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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 h-eag1.(A) Repression of h-eag1 expression by miR-34a or miR-34c, as reported by luciferase activity assay with the pMIR-REPORTTM luciferase miRNA expression reporter vector carrying the h-eag1 3′UTR in HEK293 cells. Ctl: cells transfected with the luciferase vector alone; MT-AMO: the multiple-target anti-miRNA antisense oligonucleotides to miR-34a, miR-34b and miR-34c, co-transfected with the luciferase vector and miR-34a or miR-34c. *p<0.05 vs Ctl/Lipo; φp<0.05 vs miR-34a; n = 4 for each group. (B) Western blot analysis revealing repression of h-eag1 protein by miR-34a and miR-34c in SHSY5Y cells. *p<0.05 vs Ctl/Lipo; φp<0.05 vs miR-34a alone; n = 4 for each group. The immunoblot bands shown were run on the same gel. (C) Effect of miR-34 on h-eag1 mRNA level in SHSY5Y cells. *p<0.05 vs Ctl/Lipo; φp<0.05 vs miR-34a alone; n = 4 for each group.
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pone-0020362-g002: miR-34 as a post-transcriptional repressor of h-eag1.(A) Repression of h-eag1 expression by miR-34a or miR-34c, as reported by luciferase activity assay with the pMIR-REPORTTM luciferase miRNA expression reporter vector carrying the h-eag1 3′UTR in HEK293 cells. Ctl: cells transfected with the luciferase vector alone; MT-AMO: the multiple-target anti-miRNA antisense oligonucleotides to miR-34a, miR-34b and miR-34c, co-transfected with the luciferase vector and miR-34a or miR-34c. *p<0.05 vs Ctl/Lipo; φp<0.05 vs miR-34a; n = 4 for each group. (B) Western blot analysis revealing repression of h-eag1 protein by miR-34a and miR-34c in SHSY5Y cells. *p<0.05 vs Ctl/Lipo; φp<0.05 vs miR-34a alone; n = 4 for each group. The immunoblot bands shown were run on the same gel. (C) Effect of miR-34 on h-eag1 mRNA level in SHSY5Y cells. *p<0.05 vs Ctl/Lipo; φp<0.05 vs miR-34a alone; n = 4 for each group.

Mentions: We then investigated if h-eag1 is also regulated at the post-transcriptional level by miRNAs. We first performed computational prediction of h-eag1 as a target for miRNA regulation. And we identified multiple binding sites for a tumor-suppressor miRNA subfamily miR-34 (including miR-34a, miR-34b and miR-34c) in the 3′UTR of h-eag1 mRNA (Figure S4). To experimentally establish miR-34:h-eag1 interaction, we inserted a fragment of 3′UTR of h-eag1 containing the miR-34 target sites into the position downstream the luciferase gene in the pMIR-REPORTTM vector. Transfection of miR-34a markedly suppressed the luciferase activities and the effect was reversed by their multiple-target anti-miRNA antisense oligonucleotides (MT-AMO) (Fig. 2A; Supporting Figures online; Figure S5), a single oligomer capable of targeting all three members of the miR-34 subfamily [28]. Consistently, miR-34a decreased the protein level of h-eag1 by 70% in SHSY5Y cells, as assessed by Western blot analysis, whereas the MT-AMO increased it, presumably through downregulating the endogenous miR-34a/b/c (Fig. 2B). The h-eag1 antibody recognized two bands of 110 and 125 kDa, respectively, which according to the study by Napp et al [29] represent core-glycosylated form of eag1 protein and the complex N-Linked-glycosylated form of eag1, respectively. We analyzed the summation of the two bands to represent the total h-eag1 protein level and both bands were found affected by miR-34 and MT-AMO. The same observations were expanded to miR-34b and miR-34c and to MCF-7 cells (Figure S6 & S7). Reduction of h-eag1 expression was also seen at the mRNA level (Fig. 2C). Similar results were observed in MCF-7 cells (Figure S7). As a negative control, miR-1 did not produce any appreciable effects on h-eag1 expression.


