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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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FACs analysis of annexin V staining in Kelly cells. (A) transfection with negative control oligonucleotide and (B) siRNA mediated knock down of AKT2 using siRNA. Cells were harvested for detection of externalised annexin V (apoptotic cells) and PI staining of DNA (necrotic cells). Double-stained cells (upper right quadrant) indicate cells in late apoptosis. The resultant increase in apoptosis caused by siRNA inhibition of AKT2 was highly significant (P < 0.0001). Analysis of caspase 3/7 activity in Kelly (C) and SK-N-AS (D) cells following siRNA knockdown of AKT2 relative to negative control (NC).
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Figure 6: FACs analysis of annexin V staining in Kelly cells. (A) transfection with negative control oligonucleotide and (B) siRNA mediated knock down of AKT2 using siRNA. Cells were harvested for detection of externalised annexin V (apoptotic cells) and PI staining of DNA (necrotic cells). Double-stained cells (upper right quadrant) indicate cells in late apoptosis. The resultant increase in apoptosis caused by siRNA inhibition of AKT2 was highly significant (P < 0.0001). Analysis of caspase 3/7 activity in Kelly (C) and SK-N-AS (D) cells following siRNA knockdown of AKT2 relative to negative control (NC).

Mentions: MiR-184 has many computationally predicted targets, so in order to determine if the anti-proliferative effects of miR-184 could be attributable to targeting AKT2, we transfected both Kelly and SK-N-AS neuroblastoma cell lines with three different siRNAs to AKT2 and examined the effects on the rate of accumulation of cell numbers. AKT2 mRNA knockdown ranged from 68 to 98% by 48 hrs, depending on the siRNA (Additional File 4a and 4c), with AKT2 protein being proportionally reduced (Additional File 4b and 4d). Both cell lines exhibited a marked decline in cell numbers for each siRNA relative to the negative control (Figure 5a and 5b), along with a highly significant increase (3.9 fold; P < 0.0001) in the late apoptotic cell fraction assessed by FACs analysis of an annexin V staining assay (Figure 6a and 6b). A statistically significant increase in caspase 3/7 activation in both Kelly and SK-N-AS cells following siRNA mediated AKT2 knock down also occurred (Figure 6c and 6d). Consistent with expectations, AKT1 mRNA levels remained constant when AKT2 siRNA was transfected into the cell lines (Additional File 3b).


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)

FACs analysis of annexin V staining in Kelly cells. (A) transfection with negative control oligonucleotide and (B) siRNA mediated knock down of AKT2 using siRNA. Cells were harvested for detection of externalised annexin V (apoptotic cells) and PI staining of DNA (necrotic cells). Double-stained cells (upper right quadrant) indicate cells in late apoptosis. The resultant increase in apoptosis caused by siRNA inhibition of AKT2 was highly significant (P < 0.0001). Analysis of caspase 3/7 activity in Kelly (C) and SK-N-AS (D) cells following siRNA knockdown of AKT2 relative to negative control (NC).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: FACs analysis of annexin V staining in Kelly cells. (A) transfection with negative control oligonucleotide and (B) siRNA mediated knock down of AKT2 using siRNA. Cells were harvested for detection of externalised annexin V (apoptotic cells) and PI staining of DNA (necrotic cells). Double-stained cells (upper right quadrant) indicate cells in late apoptosis. The resultant increase in apoptosis caused by siRNA inhibition of AKT2 was highly significant (P < 0.0001). Analysis of caspase 3/7 activity in Kelly (C) and SK-N-AS (D) cells following siRNA knockdown of AKT2 relative to negative control (NC).
Mentions: MiR-184 has many computationally predicted targets, so in order to determine if the anti-proliferative effects of miR-184 could be attributable to targeting AKT2, we transfected both Kelly and SK-N-AS neuroblastoma cell lines with three different siRNAs to AKT2 and examined the effects on the rate of accumulation of cell numbers. AKT2 mRNA knockdown ranged from 68 to 98% by 48 hrs, depending on the siRNA (Additional File 4a and 4c), with AKT2 protein being proportionally reduced (Additional File 4b and 4d). Both cell lines exhibited a marked decline in cell numbers for each siRNA relative to the negative control (Figure 5a and 5b), along with a highly significant increase (3.9 fold; P < 0.0001) in the late apoptotic cell fraction assessed by FACs analysis of an annexin V staining assay (Figure 6a and 6b). A statistically significant increase in caspase 3/7 activation in both Kelly and SK-N-AS cells following siRNA mediated AKT2 knock down also occurred (Figure 6c and 6d). Consistent with expectations, AKT1 mRNA levels remained constant when AKT2 siRNA was transfected into the cell lines (Additional File 3b).

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