Functional exploration of CCNE1 splicing forms as a possible link to bladder cancer susceptibility

Background

A recent genome-wide association study (GWAS) identified a single nucleotide polymorphism, rs8102137, located 6 kb upstream of the cyclin E1 gene (CCNE1) on chromosome 19q12, as a risk factor for bladder cancer (odds ratio (OR) = 1.13, P = 1.7 × 10^–11) [1]. CCNE1 encodes a cell cycle protein that regulates cyclin-dependent kinases and is therefore an important cancer susceptibility gene.

Methods

This study used 42 bladder tumor samples and 41 normal bladder tissue samples (24 matched normal-tumor pairs), HeLa cells and several prostate and bladder cancer cell lines. Genotyping of rs8102137 in DNA and rs7257694 in both DNA and cDNA samples was performed using an allelic discrimination genotyping assay. TaqMan and SYBR Green assays were used to measure the expression of the different CCNE1 isoforms. The CCNE1 isoforms were cloned into a pFC14A (HaloTag) CMV Flexi Vector. Protein expression of CCNE1 isoforms in normal and tumor bladder tissues and transfected cells was analyzed by western blotting. Subcellular localization of recombinant CCNE1 splicing forms was analyzed by confocal microscopy.

Results

CCNE1 mRNA was expressed at a higher level in bladder tumors (n = 42) than in adjacent normal bladder tissue samples (n = 41, 3.7fold, P = 2.7 × 10^–12). However, no association was found between mRNA expression level and the genotype of rs8102137. We observed strong allelic expression imbalance for a synonymous coding variation located in the last exon (rs7257694, Ser390Ser), which is in high linkage disequilibrium with rs8102137 (normal bladder tissue samples, n = 41, D′ = 1.0, r² = 0.815; HapMap CEU samples, n = 60, D′ = 0.95, r² = 0.68). In normal and tumor tissue samples heterozygous for both single nucleotide polymorphisms, the risk variant of rs8102137 was associated with lower expression of allele T of rs7257694 (normal samples, P = 2.2 × 10^–4; tumor samples, P = 1.11 × 10^–10). Western blotting analysis of bladder tissue and prostate cell line lysates revealed that the allelic expression imbalance is likely to be related to two CCNE1 protein isoforms that showed a differential pattern of expression dependent on the rs8102137 and rs7257694 genotype. We have cloned the alternative splicing forms of CCNE1 and are currently evaluating their functional relevance.

Conclusions

Our results suggest that bladder-cancer-associated genetic variants of the CCNE1 gene might contribute to altered cell cycle regulation, owing to differential mRNA splicing producing different protein isoforms of CCNE1.