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MicroRNA-184 inhibits neuroblastoma cell survival through targeting the serine/threonine kinase AKT2.

Foley NH, Bray IM, Tivnan A, Bryan K, Murphy DM, Buckley PG, Ryan J, O'Meara A, O'Sullivan M, Stallings RL - Mol. Cancer (2010)

Bottom Line: The purpose of this study was to elucidate the molecular mechanism by which miR-184 conveys pro-apoptotic effects.Moreover, co-transfection of miR-184 with an AKT2 expression vector lacking the miR-184 target site in the 3'UTR rescues cells from the pro-apoptotic effects of miR-184.Thus, two important genes with positive effects on cell growth and survival, MYCN and AKT2, can be linked into a common genetic pathway through the actions of miR-184.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Genetics, Royal College of Surgeons in Ireland, York House, York Street, Dublin 2, Ireland.

ABSTRACT

Background: Neuroblastoma is a paediatric cancer of the sympathetic nervous system. The single most important genetic indicator of poor clinical outcome is amplification of the MYCN transcription factor. One of many down-stream MYCN targets is miR-184, which is either directly or indirectly repressed by this transcription factor, possibly due to its pro-apoptotic effects when ectopically over-expressed in neuroblastoma cells. The purpose of this study was to elucidate the molecular mechanism by which miR-184 conveys pro-apoptotic effects.

Results: We demonstrate that the knock-down of endogenous miR-184 has the opposite effect of ectopic up-regulation, leading to enhanced neuroblastoma cell numbers. As a mechanism of how miR-184 causes apoptosis when over-expressed, and increased cell numbers when inhibited, we demonstrate direct targeting and degradation of AKT2, a major downstream effector of the phosphatidylinositol 3-kinase (PI3K) pathway, one of the most potent pro-survival pathways in cancer. The pro-apoptotic effects of miR-184 ectopic over-expression in neuroblastoma cell lines is reproduced by siRNA inhibition of AKT2, while a positive effect on cell numbers similar to that obtained by the knock-down of endogenous miR-184 can be achieved by ectopic up-regulation of AKT2. Moreover, co-transfection of miR-184 with an AKT2 expression vector lacking the miR-184 target site in the 3'UTR rescues cells from the pro-apoptotic effects of miR-184.

Conclusions: MYCN contributes to tumorigenesis, in part, by repressing miR-184, leading to increased levels of AKT2, a direct target of miR-184. Thus, two important genes with positive effects on cell growth and survival, MYCN and AKT2, can be linked into a common genetic pathway through the actions of miR-184. As an inhibitor of AKT2, miR-184 could be of potential benefit in miRNA mediated therapeutics of MYCN amplified neuroblastoma and other forms of cancer.

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Growth curves for Kelly (A) and SK-N-AS (B) following transfection with the anti-miR-184 (×3 biological replicates). A scrambled anti-miR was used as negative control.
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Figure 1: Growth curves for Kelly (A) and SK-N-AS (B) following transfection with the anti-miR-184 (×3 biological replicates). A scrambled anti-miR was used as negative control.

Mentions: It was previously reported that transfection of miR-184 mimics into neuroblastoma cell lines causes a decrease in cell numbers and increase in apoptosis in neuroblastoma cell lines [3]. Here, we first assessed whether the reciprocal experiment of knocking down endogenous miR-184 in Kelly and SK-N-AS cell lines following transfection with an anti-miR-184 also has a detectable biological effect. As illustrated in Figure 1a and 1b, the inhibition of miR-184 results in a reproducible and statistically significant increase in cell numbers (~1.6 fold in Kelly; p < 0.0001; and 1.3 fold in SK-N-AS; p < 0.0001) (Figure 1a and 1b). In addition, we also determined that ectopic over-expression of miR-184 at physiological levels using an expression plasmid has similar biological effects on Kelly and SK-N-AS cells as the mature miR-184 mimics which were introduced at supra-physiological levels (Additional File 2a-g). The molecular mechanism by which miR-184 exerts effects on cell numbers and apoptosis of neuroblastoma cells, however, is unknown.


