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


CONCLUSIONS
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.
Department of Cancer Genetics, Royal College of Surgeons in Ireland, York House, York Street, Dublin 2, Ireland.
Amanda
A
Tivnan
Lorraine
L
Tracey
Patrick G
PG
Buckley
Leah C
LC
Alcock
Andrew M
AM
Davidoff
Raymond L
RL
Stallings
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D016147:Genes, Tumor Suppressor; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements; D020930:MAP Kinase Kinase Kinases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D035683:MicroRNAs / Q000008:administration & dosage / Q000235:genetics*; D009447:Neuroblastoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D010750:Phosphoproteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D047368:Tumor Burden / Q000235:genetics; D023041:Xenograft Model Antitumor Assays
C522425:MIRN34 microRNA, human; D035683:MicroRNAs; D010750:Phosphoproteins; D008156:Luciferases; D020930:MAP Kinase Kinase Kinases; C433243:MAP3K9 protein, human
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