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Transcriptional and post-transcriptional mechanisms for oncogenic overexpression of ether à go-go K+ channel.

Lin H, Li Z, Chen C, Luo X, Xiao J, Dong D, Lu Y, Yang B, Wang Z - PLoS ONE (2011)

Bottom Line: It was found to be necessary for cell cycle progression and tumorigenesis.H-eag1antisense antagonized the growth-stimulating effects and the upregulation of h-eag1 expression in SHSY5Y cells, induced by knockdown of miR-34, E2F1 overexpression, or inhibition of p53 activity.Moreover, these findings place h-eag1 in the p53-miR-34-E2F1-h-eag1 pathway with h-eag as a terminal effecter component and with miR-34 (and E2F1) as a linker between p53 and h-eag1.

View Article: PubMed Central - PubMed

Affiliation: Research Center, Montreal Heart Institute, Montreal, Quebec, Canada.

ABSTRACT
The human ether-à-go-go-1 (h-eag1) K(+) channel is expressed in a variety of cell lines derived from human malignant tumors and in clinical samples of several different cancers, but is otherwise absent in normal tissues. It was found to be necessary for cell cycle progression and tumorigenesis. Specific inhibition of h-eag1 expression leads to inhibition of tumor cell proliferation. We report here that h-eag1 expression is controlled by the p53-miR-34-E2F1 pathway through a negative feed-forward mechanism. We first established E2F1 as a transactivator of h-eag1 gene through characterizing its promoter region. We then revealed that miR-34, a known transcriptional target of p53, is an important negative regulator of h-eag1 through dual mechanisms by directly repressing h-eag1 at the post-transcriptional level and indirectly silencing h-eag1 at the transcriptional level via repressing E2F1. There is a strong inverse relationship between the expression levels of miR-34 and h-eag1 protein. H-eag1antisense antagonized the growth-stimulating effects and the upregulation of h-eag1 expression in SHSY5Y cells, induced by knockdown of miR-34, E2F1 overexpression, or inhibition of p53 activity. Therefore, p53 negatively regulates h-eag1 expression by a negative feed-forward mechanism through the p53-miR-34-E2F1 pathway. Inactivation of p53 activity, as is the case in many cancers, can thus cause oncogenic overexpression of h-eag1 by relieving the negative feed-forward regulation. These findings not only help us understand the molecular mechanisms for oncogenic overexpression of h-eag1 in tumorigenesis but also uncover the cell-cycle regulation through the p53-miR-34-E2F1-h-eag1 pathway. Moreover, these findings place h-eag1 in the p53-miR-34-E2F1-h-eag1 pathway with h-eag as a terminal effecter component and with miR-34 (and E2F1) as a linker between p53 and h-eag1. Our study therefore fills the gap between p53 pathway and its cellular function mediated by h-eag1.

