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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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Control of h-eag1 expression by E2F1.(A & B) Effect of E2F1 inhibition by its decoy oligodeoxynucleotides (E2F1-dODN) on h-eag1 expression at both mRNA and protein levels in the presence of both p53 inhibitor and the MT-AMO to miR-34. SHSY5Y cells were pretreated with PFT (30 µM) and MT-AMO and then transfected with E2F1-dODN to sequestrate E2F1. *p<0.05 vs Ctl/Lipo; φp<0.05 vs PFT+MT-AMO; n = 4 for each group. (C & D) Effect of E2F1 overexpression on h-eag1 expression in the presence of both p53 activator and miR-34a. SHSY5Y cells were pretreated with nutlin-3 (1 µM) and miR-34a and then transfected with the plasmid to overexpress E2F1. *p<0.05 vs Ctl/Lipo; φp<0.05 vs Nutlin-3+miR-34a; n = 4 for each group.
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pone-0020362-g005: Control of h-eag1 expression by E2F1.(A & B) Effect of E2F1 inhibition by its decoy oligodeoxynucleotides (E2F1-dODN) on h-eag1 expression at both mRNA and protein levels in the presence of both p53 inhibitor and the MT-AMO to miR-34. SHSY5Y cells were pretreated with PFT (30 µM) and MT-AMO and then transfected with E2F1-dODN to sequestrate E2F1. *p<0.05 vs Ctl/Lipo; φp<0.05 vs PFT+MT-AMO; n = 4 for each group. (C & D) Effect of E2F1 overexpression on h-eag1 expression in the presence of both p53 activator and miR-34a. SHSY5Y cells were pretreated with nutlin-3 (1 µM) and miR-34a and then transfected with the plasmid to overexpress E2F1. *p<0.05 vs Ctl/Lipo; φp<0.05 vs Nutlin-3+miR-34a; n = 4 for each group.

Mentions: miR-34 has been known to be a direct transcriptional target of p53 [33]–[36] and to mediate the apoptotic action of p53. Thus, changes of p53 activity are deemed to change the level of miR-34 thereby those of E2F1 and h-eag1 as well. Indeed, p53 activation by Mdm2 inhibitor nutlin-3 (1 µM) increased miR-34 level (Fig. 4A), and simultaneously decreased E2F1 and h-eag1 mRNA concentrations (Fig. 4A) and protein levels (Fig. 4B). These changes were abrogated when the MT-AMO was co-applied with Nutlin-3. Pifithrin-alpha (PFT-α; 30 µM), the p53 inhibitor, produced exactly the opposite effects to p53 activator. Evidently, p53 negatively regulates expression of h-eag1. Moreover, in the presence of p53 inhibitor, exogenously applied miR-34a retained the full ability to downregulate E2F1 (Fig. 4C & 4D) and h-eag1 (Fig. 4E & 4F), suggesting that miR-34 mediates the regulatory role of p53 on E2F1 and h-eag1. Furthermore, in the presence of both p53 inhibitor and the MT-AMO, h-eag1 expression was markedly upregulated at both mRNA and protein levels, but the E2F1-dODN abolished these increases (Fig. 5A & 5B). On the other hand, the downregulation of h-eag1 expression induced by co-application of p53 activator and miR-34a was abolished by E2F1 overexpression (Fig. 5C & 5D).


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)

Control of h-eag1 expression by E2F1.(A & B) Effect of E2F1 inhibition by its decoy oligodeoxynucleotides (E2F1-dODN) on h-eag1 expression at both mRNA and protein levels in the presence of both p53 inhibitor and the MT-AMO to miR-34. SHSY5Y cells were pretreated with PFT (30 µM) and MT-AMO and then transfected with E2F1-dODN to sequestrate E2F1. *p<0.05 vs Ctl/Lipo; φp<0.05 vs PFT+MT-AMO; n = 4 for each group. (C & D) Effect of E2F1 overexpression on h-eag1 expression in the presence of both p53 activator and miR-34a. SHSY5Y cells were pretreated with nutlin-3 (1 µM) and miR-34a and then transfected with the plasmid to overexpress E2F1. *p<0.05 vs Ctl/Lipo; φp<0.05 vs Nutlin-3+miR-34a; n = 4 for each group.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105031&req=5

pone-0020362-g005: Control of h-eag1 expression by E2F1.(A & B) Effect of E2F1 inhibition by its decoy oligodeoxynucleotides (E2F1-dODN) on h-eag1 expression at both mRNA and protein levels in the presence of both p53 inhibitor and the MT-AMO to miR-34. SHSY5Y cells were pretreated with PFT (30 µM) and MT-AMO and then transfected with E2F1-dODN to sequestrate E2F1. *p<0.05 vs Ctl/Lipo; φp<0.05 vs PFT+MT-AMO; n = 4 for each group. (C & D) Effect of E2F1 overexpression on h-eag1 expression in the presence of both p53 activator and miR-34a. SHSY5Y cells were pretreated with nutlin-3 (1 µM) and miR-34a and then transfected with the plasmid to overexpress E2F1. *p<0.05 vs Ctl/Lipo; φp<0.05 vs Nutlin-3+miR-34a; n = 4 for each group.
Mentions: miR-34 has been known to be a direct transcriptional target of p53 [33]–[36] and to mediate the apoptotic action of p53. Thus, changes of p53 activity are deemed to change the level of miR-34 thereby those of E2F1 and h-eag1 as well. Indeed, p53 activation by Mdm2 inhibitor nutlin-3 (1 µM) increased miR-34 level (Fig. 4A), and simultaneously decreased E2F1 and h-eag1 mRNA concentrations (Fig. 4A) and protein levels (Fig. 4B). These changes were abrogated when the MT-AMO was co-applied with Nutlin-3. Pifithrin-alpha (PFT-α; 30 µM), the p53 inhibitor, produced exactly the opposite effects to p53 activator. Evidently, p53 negatively regulates expression of h-eag1. Moreover, in the presence of p53 inhibitor, exogenously applied miR-34a retained the full ability to downregulate E2F1 (Fig. 4C & 4D) and h-eag1 (Fig. 4E & 4F), suggesting that miR-34 mediates the regulatory role of p53 on E2F1 and h-eag1. Furthermore, in the presence of both p53 inhibitor and the MT-AMO, h-eag1 expression was markedly upregulated at both mRNA and protein levels, but the E2F1-dODN abolished these increases (Fig. 5A & 5B). On the other hand, the downregulation of h-eag1 expression induced by co-application of p53 activator and miR-34a was abolished by E2F1 overexpression (Fig. 5C & 5D).

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