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

Akdeli N, Riemann K, Westphal J, Hess J, Siffert W, Bachmann HS - Mol. Cancer (2014)

Bottom Line: 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).The present study provides evidence that at least one genomic variant of PLK1 has functional properties and influences expression of PLK1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Pharmacogenetics, University Hospital Essen, Hufelandstr, 55, 45147 Essen, Germany. hagen.bachmann@uk-essen.de.

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.

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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).
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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).

Mentions: Functional polymorphisms in the silencer and promoter region may impair mRNA levels by changing transcription factor (TF) binding sites and concurrent modulation of the promoter activity. We predicted potential allele-dependent TF binding using MatInspector, Alibaba2 and Consite (Figure 2A) [29-31]. For rs57973275 a putative binding site of the TF Activator protein 1 (AP1) emerged for the G allele and C-Rel for the A allele. In the presence of the A allele, rs16972787 created binding sites for PAX-2 (Paired box gene 2), PAX-6 (Paired box gene 6) and Snail, as well as a MEF3 binding motif, whereas the same binding sites are not present for the G allele, which instead carries a putative binding site for KLF7 (Krueppel like transcription factor 7). We used these in silico results to design oligonucleotides which comprise the putative binding sites of the different TFs. To analyze differences between the alleles we performed electrophoretic mobility shift assays (EMSA) with these oligonucleotides. Nuclear extracts of three different cell lines (HEK293, HepG2 and HeLa) were used for both SNPs to cover the diversity of the predicted TFs. All three cell lines are known to express (HEK293) and overexpress (HepG2 and HeLa) PLK1 [32-34]. Figure 2B shows two representative results of these experiments. In contrast to the in silico results we were not able to detect any specific transcription factor binding to the alleles of both polymorphisms.


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

Akdeli N, Riemann K, Westphal J, Hess J, Siffert W, Bachmann HS - Mol. Cancer (2014)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4020576&req=5

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).
Mentions: Functional polymorphisms in the silencer and promoter region may impair mRNA levels by changing transcription factor (TF) binding sites and concurrent modulation of the promoter activity. We predicted potential allele-dependent TF binding using MatInspector, Alibaba2 and Consite (Figure 2A) [29-31]. For rs57973275 a putative binding site of the TF Activator protein 1 (AP1) emerged for the G allele and C-Rel for the A allele. In the presence of the A allele, rs16972787 created binding sites for PAX-2 (Paired box gene 2), PAX-6 (Paired box gene 6) and Snail, as well as a MEF3 binding motif, whereas the same binding sites are not present for the G allele, which instead carries a putative binding site for KLF7 (Krueppel like transcription factor 7). We used these in silico results to design oligonucleotides which comprise the putative binding sites of the different TFs. To analyze differences between the alleles we performed electrophoretic mobility shift assays (EMSA) with these oligonucleotides. Nuclear extracts of three different cell lines (HEK293, HepG2 and HeLa) were used for both SNPs to cover the diversity of the predicted TFs. All three cell lines are known to express (HEK293) and overexpress (HepG2 and HeLa) PLK1 [32-34]. Figure 2B shows two representative results of these experiments. In contrast to the in silico results we were not able to detect any specific transcription factor binding to the alleles of both polymorphisms.

Bottom Line: 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).The present study provides evidence that at least one genomic variant of PLK1 has functional properties and influences expression of PLK1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Pharmacogenetics, University Hospital Essen, Hufelandstr, 55, 45147 Essen, Germany. hagen.bachmann@uk-essen.de.

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.

Show MeSH
Related in: MedlinePlus