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E2F1 as a transactivator of h-eag1 in SHSY5Y human neuroblastoma cells.(A) Role of E2F1 in driving the h-eag1 core promoter activity. pGL3-Base: h-eag1 promoter-free pGL3 vector for control; pGL3-Core: pGL3 vector carrying the h-eag1 core promoter (a fragment spanning -630/+114); E2F1-dODN, SP1-dODN, and AP2-dODN: the decoy oligodeoxynucleotides targeting E2F1, SP1, and AP2 transcription factors, respectively, co-transfected with pGL3-Core; pGL3-Mutant: pGL3 vector carrying a mutated h-eag1 core promoter. Transfection was carried out using lipofectamine 2000. *p<0.05 vs pGL3-Core; n = 5 for each group. (B) Changes of h-eag1 mRNA level determined by real-time quantitative RT-PCR (qPCR) in SHSY5Y cells. E2F1-dODN, E2F1-MT dODN, SP1-dODN, or AP2-dODN was transfected alone. Ctl/Lipo: cells mock-treated with lipofectamine 2000; E2F1-MT dODN: the decoy oligodeoxynucleotides targeting E2F1 with mutation at the core region. *p<0.05 vs Ctl/Lipo; n = 5 for each group. (C) Increase in h-eag1 mRNA level by overexpression of E2F1 in SHSY5Y cells transfected with the plasmid expressing the E2F1 gene. E2F1-P: pRcCMV-E2F1 expression vector (Invitrogen), the plasmid carrying the E2F1 cDNA. *p<0.05 vs Ctl/Lipo; n = 5 for each group. (D) Chromatin immunoprecipitation assay (ChIP) assay for the presence of E2F1 on its cis-acting elements in the h-eag1 promoter region in SHSY5Y cells. Left panel: the bands of PCR products of the 5′-flanking region encompassing E2F1 binding sites following immunoprecipitation with the anti-E2F1 antibody or the anti-lamin A antibody for a negative control. Right panel: averaged data on the recovered DNA by anti-E2F1 expressed as fold changes over anti-lamin A band. Input: the input representing genomic DNA prior to immunoprecipitation. (E) Electrophoresis mobility shift assay (EMSA) for the fragment encompassing the putative E2F1 cis-acting element in the h-eag1 promoter region to bind E2F1 protein in the nuclear extract from SHSY5Y cells. Probe: digoxigenin (DIG)-labeled oligonucleotides fragment containing E2F1 binding site; MT Probe: DIG-labeled fragment containing mutated E2F1 site at the core motif; NE: nuclear extract from SHSY5Y cells. Solid arrowhead points to the shifted band representing the DNA-protein complex. Note that the shifted band is weakened by anti-E2F1 antibody or with the mutant E2F1 binding motif.
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pone-0020362-g001: E2F1 as a transactivator of h-eag1 in SHSY5Y human neuroblastoma cells.(A) Role of E2F1 in driving the h-eag1 core promoter activity. pGL3-Base: h-eag1 promoter-free pGL3 vector for control; pGL3-Core: pGL3 vector carrying the h-eag1 core promoter (a fragment spanning -630/+114); E2F1-dODN, SP1-dODN, and AP2-dODN: the decoy oligodeoxynucleotides targeting E2F1, SP1, and AP2 transcription factors, respectively, co-transfected with pGL3-Core; pGL3-Mutant: pGL3 vector carrying a mutated h-eag1 core promoter. Transfection was carried out using lipofectamine 2000. *p<0.05 vs pGL3-Core; n = 5 for each group. (B) Changes of h-eag1 mRNA level determined by real-time quantitative RT-PCR (qPCR) in SHSY5Y cells. E2F1-dODN, E2F1-MT dODN, SP1-dODN, or AP2-dODN was transfected alone. Ctl/Lipo: cells mock-treated with lipofectamine 2000; E2F1-MT dODN: the decoy oligodeoxynucleotides targeting E2F1 with mutation at the core region. *p<0.05 vs Ctl/Lipo; n = 5 for each group. (C) Increase in h-eag1 mRNA level by overexpression of E2F1 in SHSY5Y cells transfected with the plasmid expressing the E2F1 gene. E2F1-P: pRcCMV-E2F1 expression vector (Invitrogen), the plasmid carrying the E2F1 cDNA. *p<0.05 vs Ctl/Lipo; n = 5 for each group. (D) Chromatin immunoprecipitation assay (ChIP) assay for the presence of E2F1 on its cis-acting elements in the h-eag1 promoter region in SHSY5Y cells. Left panel: the bands of PCR products of the 5′-flanking region encompassing E2F1 binding sites following immunoprecipitation with the anti-E2F1 antibody or the anti-lamin A antibody for a negative control. Right panel: averaged data on the recovered DNA by anti-E2F1 expressed as fold changes over anti-lamin A band. Input: the input representing genomic DNA prior to immunoprecipitation. (E) Electrophoresis mobility shift assay (EMSA) for the fragment encompassing the putative E2F1 cis-acting element in the h-eag1 promoter region to bind E2F1 protein in the nuclear extract from SHSY5Y cells. Probe: digoxigenin (DIG)-labeled oligonucleotides fragment containing E2F1 binding site; MT Probe: DIG-labeled fragment containing mutated E2F1 site at the core motif; NE: nuclear extract from SHSY5Y cells. Solid arrowhead points to the shifted band representing the DNA-protein complex. Note that the shifted band is weakened by anti-E2F1 antibody or with the mutant E2F1 binding motif.

