Targeting microRNA to induce cell death in Medulloblastoma


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Yuen Ngan Fan (1), Daniel Meley (1), Dave G Spiller (1), Mike RH White (1), Barry Pizer (2), Violaine See (1)
University of Liverpool, Liverpool, United Kingdom (1), Alder Hey Hospital, Liverpool, United Kingdom (2)

Background

Neuronal proliferation, differentiation and migration are coordinated during cerebellar development. Disruption of these processes can lead to Medulloblastoma (MB), the most common malignant paediatric brain tumour. Etoposide inhibits the DNA repair mechanism thus activating the p53 apoptotic pathway. However, some MB cells are resistant to etoposide, and we have previously shown a correlation between p53 activity in brain tumour cells and their sensitivity to chemotherapeutic intervention. Our aim is to further elucidate the molecular mechanisms underlying brain tumour resistance to chemotherapeutic agents. Here we show that the p53-dependent microRNA, miR-34a, is induced upon etoposide treatment in p53 WT cells and participate to the cell death program.

Method

We used two medulloblastoma cells lines, D-283 (p53 WT) and MEB-Med-8A (p53 mutated).  We performed a combination of single cell imaging, real time PCR and survival assays. We used time-lapse confocal microscopy in single living cells to assess p53 activity. Transcriptional activity subsequent of p53 activation was detected by real-time PCR. Cell survival was measured by MTS assay (Promega).

Results

We observed p53 nuclear accumulation in D-283 cells treated with etoposide but not observed in the p53 mutated cell type Med-8. We have then shown that etoposide induces a p53-dependent miR34a transcription in D283 cells. This was correlated with decreasing level of the NAD-dependent deacetylase Sirtuin1 (SIRT1). We have further demonstrated that the activation of miR34a and the subsequent the loss of SIRT-1 is sufficient to induce cell death in p53 mutated cells.

Conclusion

We have demonstrated that in p53 WT cells, etoposide induces miR34a transcription that participates to the death signalling. More importantly, in p53 mutated cells, we can bypass p53 mutation by directly activating miR34a, which is enough to trigger cell death. In conclusion, mir34a and its target SIRT1 could constitute new molecular targets for brain tumours.

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