MicroRNA-184 inhibits neuroblastoma cell survival through targeting the serine/threonine kinase AKT2.

Foley NH, Bray IM, Tivnan A, Bryan K, Murphy DM, Buckley PG, Ryan J, O'Meara A, O'Sullivan M, Stallings RL - Mol. Cancer (2010)

Growth curves for Kelly (A) and SK-N-AS (B) following transfection with the anti-miR-184 (×3 biological replicates). A scrambled anti-miR was used as negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2864218&req=5

Figure 1: Growth curves for Kelly (A) and SK-N-AS (B) following transfection with the anti-miR-184 (×3 biological replicates). A scrambled anti-miR was used as negative control.
Mentions: It was previously reported that transfection of miR-184 mimics into neuroblastoma cell lines causes a decrease in cell numbers and increase in apoptosis in neuroblastoma cell lines [3]. Here, we first assessed whether the reciprocal experiment of knocking down endogenous miR-184 in Kelly and SK-N-AS cell lines following transfection with an anti-miR-184 also has a detectable biological effect. As illustrated in Figure 1a and 1b, the inhibition of miR-184 results in a reproducible and statistically significant increase in cell numbers (~1.6 fold in Kelly; p < 0.0001; and 1.3 fold in SK-N-AS; p < 0.0001) (Figure 1a and 1b). In addition, we also determined that ectopic over-expression of miR-184 at physiological levels using an expression plasmid has similar biological effects on Kelly and SK-N-AS cells as the mature miR-184 mimics which were introduced at supra-physiological levels (Additional File 2a-g). The molecular mechanism by which miR-184 exerts effects on cell numbers and apoptosis of neuroblastoma cells, however, is unknown.

Bottom Line: The purpose of this study was to elucidate the molecular mechanism by which miR-184 conveys pro-apoptotic effects.Moreover, co-transfection of miR-184 with an AKT2 expression vector lacking the miR-184 target site in the 3'UTR rescues cells from the pro-apoptotic effects of miR-184.Thus, two important genes with positive effects on cell growth and survival, MYCN and AKT2, can be linked into a common genetic pathway through the actions of miR-184.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Genetics, Royal College of Surgeons in Ireland, York House, York Street, Dublin 2, Ireland.

ABSTRACT

Background: Neuroblastoma is a paediatric cancer of the sympathetic nervous system. The single most important genetic indicator of poor clinical outcome is amplification of the MYCN transcription factor. One of many down-stream MYCN targets is miR-184, which is either directly or indirectly repressed by this transcription factor, possibly due to its pro-apoptotic effects when ectopically over-expressed in neuroblastoma cells. The purpose of this study was to elucidate the molecular mechanism by which miR-184 conveys pro-apoptotic effects.

Results: We demonstrate that the knock-down of endogenous miR-184 has the opposite effect of ectopic up-regulation, leading to enhanced neuroblastoma cell numbers. As a mechanism of how miR-184 causes apoptosis when over-expressed, and increased cell numbers when inhibited, we demonstrate direct targeting and degradation of AKT2, a major downstream effector of the phosphatidylinositol 3-kinase (PI3K) pathway, one of the most potent pro-survival pathways in cancer. The pro-apoptotic effects of miR-184 ectopic over-expression in neuroblastoma cell lines is reproduced by siRNA inhibition of AKT2, while a positive effect on cell numbers similar to that obtained by the knock-down of endogenous miR-184 can be achieved by ectopic up-regulation of AKT2. Moreover, co-transfection of miR-184 with an AKT2 expression vector lacking the miR-184 target site in the 3'UTR rescues cells from the pro-apoptotic effects of miR-184.

Conclusions: MYCN contributes to tumorigenesis, in part, by repressing miR-184, leading to increased levels of AKT2, a direct target of miR-184. Thus, two important genes with positive effects on cell growth and survival, MYCN and AKT2, can be linked into a common genetic pathway through the actions of miR-184. As an inhibitor of AKT2, miR-184 could be of potential benefit in miRNA mediated therapeutics of MYCN amplified neuroblastoma and other forms of cancer.

Show MeSH
Related in: MedlinePlus