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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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miR-34 as a post-transcriptional repressor of E2F1.(A) Effect of miR-34a on E2F1 protein levels in SHSY5Y cells. *p<0.05 vs Ctl/Lipo; φp<0.05 vs miR-34a alone; n = 6 for each group. (B) Inability of miR-34 to affect the overexpression of h-eag1 mRNA induced by transfection of the plasmid expressing the E2F1 cDNA. *p<0.05 vs Ctl/Lipo; n = 4 for each group. (C) Repression of h = eag1 protein levels by miR-34a in the presence of E1F1 overexpression by the vector containing the E2F1 cDNA. *p<0.05 vs Ctl/Lipo; φp<0.05 vs miR-34a alone; n = 4 for each group.
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pone-0020362-g003: miR-34 as a post-transcriptional repressor of E2F1.(A) Effect of miR-34a on E2F1 protein levels in SHSY5Y cells. *p<0.05 vs Ctl/Lipo; φp<0.05 vs miR-34a alone; n = 6 for each group. (B) Inability of miR-34 to affect the overexpression of h-eag1 mRNA induced by transfection of the plasmid expressing the E2F1 cDNA. *p<0.05 vs Ctl/Lipo; n = 4 for each group. (C) Repression of h = eag1 protein levels by miR-34a in the presence of E1F1 overexpression by the vector containing the E2F1 cDNA. *p<0.05 vs Ctl/Lipo; φp<0.05 vs miR-34a alone; n = 4 for each group.

Mentions: It has been documented that miR-34a directly targets the mRNA encoding E2F1 and significantly reduces the levels of E2F1 and E2F3 proteins [30]–[32]. We confirmed that transfection of miR-34a reduced E2F1 protein levels by ∼68% in SHSY5Y cells (Fig. 3A) and the same results were obtained with miR-34b and miR-34c (Figure S6 & S8). Moreover, application of the MT-AMO caused significant increases in the protein levels of E2F1 (Fig. 3A) and h-eag1 (Fig. 2B). These results indicate that miR-34 regulates h-eag1 expression through at least two mechanisms. First, miR-34 directly represses h-eag1 protein. Second, miR-34 represses E2F1 protein, leading to reduced transcription of h-eag1. This latter effect also explains partially the effectiveness of miR-34 to decrease h-eag1 mRNA. This is supported by the experiments showing the lack of effects of miR-34a on h-eag1 transcript level in cells co-transfected with the E2F1-carrying vector that does not contain the 3′UTR of E2F1 gene (Fig. 3B). By comparison, miR-34a retained its ability to repress h-eag1 protein in the presence of E2F1 overexpression (Fig. 3C).


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)

miR-34 as a post-transcriptional repressor of E2F1.(A) Effect of miR-34a on E2F1 protein levels in SHSY5Y cells. *p<0.05 vs Ctl/Lipo; φp<0.05 vs miR-34a alone; n = 6 for each group. (B) Inability of miR-34 to affect the overexpression of h-eag1 mRNA induced by transfection of the plasmid expressing the E2F1 cDNA. *p<0.05 vs Ctl/Lipo; n = 4 for each group. (C) Repression of h = eag1 protein levels by miR-34a in the presence of E1F1 overexpression by the vector containing the E2F1 cDNA. *p<0.05 vs Ctl/Lipo; φp<0.05 vs miR-34a alone; n = 4 for each group.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105031&req=5

pone-0020362-g003: miR-34 as a post-transcriptional repressor of E2F1.(A) Effect of miR-34a on E2F1 protein levels in SHSY5Y cells. *p<0.05 vs Ctl/Lipo; φp<0.05 vs miR-34a alone; n = 6 for each group. (B) Inability of miR-34 to affect the overexpression of h-eag1 mRNA induced by transfection of the plasmid expressing the E2F1 cDNA. *p<0.05 vs Ctl/Lipo; n = 4 for each group. (C) Repression of h = eag1 protein levels by miR-34a in the presence of E1F1 overexpression by the vector containing the E2F1 cDNA. *p<0.05 vs Ctl/Lipo; φp<0.05 vs miR-34a alone; n = 4 for each group.
Mentions: It has been documented that miR-34a directly targets the mRNA encoding E2F1 and significantly reduces the levels of E2F1 and E2F3 proteins [30]–[32]. We confirmed that transfection of miR-34a reduced E2F1 protein levels by ∼68% in SHSY5Y cells (Fig. 3A) and the same results were obtained with miR-34b and miR-34c (Figure S6 & S8). Moreover, application of the MT-AMO caused significant increases in the protein levels of E2F1 (Fig. 3A) and h-eag1 (Fig. 2B). These results indicate that miR-34 regulates h-eag1 expression through at least two mechanisms. First, miR-34 directly represses h-eag1 protein. Second, miR-34 represses E2F1 protein, leading to reduced transcription of h-eag1. This latter effect also explains partially the effectiveness of miR-34 to decrease h-eag1 mRNA. This is supported by the experiments showing the lack of effects of miR-34a on h-eag1 transcript level in cells co-transfected with the E2F1-carrying vector that does not contain the 3′UTR of E2F1 gene (Fig. 3B). By comparison, miR-34a retained its ability to repress h-eag1 protein in the presence of E2F1 overexpression (Fig. 3C).

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