A 3'UTR polymorphism modulates mRNA stability of the oncogene and drug target Polo-like Kinase 1

Abstract

Background: The Polo-like Kinase 1 (PLK1) protein regulates cell cycle progression and is overexpressed in many malignant tissues. Overexpression is associated with poor prognosis in several cancer entities, whereby expression of PLK1 shows high inter-individual variability. Although PLK1 is extensively studied, not much is known about the genetic variability of the PLK1 gene. The function of PLK1 and the expression of the corresponding gene could be influenced by genomic variations. Hence, we investigated the gene for functional polymorphisms. Such polymorphisms could be useful to investigate whether PLK1 alters the risk for and the course of cancer and they could have an impact on the response to PLK1 inhibitors.

Methods: The coding region, the 5' and 3'UTRs and the regulatory regions of PLK1 were systematically sequenced. We determined the allele frequencies and genotype distributions of putatively functional SNPs in 120 Caucasians and analyzed the linkage and haplotype structure using Haploview. The functional analysis included electrophoretic mobility shift assay (EMSA) for detected variants of the silencer and promoter regions and reporter assays for a 3'UTR polymorphism.

Results: Four putatively functional polymorphisms were detected and further analyzed, one in the silencer region (rs57973275), one in the core promoter region (rs16972787), one in intron 3 (rs40076) and one polymorphism in the 3'untranslated region (3'UTR) of PLK1 (rs27770). Alleles of rs27770 display different secondary mRNA structures and showed a distinct allele-dependent difference in mRNA stability with a significantly higher reporter activity of the A allele (p < 0.01).

Conclusion: The present study provides evidence that at least one genomic variant of PLK1 has functional properties and influences expression of PLK1. This suggests polymorphisms of the PLK1 gene as an interesting target for further studies that might affect cancer risk, tumor progression as well as the response to PLK1 inhibitors.

Figure 1

The PLK1 gene locus and genetic variants. Structure of the PLK1 gene (panel A). Black boxes represent exons, the size of exons and regulatory regions are given in base pairs (bp), they are not drawn to scale. The 5′ and 3′UTR are highlighted in white. Sequenced regions are depicted by dashed lines. Positions of PLK1 polymorphisms are given according to the nomenclature of the Human Genome Variation Society [26]. Haploview plot of the linkage disequilibrium analysis of PLK1 polymorphisms in 120 healthy Caucasians (panel B). Numbers in squares are D’ values in percent. If no number is given for a pairwise comparison D’ is 100 percent. Black color indicates an r² of 1, shades of grey/black indicate an r² between 1 and 0. White indicates an r² of 0. Haplotypes of the two haplotype blocks (panel C) and across the PLK1 gene (panel D). The haplotype frequencies are shown to the right of each haplotype. Only haplotypes having a frequency > =1% are shown. Below the crossing area the multi-allelic D’, which indicates the level of recombination between the blocks, is shown. Connecting lines from one block to the other are shown for haplotypes with a frequency of at least 10% (thick lines) and with a frequency of at least 1% (thin lines), respectively.

Figure 2

In silico transcription factor binding sites and electrophoretic mobility shift assay. Schematic representation of the PLK1 silencer and core promoter regions (panel A). Alleles and the surrounding sequence of the silencer SNP rs57973275 (c.-1706G > A) and the core promoter SNP rs16972787 (c.-233G > A) are shown. Transcription factors and their putative binding sites are shown above and below the corresponding PLK1 alleles, respectively. Representative electrophoretic mobility shift assays for both SNPs that failed to show genotype-dependent binding of nuclear extracts of different cell lines (panel B).

Figure 3

Secondary PLK1 mRNA structure and reporter assays for allele-dependent mRNA stability. Mfold-predicted most favorable secondary PLK1 mRNA structure dependent on rs27770 (c.*154A > G) alleles (panel A). Prediction is based on PLK1 mRNA sequence according to NCBI accession number NM005030. Arrows indicate position of the polymorphism within the secondary structures. Allele-dependent activity of PLK1 3′UTR reporter constructs expressed in HEK293 cells (panel B). Structures of the constructs used for the reporter assay are depicted on the left side. Reporter activity was quantified by measuring Firefly luciferase activity normalized to Renilla luciferase activity. Data are mean ± SE of five independent experiments, ***P < 0.001, **P < 0.01, one-way ANOVA and Holm-Sidak’s post-hoc multiple comparisons test.