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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.

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Role of MAP3K9 in miR-34a-mediated apoptosis. The 3'UTR of MAP3K9 was identified as a putative target of miR-34a with a 7-mer complementarity region (Figure 3A). Quantitative PCR and Western Blotting of miR-34a- treated NB1691luc and SK-N-ASluc cells was carried out and both MAP3K9 mRNA transcript and protein levels were shown to significantly decrease relative to premiR-negative control-treated samples in both cells lines analysed (n = 3). Figure 3C outlines the possible mechanism through which miR-34a might mediate cellular apoptosis, targeting and suppressing MAP3K9 expression, leading to downstream reduction in JNK, p38, CREB and STAT3 activated protein levels (verified by phosphoprotein analysis Figure 2)
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Figure 3: Role of MAP3K9 in miR-34a-mediated apoptosis. The 3'UTR of MAP3K9 was identified as a putative target of miR-34a with a 7-mer complementarity region (Figure 3A). Quantitative PCR and Western Blotting of miR-34a- treated NB1691luc and SK-N-ASluc cells was carried out and both MAP3K9 mRNA transcript and protein levels were shown to significantly decrease relative to premiR-negative control-treated samples in both cells lines analysed (n = 3). Figure 3C outlines the possible mechanism through which miR-34a might mediate cellular apoptosis, targeting and suppressing MAP3K9 expression, leading to downstream reduction in JNK, p38, CREB and STAT3 activated protein levels (verified by phosphoprotein analysis Figure 2)

Mentions: Based upon the noted alterations in phosphoprotein activation levels, as discussed above, we examined the TargetScan miRNA prediction database [30] for potential kinases that might be direct targets of miR-34a that could account for these alterations. As illustrated in Figure 3A, the 3'UTR of MAP3K9 (MLK1) has a 7-mer complementarity region with the miR-34a seed region, leading us to examine the effects of miR-34a over expression on MAP3K9 mRNA transcripts and protein expression in NB1691luc and SK-N-ASluc cells. Notably, as shown in Figure 3B, the presence of miR-34a led to a significant reduction in MAP3K9 mRNA and protein expression in both neuroblastoma cell lines relative to premiR-negative control treated samples (n = 3). In order to validate that the 3' UTR of MAP3K9 is a direct target of miR-34a, a 1,140 base pair segment of the MAP3K9 3' UTR, inclusive of the miR-34a target site, was cloned into the 3' region of the luciferase gene in the PsiCheck2 vector (Psi/miR-34a, Additional File 2, Figure S2). In addition, a second construct was created with a 5 base pair mutation within the target seed site (Psi/miR34amut). As illustrated in Additional File 3, Figure S3, co-transfection of Psi/miR-34a with mature miR-34a mimics did not decrease luciferase activity relative to the negative control. Negative results for these experiments were obtained at different time points (24, 48 and 72 hrs) and with two cell lines (SK-N-AS and NB1691), indicating that either the miR-34a affect on MAP3K9 is not a direct effect, or that there is some conformational structural difference between the 3' UTR of the reporter versus the native 3' UTR, which inhibits miR-34a targeting of the reporter.


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)

Role of MAP3K9 in miR-34a-mediated apoptosis. The 3'UTR of MAP3K9 was identified as a putative target of miR-34a with a 7-mer complementarity region (Figure 3A). Quantitative PCR and Western Blotting of miR-34a- treated NB1691luc and SK-N-ASluc cells was carried out and both MAP3K9 mRNA transcript and protein levels were shown to significantly decrease relative to premiR-negative control-treated samples in both cells lines analysed (n = 3). Figure 3C outlines the possible mechanism through which miR-34a might mediate cellular apoptosis, targeting and suppressing MAP3K9 expression, leading to downstream reduction in JNK, p38, CREB and STAT3 activated protein levels (verified by phosphoprotein analysis Figure 2)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Role of MAP3K9 in miR-34a-mediated apoptosis. The 3'UTR of MAP3K9 was identified as a putative target of miR-34a with a 7-mer complementarity region (Figure 3A). Quantitative PCR and Western Blotting of miR-34a- treated NB1691luc and SK-N-ASluc cells was carried out and both MAP3K9 mRNA transcript and protein levels were shown to significantly decrease relative to premiR-negative control-treated samples in both cells lines analysed (n = 3). Figure 3C outlines the possible mechanism through which miR-34a might mediate cellular apoptosis, targeting and suppressing MAP3K9 expression, leading to downstream reduction in JNK, p38, CREB and STAT3 activated protein levels (verified by phosphoprotein analysis Figure 2)
Mentions: Based upon the noted alterations in phosphoprotein activation levels, as discussed above, we examined the TargetScan miRNA prediction database [30] for potential kinases that might be direct targets of miR-34a that could account for these alterations. As illustrated in Figure 3A, the 3'UTR of MAP3K9 (MLK1) has a 7-mer complementarity region with the miR-34a seed region, leading us to examine the effects of miR-34a over expression on MAP3K9 mRNA transcripts and protein expression in NB1691luc and SK-N-ASluc cells. Notably, as shown in Figure 3B, the presence of miR-34a led to a significant reduction in MAP3K9 mRNA and protein expression in both neuroblastoma cell lines relative to premiR-negative control treated samples (n = 3). In order to validate that the 3' UTR of MAP3K9 is a direct target of miR-34a, a 1,140 base pair segment of the MAP3K9 3' UTR, inclusive of the miR-34a target site, was cloned into the 3' region of the luciferase gene in the PsiCheck2 vector (Psi/miR-34a, Additional File 2, Figure S2). In addition, a second construct was created with a 5 base pair mutation within the target seed site (Psi/miR34amut). As illustrated in Additional File 3, Figure S3, co-transfection of Psi/miR-34a with mature miR-34a mimics did not decrease luciferase activity relative to the negative control. Negative results for these experiments were obtained at different time points (24, 48 and 72 hrs) and with two cell lines (SK-N-AS and NB1691), indicating that either the miR-34a affect on MAP3K9 is not a direct effect, or that there is some conformational structural difference between the 3' UTR of the reporter versus the native 3' UTR, which inhibits miR-34a targeting of the reporter.

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