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 h-eag1.(A) Repression of h-eag1 expression by miR-34a or miR-34c, as reported by luciferase activity assay with the pMIR-REPORTTM luciferase miRNA expression reporter vector carrying the h-eag1 3′UTR in HEK293 cells. Ctl: cells transfected with the luciferase vector alone; MT-AMO: the multiple-target anti-miRNA antisense oligonucleotides to miR-34a, miR-34b and miR-34c, co-transfected with the luciferase vector and miR-34a or miR-34c. *p<0.05 vs Ctl/Lipo; φp<0.05 vs miR-34a; n = 4 for each group. (B) Western blot analysis revealing repression of h-eag1 protein by miR-34a and miR-34c in SHSY5Y cells. *p<0.05 vs Ctl/Lipo; φp<0.05 vs miR-34a alone; n = 4 for each group. The immunoblot bands shown were run on the same gel. (C) Effect of miR-34 on h-eag1 mRNA level in SHSY5Y cells. *p<0.05 vs Ctl/Lipo; φp<0.05 vs miR-34a alone; n = 4 for each group.
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Related In: Results  -  Collection

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

pone-0020362-g002: miR-34 as a post-transcriptional repressor of h-eag1.(A) Repression of h-eag1 expression by miR-34a or miR-34c, as reported by luciferase activity assay with the pMIR-REPORTTM luciferase miRNA expression reporter vector carrying the h-eag1 3′UTR in HEK293 cells. Ctl: cells transfected with the luciferase vector alone; MT-AMO: the multiple-target anti-miRNA antisense oligonucleotides to miR-34a, miR-34b and miR-34c, co-transfected with the luciferase vector and miR-34a or miR-34c. *p<0.05 vs Ctl/Lipo; φp<0.05 vs miR-34a; n = 4 for each group. (B) Western blot analysis revealing repression of h-eag1 protein by miR-34a and miR-34c in SHSY5Y cells. *p<0.05 vs Ctl/Lipo; φp<0.05 vs miR-34a alone; n = 4 for each group. The immunoblot bands shown were run on the same gel. (C) Effect of miR-34 on h-eag1 mRNA level in SHSY5Y cells. *p<0.05 vs Ctl/Lipo; φp<0.05 vs miR-34a alone; n = 4 for each group.
Mentions: We then investigated if h-eag1 is also regulated at the post-transcriptional level by miRNAs. We first performed computational prediction of h-eag1 as a target for miRNA regulation. And we identified multiple binding sites for a tumor-suppressor miRNA subfamily miR-34 (including miR-34a, miR-34b and miR-34c) in the 3′UTR of h-eag1 mRNA (Figure S4). To experimentally establish miR-34:h-eag1 interaction, we inserted a fragment of 3′UTR of h-eag1 containing the miR-34 target sites into the position downstream the luciferase gene in the pMIR-REPORTTM vector. Transfection of miR-34a markedly suppressed the luciferase activities and the effect was reversed by their multiple-target anti-miRNA antisense oligonucleotides (MT-AMO) (Fig. 2A; Supporting Figures online; Figure S5), a single oligomer capable of targeting all three members of the miR-34 subfamily [28]. Consistently, miR-34a decreased the protein level of h-eag1 by 70% in SHSY5Y cells, as assessed by Western blot analysis, whereas the MT-AMO increased it, presumably through downregulating the endogenous miR-34a/b/c (Fig. 2B). The h-eag1 antibody recognized two bands of 110 and 125 kDa, respectively, which according to the study by Napp et al [29] represent core-glycosylated form of eag1 protein and the complex N-Linked-glycosylated form of eag1, respectively. We analyzed the summation of the two bands to represent the total h-eag1 protein level and both bands were found affected by miR-34 and MT-AMO. The same observations were expanded to miR-34b and miR-34c and to MCF-7 cells (Figure S6 & S7). Reduction of h-eag1 expression was also seen at the mRNA level (Fig. 2C). Similar results were observed in MCF-7 cells (Figure S7). As a negative control, miR-1 did not produce any appreciable effects on h-eag1 expression.

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