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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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(A) Predicted alignment of miR-184 to the mRNA 3'UTR region of AKT2, as predicted by the Sanger miR Registry. (B) AKT2 mRNA levels were significantly different (p < 0.002) between tumours with high miR-184 levels (predominantly MNA tumors)(n = 10) and tumours with low miR-184 levels (predominantly non-MNA)(n = 9). AKT2 mRNA levels were also significantly different (p < 0.035) between tumours with MNA (n = 10) versus non-MNA (n = 9).
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Figure 2: (A) Predicted alignment of miR-184 to the mRNA 3'UTR region of AKT2, as predicted by the Sanger miR Registry. (B) AKT2 mRNA levels were significantly different (p < 0.002) between tumours with high miR-184 levels (predominantly MNA tumors)(n = 10) and tumours with low miR-184 levels (predominantly non-MNA)(n = 9). AKT2 mRNA levels were also significantly different (p < 0.035) between tumours with MNA (n = 10) versus non-MNA (n = 9).

Mentions: An examination of the Sanger microcosm database http://microrna.sanger.ac.uk/sequences/ indicated that miR-184 has a very large number of computationally predicted mRNA targets. Among the top 3% (n = 30) of miR-184 predicted targets was the 3'UTR of AKT2, which had a high level of sequence identity with the miR-184 seed region, a 13 base pair match (Figure 2a). We focused our studies on AKT2 as a potential miR-184 target given that this was the only gene in the top 3% whose function might account for the apoptotic phenotype induced by miR-184. AKT2 is a well documented pro-survival proteinFor an initial assessment of whether AKT2 mRNA levels and miR-184 levels might be inversely related, qPCR analysis of AKT2 mRNA was carried out on 10 tumors with low miR-184 and 10 with high levels. As illustrated in Figure 2b, AKT2 mRNA levels are significantly lower in tumors with higher miR-184 (P < 0.002). As one might expect, AKT2 was expressed at higher levels in MNA tumors relative to non-MNA tumors (P < 0.035), as MYCN suppresses miR-184 transcript quantities (Figure 2c). An inverse relationship between miR-184 and AKT2 mRNA levels was also determined to exist in neuroblastoma cell lines (Figure 3a and 3b). Levels of AKT2 protein corresponded to the levels of mRNA in the cell lines (Figure 3c).


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)

(A) Predicted alignment of miR-184 to the mRNA 3'UTR region of AKT2, as predicted by the Sanger miR Registry. (B) AKT2 mRNA levels were significantly different (p < 0.002) between tumours with high miR-184 levels (predominantly MNA tumors)(n = 10) and tumours with low miR-184 levels (predominantly non-MNA)(n = 9). AKT2 mRNA levels were also significantly different (p < 0.035) between tumours with MNA (n = 10) versus non-MNA (n = 9).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: (A) Predicted alignment of miR-184 to the mRNA 3'UTR region of AKT2, as predicted by the Sanger miR Registry. (B) AKT2 mRNA levels were significantly different (p < 0.002) between tumours with high miR-184 levels (predominantly MNA tumors)(n = 10) and tumours with low miR-184 levels (predominantly non-MNA)(n = 9). AKT2 mRNA levels were also significantly different (p < 0.035) between tumours with MNA (n = 10) versus non-MNA (n = 9).
Mentions: An examination of the Sanger microcosm database http://microrna.sanger.ac.uk/sequences/ indicated that miR-184 has a very large number of computationally predicted mRNA targets. Among the top 3% (n = 30) of miR-184 predicted targets was the 3'UTR of AKT2, which had a high level of sequence identity with the miR-184 seed region, a 13 base pair match (Figure 2a). We focused our studies on AKT2 as a potential miR-184 target given that this was the only gene in the top 3% whose function might account for the apoptotic phenotype induced by miR-184. AKT2 is a well documented pro-survival proteinFor an initial assessment of whether AKT2 mRNA levels and miR-184 levels might be inversely related, qPCR analysis of AKT2 mRNA was carried out on 10 tumors with low miR-184 and 10 with high levels. As illustrated in Figure 2b, AKT2 mRNA levels are significantly lower in tumors with higher miR-184 (P < 0.002). As one might expect, AKT2 was expressed at higher levels in MNA tumors relative to non-MNA tumors (P < 0.035), as MYCN suppresses miR-184 transcript quantities (Figure 2c). An inverse relationship between miR-184 and AKT2 mRNA levels was also determined to exist in neuroblastoma cell lines (Figure 3a and 3b). Levels of AKT2 protein corresponded to the levels of mRNA in the cell lines (Figure 3c).

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