Mentions: In an initial effort to understand the molecular mechanisms for oncogenic overexpression of eag1 in cancer cells, we characterized the promoter region of the gene. We used 5′RACE to identify the transcription start site (TSS) which was found located to 152 bp upstream the translation start codon (ATG) of h-eag1 (GenBank accession No. DQ120124) (Figure S1). We then defined the minimal promoter region by luciferase reporter gene assay (Figure S2). Computer analysis revealed consensus sequences for E2F1, AP2, and SP1 within the core promoter region (position −630/+114), which might act as transactivators of h-eag1 gene. Using the decoy oligodeoxynucleotide (dODN) approach [20], [22], [23], which contains the perfect binding site for the target transcription factor and can sequestrate the target leading to reduction of transcriptional activity (Supporting Figures online), we revealed a significant role of E2F1, but not of SP1 and AP2, in driving the core promoter activity (Fig. 1A). Mutation of the E2F1 cis-element rendered a loss of luciferase activity of the core promoter. We further verified E2F1 as a key factor in activating h-eag1 transcription: E2F1-dODN decreased h-eag1 mRNA level by ∼80% in SHSY5Y human neuroblastoma cells (Fig. 1B) and MCF-1 human breast cancer cells (Figure S3). With qPCR, we have also ruled out the role of SP1 and AP2 in transcriptional activation of h-eag1 (Fig. 1B). Transfection of E2F1 plasmid, on the other hand, increased h-eag1 mRNA level by as much as 8-fold, which was diminished by E2F1-dODN. As a negative control, transfection of SP1 plasmid did not significantly alter h-eag1 mRNA level (Fig. 1C) despite that this maneuver was able to enhance expression of h-erg1 at the mRNA level (Fig. 1C), another member of the eag K+ channel gene family, as already established in our previous study [24]. The ability of E2F1 to bind its cis-acting elements in the promoter region of h-eag1 was verified using ChIP and EMSA (Fig. 1D & 1E). The transcription factor E2F1 plays a pivotal role in the coordinated expression of genes necessary for cell cycle progression and division, and is known to be an oncoprotein critical for the transcriptional activation of genes that control the rate of tumor cell proliferation [25]–[27]. Our finding thus indicates a role of E2F1 in oncogenic upregulation of h-eag1 expression at the transcriptional level.


Transcriptional and post-transcriptional mechanisms for oncogenic overexpression of ether à go-go K+ channel.

Lin H, Li Z, Chen C, Luo X, Xiao J, Dong D, Lu Y, Yang B, Wang Z - PLoS ONE (2011)

E2F1 as a transactivator of h-eag1 in SHSY5Y human neuroblastoma cells.(A) Role of E2F1 in driving the h-eag1 core promoter activity. pGL3-Base: h-eag1 promoter-free pGL3 vector for control; pGL3-Core: pGL3 vector carrying the h-eag1 core promoter (a fragment spanning -630/+114); E2F1-dODN, SP1-dODN, and AP2-dODN: the decoy oligodeoxynucleotides targeting E2F1, SP1, and AP2 transcription factors, respectively, co-transfected with pGL3-Core; pGL3-Mutant: pGL3 vector carrying a mutated h-eag1 core promoter. Transfection was carried out using lipofectamine 2000. *p<0.05 vs pGL3-Core; n = 5 for each group. (B) Changes of h-eag1 mRNA level determined by real-time quantitative RT-PCR (qPCR) in SHSY5Y cells. E2F1-dODN, E2F1-MT dODN, SP1-dODN, or AP2-dODN was transfected alone. Ctl/Lipo: cells mock-treated with lipofectamine 2000; E2F1-MT dODN: the decoy oligodeoxynucleotides targeting E2F1 with mutation at the core region. *p<0.05 vs Ctl/Lipo; n = 5 for each group. (C) Increase in h-eag1 mRNA level by overexpression of E2F1 in SHSY5Y cells transfected with the plasmid expressing the E2F1 gene. E2F1-P: pRcCMV-E2F1 expression vector (Invitrogen), the plasmid carrying the E2F1 cDNA. *p<0.05 vs Ctl/Lipo; n = 5 for each group. (D) Chromatin immunoprecipitation assay (ChIP) assay for the presence of E2F1 on its cis-acting elements in the h-eag1 promoter region in SHSY5Y cells. Left panel: the bands of PCR products of the 5′-flanking region encompassing E2F1 binding sites following immunoprecipitation with the anti-E2F1 antibody or the anti-lamin A antibody for a negative control. Right panel: averaged data on the recovered DNA by anti-E2F1 expressed as fold changes over anti-lamin A band. Input: the input representing genomic DNA prior to immunoprecipitation. (E) Electrophoresis mobility shift assay (EMSA) for the fragment encompassing the putative E2F1 cis-acting element in the h-eag1 promoter region to bind E2F1 protein in the nuclear extract from SHSY5Y cells. Probe: digoxigenin (DIG)-labeled oligonucleotides fragment containing E2F1 binding site; MT Probe: DIG-labeled fragment containing mutated E2F1 site at the core motif; NE: nuclear extract from SHSY5Y cells. Solid arrowhead points to the shifted band representing the DNA-protein complex. Note that the shifted band is weakened by anti-E2F1 antibody or with the mutant E2F1 binding motif.
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Related In: Results  -  Collection

