Impact of targeting the AU-rich element of bcl-2 mRNA with oligoribonucleotides on apoptosis, cell cycle and neuronal differentiation in SHSY-5Y cells

We have previously identified a destabilizing AU-rich element (ARE) in the 3 ′ UTR of bcl-2 mRNA that interacted with ARE-binding proteins (AUBPs) to down-regulate bcl-2 gene expression in response to apoptotic stimuli. We have also described three contiguous 2 ′ -O-methyl oligoribonucleotides both in sense and in antisense orientation with respect to the bcl-2 ARE that are able to regulate the bcl-2 mRNA half-life and Bcl-2 protein level in two different cell lines. Here we show that treatment of neuronal cell line (SHSY-5Y) with antisense ORNs targeting the bcl-2 ARE ( bcl-2 ARE asORNs) prevents bcl-2 down-regulation in response to apoptotic stimuli with glucose/growth factor starvation (Locke medium) or oxygen deprivation, and enhances the apoptotic threshold as evaluated by Time lapse videomicroscopy, FACS analysis and caspase-3 activation. Additional effects of bcl-2 ARE asORNs included inhibition of cell cycle entry and a marked increase of cellular neurite number and length, a hallmark of neuronal differentiation resulting from bcl-2 up-regulation. The ability of bcl-2 ARE asORNs to enhance the apoptotic threshold and to induce neuronal differentiation implies their potential application as a novel informational tool to protect cells from ischemic damage and to prevent neuronal degeneration. Annexin cells into early and late stage of excluded from cells and early apoptotic cells, but permeates late stage apoptotic and dead cells. assay was manufacturer’s Statistical Analysis. The statistical evaluation of the data was performed with the two-tailed Student’s t test for unpaired values. Differences were considered statistically significant when p ≤ 0.05. The data are reported as percentage of the maximal value. 24 th and 48 th hours, respectively. Treatment with the degORN did not have any significant effect on the viability as compared to the untreated control.

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