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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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Relative AKT2 mRNA levels following transfection of miR-184 mimics (pre-miR-184), anti-miR-184, or a scrambled oligo (NC) into (A) Kelly and (B) SK-N-AS cells at 24, 48 and 72 hrs after transfection. RT-qPCR results were normalised to RPLPO ribosomal protein and are relative to the negative control at 24 hrs. Western Blot (C) representing protein levels of AKT2 in Kelly cells 72 hrs after transfection with the miR-184 mimics, a negative control oligo, or anti-miR-184. β-Actin was used for the endogenous control. (D) pMir-Reporter containing the AKT2 binding site for miR-184 in the 3' UTR region of the luciferase gene co-transfected into Kelly cells with a scrambled negative control oligonucleotide (left) or mature miR-184 mimic (middle). The right bar shows the result of co-transfection with the pMir-Reporter containing the mutated AKT2 binding site for miR-184 co-transfected into Kelly cells with mature miR-184 mimic. Luciferase values were normalized to B-galactosidase on a second plasmid that was co-transfected into each cell line and all values are relative to the negative control (left).
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Figure 4: Relative AKT2 mRNA levels following transfection of miR-184 mimics (pre-miR-184), anti-miR-184, or a scrambled oligo (NC) into (A) Kelly and (B) SK-N-AS cells at 24, 48 and 72 hrs after transfection. RT-qPCR results were normalised to RPLPO ribosomal protein and are relative to the negative control at 24 hrs. Western Blot (C) representing protein levels of AKT2 in Kelly cells 72 hrs after transfection with the miR-184 mimics, a negative control oligo, or anti-miR-184. β-Actin was used for the endogenous control. (D) pMir-Reporter containing the AKT2 binding site for miR-184 in the 3' UTR region of the luciferase gene co-transfected into Kelly cells with a scrambled negative control oligonucleotide (left) or mature miR-184 mimic (middle). The right bar shows the result of co-transfection with the pMir-Reporter containing the mutated AKT2 binding site for miR-184 co-transfected into Kelly cells with mature miR-184 mimic. Luciferase values were normalized to B-galactosidase on a second plasmid that was co-transfected into each cell line and all values are relative to the negative control (left).

Mentions: To confirm that AKT2 was indeed regulated by miR-184 in neuroblastoma, the miR-184 mimic and the anti-miR-184 were individually transfected into Kelly and SK-N-AS cell lines. A significant decrease of AKT2 mRNA was observed over three time points (24, 48 and 72 hrs) following transfection of miR-184 mimics into Kelly (P = 0.004)(Figure 4a) and SK-N-AS (P = 0.003)(Figure 4b), and conversely, the suppression of miR-184 using the anti-mir-184 caused an increase in mRNA for AKT2 across all time points in both cell lines (P = 0.009 and 0.01 respectively). These results were also observed at protein level (Figure 4c). MiR-184 is not predicted to target the 3'UTRs of related AKT family members, AKT1 nor AKT3. Consistent with expectations, AKT1 mRNA levels remained constant when miR-184 was transfected into Kelly and SK-N-AS (Additional File 3a), while AKT3 mRNA was undetectable in both cell lines by TaqMan qPCR.


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)

Relative AKT2 mRNA levels following transfection of miR-184 mimics (pre-miR-184), anti-miR-184, or a scrambled oligo (NC) into (A) Kelly and (B) SK-N-AS cells at 24, 48 and 72 hrs after transfection. RT-qPCR results were normalised to RPLPO ribosomal protein and are relative to the negative control at 24 hrs. Western Blot (C) representing protein levels of AKT2 in Kelly cells 72 hrs after transfection with the miR-184 mimics, a negative control oligo, or anti-miR-184. β-Actin was used for the endogenous control. (D) pMir-Reporter containing the AKT2 binding site for miR-184 in the 3' UTR region of the luciferase gene co-transfected into Kelly cells with a scrambled negative control oligonucleotide (left) or mature miR-184 mimic (middle). The right bar shows the result of co-transfection with the pMir-Reporter containing the mutated AKT2 binding site for miR-184 co-transfected into Kelly cells with mature miR-184 mimic. Luciferase values were normalized to B-galactosidase on a second plasmid that was co-transfected into each cell line and all values are relative to the negative control (left).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Relative AKT2 mRNA levels following transfection of miR-184 mimics (pre-miR-184), anti-miR-184, or a scrambled oligo (NC) into (A) Kelly and (B) SK-N-AS cells at 24, 48 and 72 hrs after transfection. RT-qPCR results were normalised to RPLPO ribosomal protein and are relative to the negative control at 24 hrs. Western Blot (C) representing protein levels of AKT2 in Kelly cells 72 hrs after transfection with the miR-184 mimics, a negative control oligo, or anti-miR-184. β-Actin was used for the endogenous control. (D) pMir-Reporter containing the AKT2 binding site for miR-184 in the 3' UTR region of the luciferase gene co-transfected into Kelly cells with a scrambled negative control oligonucleotide (left) or mature miR-184 mimic (middle). The right bar shows the result of co-transfection with the pMir-Reporter containing the mutated AKT2 binding site for miR-184 co-transfected into Kelly cells with mature miR-184 mimic. Luciferase values were normalized to B-galactosidase on a second plasmid that was co-transfected into each cell line and all values are relative to the negative control (left).
Mentions: To confirm that AKT2 was indeed regulated by miR-184 in neuroblastoma, the miR-184 mimic and the anti-miR-184 were individually transfected into Kelly and SK-N-AS cell lines. A significant decrease of AKT2 mRNA was observed over three time points (24, 48 and 72 hrs) following transfection of miR-184 mimics into Kelly (P = 0.004)(Figure 4a) and SK-N-AS (P = 0.003)(Figure 4b), and conversely, the suppression of miR-184 using the anti-mir-184 caused an increase in mRNA for AKT2 across all time points in both cell lines (P = 0.009 and 0.01 respectively). These results were also observed at protein level (Figure 4c). MiR-184 is not predicted to target the 3'UTRs of related AKT family members, AKT1 nor AKT3. Consistent with expectations, AKT1 mRNA levels remained constant when miR-184 was transfected into Kelly and SK-N-AS (Additional File 3a), while AKT3 mRNA was undetectable in both cell lines by TaqMan qPCR.

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