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pone-0020362-g001: E2F1 as a transactivator of h-eag1 in SHSY5Y human neuroblastoma cells.(A) Role of E2F1 in driving the h-eag1 core promoter activity. pGL3-Base: h-eag1 promoter-free pGL3 vector for control; pGL3-Core: pGL3 vector carrying the h-eag1 core promoter (a fragment spanning -630/+114); E2F1-dODN, SP1-dODN, and AP2-dODN: the decoy oligodeoxynucleotides targeting E2F1, SP1, and AP2 transcription factors, respectively, co-transfected with pGL3-Core; pGL3-Mutant: pGL3 vector carrying a mutated h-eag1 core promoter. Transfection was carried out using lipofectamine 2000. *p<0.05 vs pGL3-Core; n = 5 for each group. (B) Changes of h-eag1 mRNA level determined by real-time quantitative RT-PCR (qPCR) in SHSY5Y cells. E2F1-dODN, E2F1-MT dODN, SP1-dODN, or AP2-dODN was transfected alone. Ctl/Lipo: cells mock-treated with lipofectamine 2000; E2F1-MT dODN: the decoy oligodeoxynucleotides targeting E2F1 with mutation at the core region. *p<0.05 vs Ctl/Lipo; n = 5 for each group. (C) Increase in h-eag1 mRNA level by overexpression of E2F1 in SHSY5Y cells transfected with the plasmid expressing the E2F1 gene. E2F1-P: pRcCMV-E2F1 expression vector (Invitrogen), the plasmid carrying the E2F1 cDNA. *p<0.05 vs Ctl/Lipo; n = 5 for each group. (D) Chromatin immunoprecipitation assay (ChIP) assay for the presence of E2F1 on its cis-acting elements in the h-eag1 promoter region in SHSY5Y cells. Left panel: the bands of PCR products of the 5′-flanking region encompassing E2F1 binding sites following immunoprecipitation with the anti-E2F1 antibody or the anti-lamin A antibody for a negative control. Right panel: averaged data on the recovered DNA by anti-E2F1 expressed as fold changes over anti-lamin A band. Input: the input representing genomic DNA prior to immunoprecipitation. (E) Electrophoresis mobility shift assay (EMSA) for the fragment encompassing the putative E2F1 cis-acting element in the h-eag1 promoter region to bind E2F1 protein in the nuclear extract from SHSY5Y cells. Probe: digoxigenin (DIG)-labeled oligonucleotides fragment containing E2F1 binding site; MT Probe: DIG-labeled fragment containing mutated E2F1 site at the core motif; NE: nuclear extract from SHSY5Y cells. Solid arrowhead points to the shifted band representing the DNA-protein complex. Note that the shifted band is weakened by anti-E2F1 antibody or with the mutant E2F1 binding motif.
Mentions: In an initial effort to understand the molecular mechanisms for oncogenic overexpression of eag1 in cancer cells, we characterized the promoter region of the gene. We used 5′RACE to identify the transcription start site (TSS) which was found located to 152 bp upstream the translation start codon (ATG) of h-eag1 (GenBank accession No. DQ120124) (Figure S1). We then defined the minimal promoter region by luciferase reporter gene assay (Figure S2). Computer analysis revealed consensus sequences for E2F1, AP2, and SP1 within the core promoter region (position −630/+114), which might act as transactivators of h-eag1 gene. Using the decoy oligodeoxynucleotide (dODN) approach [20], [22], [23], which contains the perfect binding site for the target transcription factor and can sequestrate the target leading to reduction of transcriptional activity (Supporting Figures online), we revealed a significant role of E2F1, but not of SP1 and AP2, in driving the core promoter activity (Fig. 1A). Mutation of the E2F1 cis-element rendered a loss of luciferase activity of the core promoter. We further verified E2F1 as a key factor in activating h-eag1 transcription: E2F1-dODN decreased h-eag1 mRNA level by ∼80% in SHSY5Y human neuroblastoma cells (Fig. 1B) and MCF-1 human breast cancer cells (Figure S3). With qPCR, we have also ruled out the role of SP1 and AP2 in transcriptional activation of h-eag1 (Fig. 1B). Transfection of E2F1 plasmid, on the other hand, increased h-eag1 mRNA level by as much as 8-fold, which was diminished by E2F1-dODN. As a negative control, transfection of SP1 plasmid did not significantly alter h-eag1 mRNA level (Fig. 1C) despite that this maneuver was able to enhance expression of h-erg1 at the mRNA level (Fig. 1C), another member of the eag K+ channel gene family, as already established in our previous study [24]. The ability of E2F1 to bind its cis-acting elements in the promoter region of h-eag1 was verified using ChIP and EMSA (Fig. 1D & 1E). The transcription factor E2F1 plays a pivotal role in the coordinated expression of genes necessary for cell cycle progression and division, and is known to be an oncoprotein critical for the transcriptional activation of genes that control the rate of tumor cell proliferation [25]–[27]. Our finding thus indicates a role of E2F1 in oncogenic upregulation of h-eag1 expression at the transcriptional level.

