Limits...
MicroRNA-34a is a potent tumor suppressor molecule in vivo in neuroblastoma.

Tivnan A, Tracey L, Buckley PG, Alcock LC, Davidoff AM, Stallings RL - BMC Cancer (2011)

Bottom Line: As a potential mechanism of miR-34a action on phosphoprotein levels, we demonstrate that miR-34a over-expression results in a significant reduction of MAP3K9 mRNA and protein levels.Despite this fact, any functional effects of reduced MAP3K9 expression as a result of miR-34a would be expected to be similar regardless of the mechanism involved.We demonstrate for the first time that miR-34a significantly reduces tumor growth in an in vivo orthotopic murine model of neuroblastoma and identified novel effects that miR-34a has on phospho-activation of key proteins involved with apoptosis.

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 which originates from precursor cells of the sympathetic nervous system and accounts for 15% of childhood cancer mortalities. With regards to the role of miRNAs in neuroblastoma, miR-34a, mapping to a chromosome 1p36 region that is commonly deleted, has been found to act as a tumor suppressor through targeting of numerous genes associated with cell proliferation and apoptosis.

Methods: A synthetic miR-34a (or negative control) precursor molecule was transfected into NB1691luc and SK-N-ASluc neuroblastoma cells. Quantitative PCR was used to verify increased miR-34a levels in NB1691luc and SK-N-ASluc cell lines prior to in vitro and in vivo analysis. In vitro analysis of the effects of miR-34a over expression on cell growth, cell cycle and phosphoprotein activation in signal transduction pathways was performed. Neuroblastoma cells over expressing miR-34a were injected retroperitoneally into immunocompromised CB17-SCID mice and tumor burden was assessed over a 21 day period by measuring bioluminescence (photons/sec/cm²).

Results: Over expression of miR-34a in both NB1691luc and SK-N-ASluc neuroblastoma cell lines led to a significant decrease in cell number relative to premiR-negative control treated cells over a 72 hour period. Flow cytometry results indicated that miR-34a induced cell cycle arrest and subsequent apoptosis activation. Phosphoprotein analysis highlighted key elements involved in signal transduction, whose activation was dysregulated as a result of miR-34a introduction into cells. As a potential mechanism of miR-34a action on phosphoprotein levels, we demonstrate that miR-34a over-expression results in a significant reduction of MAP3K9 mRNA and protein levels. Although MAP3K9 is a predicted target of miR-34a, direct targeting could not be validated with luciferase reporter assays. Despite this fact, any functional effects of reduced MAP3K9 expression as a result of miR-34a would be expected to be similar regardless of the mechanism involved. Most notably, in vivo studies showed that tumor growth was significantly repressed after exogenous miR-34a administration in retroperitoneal neuroblastoma tumors.

Conclusion: We demonstrate for the first time that miR-34a significantly reduces tumor growth in an in vivo orthotopic murine model of neuroblastoma and identified novel effects that miR-34a has on phospho-activation of key proteins involved with apoptosis.

Show MeSH

Related in: MedlinePlus

Effects of miR-34a on cellular phosphoprotein activation in NB1691luc cells. NB1691luc (1 × 106) cells were reverse transfected with premiR-34a (30 μM) or a premiR-negative control molecule. Protein lysates were isolated after 48 hours and 10 μg of total cell protein was analysed for phosphoprotein alterations using the MILLIPLEX MAP 8-Plex Multi-Pathway Signalling Phosphoprotein Kit (Millipore Corp.,). ERK/MAP Kinase 1/2 activation significantly increased in miR-34a treated cells relative to premiR-negative control treated NB1691luc cells (p < 0.01 n = 3). Notably, activated STAT3, JNK and p38 levels tended towards a significant reduction relative to premiR-negative control- treated samples (p = 0.046, 0.07 and 0.009 respectively)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3038978&req=5

Figure 2: Effects of miR-34a on cellular phosphoprotein activation in NB1691luc cells. NB1691luc (1 × 106) cells were reverse transfected with premiR-34a (30 μM) or a premiR-negative control molecule. Protein lysates were isolated after 48 hours and 10 μg of total cell protein was analysed for phosphoprotein alterations using the MILLIPLEX MAP 8-Plex Multi-Pathway Signalling Phosphoprotein Kit (Millipore Corp.,). ERK/MAP Kinase 1/2 activation significantly increased in miR-34a treated cells relative to premiR-negative control treated NB1691luc cells (p < 0.01 n = 3). Notably, activated STAT3, JNK and p38 levels tended towards a significant reduction relative to premiR-negative control- treated samples (p = 0.046, 0.07 and 0.009 respectively)

Mentions: Over expression of miR-34a led to enhanced activation of ERK/MAP kinase 1/2 (p = 0.002, n = 3; Figure 2). Conversely, transfection of cells with synthetic miR-34a led to a significant reduction in STAT3 (p = 0.04) and p38 phosphorylation (p = 0.009) (Figure 2).


