Impact of Targeting the Adenine- and Uracil-Rich Element of bcl-2 mRNA with Oligoribonucleotides on Apoptosis, Cell Cycle, and Neuronal Differentiation in SHSY-5Y Cells

Abstract

We have identified previously a destabilizing adenine-and uracilrich element (ARE) in the 3Ј-UTR of bcl-2 mRNA that interacted with ARE-binding proteins to down-regulate bcl-2 gene expression in response to apoptotic stimuli. We have also described three contiguous 2Ј-O-methyl oligoribonucleotides (ORNs) in both sense and 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, fluorescence-activated cell sorting 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.

The Combined Effect of Retinoic Acid and LSD1 siRNA Inhibition on Cell Death in the Human Neuroblastoma Cell Line SH-SY5Y

“…The pathogenesis of a wide variety of human diseases is frequently related to impaired control of apoptosis [28]. Indeed, we observed that Bcl-2 expression was high in neuroblastic tumors, but Bax expression was relatively low or undetectable.…”

Proceedings of the discoveries on post-transcriptional Bcl-2 deregulation in human leukemias/lymphomas

“…Furthermore, Bcl-2 ORNs stabilized other ARE containing transcripts and up regulated their expression. We also demonstrated that treatment of the SHSY-5Y neuronal cells with Bcl-2 ORNs prevented Bcl-2 down-regulation in response to apoptotic stimuli, such as glucose/growth factor starvation or oxygen deprivation, inhibited cell cycle entry and induced a markedly increase of cellular neurite number and length, a hallmark of neuronal differentiation resulting from Bcl-2 up-regulation [46] . Enhancement of apoptotic threshold and induction neuronal differentiation by Bcl-2 ORNs suggested evaluating their potential application to prevent pathological apoptosis and neuronal degenerations.…”

In vitro and in vivo inhibition of proangiogenic retinal phenotype by an antisense oligonucleotide downregulating uPAR expression

“…Nonetheless, even higher benefits, mainly in term of potency, may be provided by the ASO strategy. In this respect, we have extensively applied this strategy to inhibit the expression of several genes, also in therapeutic settings [21,[25][26][27]. In addition, ASOs have been specifically applied for the treatment of eye diseases including cytomegalovirus retinitis, keratitis-induced corneal neovascularization and inherited retinal diseases [35].…”

“…More than twenty years ago, for the first time we strongly inhibited uPAR expression at the level of its mRNA in cultures of human fibroblasts using a specific ASO (ASO-uPAR) [24]. Having continuously updated our skills on ASO strategies aimed at modulating gene expression in different experimental models [21,[25][26][27], in the present study we explored the effectiveness of ASO-uPAR in inhibiting angiogenic responses both in vitro and in vivo. Therefore, we investigated the ASO-…”