulating both cell proliferation and antiproliferative processes (2), and these transcription factors regulate the expression of a panel of cellular genes that control cellular DNA synthesis and proliferation, largely in a cell cycle-dependent manner. E2F1 is also considered to be a critical modulator of cellular senescence in human cancer (14). E2F proteins and their target genes comprise a genetic pathway that is possibly the most altered pathway in human cancer (15). Deregulated expression or activity of most members of the E2F family has been detected in numerous types of human cancer (16,17). The most common genetic alteration of the E2F1 gene is amplification, as has been reported in several types of cancer, including leukemia (e.g., the HEL human erythroleukemia cell line) (18). The gene has also been found to be amplified in various cancers, and the amplification of the E2F1 gene is linked with greater aggressiveness and poorer prognosis (17) as well as acquisition of antineoplastic agent resistance (19). Furthermore, E2F1 acting as an oncogene is also found to be amplified in a number of malignancies. It was demonstrated that several common SNPs of E2F1 are significantly associated with cancer susceptibility (8,9). One study also suggested that the E2F family proteins target miRNAs by a negative feedback loop (20). It is well-known that genetic susceptibility in miRNA binding regions contributes to altered gene function, which is also supported by some evidence in the present study. We found that rs3213180 was significantly associated with E2F1 mRNA expression, but not rs3213182 or rs3213183. Furthermore, using bioinformatic analysis, the present study indicated that both rs3213180 and rs3213177 are located in the E2F1 3’UTR and have miRNA binding sites as predicted, and E2F1 rs3213182 and rs3213183 are located in the 3’-near gene. We also observed that rs3213180 has a significant association with expression, implying that this variant could contribute in part to E2F1 post-transcriptional regulation. It is also likely that genetic variants in E2F1 3’UTR may modulate its expression and the variants in E2F1 miRNA binding site are involved in carcinogenesis. Our research may provide additional insight into the molecular mechanism of E2F1 post-transcriptional regulation. It may improve understanding of the regulatory roles of miRNA variants in E2F1 3’UTR in its mRNA expression.

In summary, our results indicated the important role of the E2F1 variant in post-transcriptional regulation. However, these findings require validation by future studies investigating their effect on E2F activity. Improved understanding of how the 3’UTR variants regulate E2F1 activity will pave the way to targeting the E2F1 pathway in cancer therapy.

Acknowledgements

This study was supported by grants from the National Natural Science Foundation of China (Nos. 81272252, 81141094, to X. Guan) and the Natural Science Foundation of Jiangsu Province (BK2011656, to X. Guan).

References