MicroRNA-34a is a potent tumor suppressor molecule in vivo in neuroblastoma.

Tivnan A, Tracey L, Buckley PG, Alcock LC, Davidoff AM, Stallings RL - BMC Cancer (2011)

Effects of miR-34a on cellular phosphoprotein activation in NB1691luc cells. NB1691luc (1 × 106) cells were reverse transfected with premiR-34a (30 μM) or a premiR-negative control molecule. Protein lysates were isolated after 48 hours and 10 μg of total cell protein was analysed for phosphoprotein alterations using the MILLIPLEX MAP 8-Plex Multi-Pathway Signalling Phosphoprotein Kit (Millipore Corp.,). ERK/MAP Kinase 1/2 activation significantly increased in miR-34a treated cells relative to premiR-negative control treated NB1691luc cells (p < 0.01 n = 3). Notably, activated STAT3, JNK and p38 levels tended towards a significant reduction relative to premiR-negative control- treated samples (p = 0.046, 0.07 and 0.009 respectively)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3038978&req=5

Figure 2: Effects of miR-34a on cellular phosphoprotein activation in NB1691luc cells. NB1691luc (1 × 106) cells were reverse transfected with premiR-34a (30 μM) or a premiR-negative control molecule. Protein lysates were isolated after 48 hours and 10 μg of total cell protein was analysed for phosphoprotein alterations using the MILLIPLEX MAP 8-Plex Multi-Pathway Signalling Phosphoprotein Kit (Millipore Corp.,). ERK/MAP Kinase 1/2 activation significantly increased in miR-34a treated cells relative to premiR-negative control treated NB1691luc cells (p < 0.01 n = 3). Notably, activated STAT3, JNK and p38 levels tended towards a significant reduction relative to premiR-negative control- treated samples (p = 0.046, 0.07 and 0.009 respectively)
Mentions: Over expression of miR-34a led to enhanced activation of ERK/MAP kinase 1/2 (p = 0.002, n = 3; Figure 2). Conversely, transfection of cells with synthetic miR-34a led to a significant reduction in STAT3 (p = 0.04) and p38 phosphorylation (p = 0.009) (Figure 2).

Bottom Line: As a potential mechanism of miR-34a action on phosphoprotein levels, we demonstrate that miR-34a over-expression results in a significant reduction of MAP3K9 mRNA and protein levels.Despite this fact, any functional effects of reduced MAP3K9 expression as a result of miR-34a would be expected to be similar regardless of the mechanism involved.We demonstrate for the first time that miR-34a significantly reduces tumor growth in an in vivo orthotopic murine model of neuroblastoma and identified novel effects that miR-34a has on phospho-activation of key proteins involved with apoptosis.

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 which originates from precursor cells of the sympathetic nervous system and accounts for 15% of childhood cancer mortalities. With regards to the role of miRNAs in neuroblastoma, miR-34a, mapping to a chromosome 1p36 region that is commonly deleted, has been found to act as a tumor suppressor through targeting of numerous genes associated with cell proliferation and apoptosis.

Methods: A synthetic miR-34a (or negative control) precursor molecule was transfected into NB1691luc and SK-N-ASluc neuroblastoma cells. Quantitative PCR was used to verify increased miR-34a levels in NB1691luc and SK-N-ASluc cell lines prior to in vitro and in vivo analysis. In vitro analysis of the effects of miR-34a over expression on cell growth, cell cycle and phosphoprotein activation in signal transduction pathways was performed. Neuroblastoma cells over expressing miR-34a were injected retroperitoneally into immunocompromised CB17-SCID mice and tumor burden was assessed over a 21 day period by measuring bioluminescence (photons/sec/cm²).

Results: Over expression of miR-34a in both NB1691luc and SK-N-ASluc neuroblastoma cell lines led to a significant decrease in cell number relative to premiR-negative control treated cells over a 72 hour period. Flow cytometry results indicated that miR-34a induced cell cycle arrest and subsequent apoptosis activation. Phosphoprotein analysis highlighted key elements involved in signal transduction, whose activation was dysregulated as a result of miR-34a introduction into cells. As a potential mechanism of miR-34a action on phosphoprotein levels, we demonstrate that miR-34a over-expression results in a significant reduction of MAP3K9 mRNA and protein levels. Although MAP3K9 is a predicted target of miR-34a, direct targeting could not be validated with luciferase reporter assays. Despite this fact, any functional effects of reduced MAP3K9 expression as a result of miR-34a would be expected to be similar regardless of the mechanism involved. Most notably, in vivo studies showed that tumor growth was significantly repressed after exogenous miR-34a administration in retroperitoneal neuroblastoma tumors.

Conclusion: We demonstrate for the first time that miR-34a significantly reduces tumor growth in an in vivo orthotopic murine model of neuroblastoma and identified novel effects that miR-34a has on phospho-activation of key proteins involved with apoptosis.

Show MeSH
Related in: MedlinePlus