Bottom Line: It was found to be necessary for cell cycle progression and tumorigenesis.H-eag1antisense antagonized the growth-stimulating effects and the upregulation of h-eag1 expression in SHSY5Y cells, induced by knockdown of miR-34, E2F1 overexpression, or inhibition of p53 activity.Moreover, these findings place h-eag1 in the p53-miR-34-E2F1-h-eag1 pathway with h-eag as a terminal effecter component and with miR-34 (and E2F1) as a linker between p53 and h-eag1.

View Article: PubMed Central - PubMed

Affiliation: Research Center, Montreal Heart Institute, Montreal, Quebec, Canada.

ABSTRACT
The human ether-à-go-go-1 (h-eag1) K(+) channel is expressed in a variety of cell lines derived from human malignant tumors and in clinical samples of several different cancers, but is otherwise absent in normal tissues. It was found to be necessary for cell cycle progression and tumorigenesis. Specific inhibition of h-eag1 expression leads to inhibition of tumor cell proliferation. We report here that h-eag1 expression is controlled by the p53-miR-34-E2F1 pathway through a negative feed-forward mechanism. We first established E2F1 as a transactivator of h-eag1 gene through characterizing its promoter region. We then revealed that miR-34, a known transcriptional target of p53, is an important negative regulator of h-eag1 through dual mechanisms by directly repressing h-eag1 at the post-transcriptional level and indirectly silencing h-eag1 at the transcriptional level via repressing E2F1. There is a strong inverse relationship between the expression levels of miR-34 and h-eag1 protein. H-eag1antisense antagonized the growth-stimulating effects and the upregulation of h-eag1 expression in SHSY5Y cells, induced by knockdown of miR-34, E2F1 overexpression, or inhibition of p53 activity. Therefore, p53 negatively regulates h-eag1 expression by a negative feed-forward mechanism through the p53-miR-34-E2F1 pathway. Inactivation of p53 activity, as is the case in many cancers, can thus cause oncogenic overexpression of h-eag1 by relieving the negative feed-forward regulation. These findings not only help us understand the molecular mechanisms for oncogenic overexpression of h-eag1 in tumorigenesis but also uncover the cell-cycle regulation through the p53-miR-34-E2F1-h-eag1 pathway. Moreover, these findings place h-eag1 in the p53-miR-34-E2F1-h-eag1 pathway with h-eag as a terminal effecter component and with miR-34 (and E2F1) as a linker between p53 and h-eag1. Our study therefore fills the gap between p53 pathway and its cellular function mediated by h-eag1.

Show MeSH
Related in: MedlinePlus