Altering cellular signaling pathways enhance gene silencing activity of shRNA, shRNA.ribozyme, and shRNA.antisense in neuroblastoma cells

1. RNA interference (RNAi) is a multicomponent machinery that operates in a sequence-specific manner to repress the expression of genes in most eukaryotic cells. 2. Here we wanted to investigate in a murine neuroblastoma cell line (NBP2) (a) if replacement of the loop of the short hairpin RNA (shRNA) with a hammerhead ribozyme (shRNA.RZ) or an antisense oligonucleotide (shRNA. AS) would affect the efficacy of gene suppression, and (b) if activation or inhibition of signaling pathways would enhance the efficacy of shRNA, shRNA.RZ, and shRNA. AS complex in gene silencing. 3. We used U6-driven expression of these shRNAs to target either a short-lived green fluorescent protein (d2EGFP) or an endogenous cyclophilin A (CyP-A) gene in a d2EGFP expressing NBP2 cell line (NBP2-PN25). 4. Activation of the cAMP signaling pathway or inhibition of phosphatidylinositol 3-kinase (PI3K) enhanced the efficacy of shRNA and shRNA.RZ complex in reducing the expression of d2EGFP shRNA.RZ complex was as efficacious as shRNA in reducing the expression of d2EGFP and CyP-A shRNA. AS complex showed a slightly lower efficacy than shRNA alone in decreasing d2EGFP expression. In contrast, the U6-driven hammerhead ribozyme targeted to d2EGFP showed no gene silencing activity. 5. This report describes novel strategies of modifying shRNA and altering signaling pathways to affect siRNA-mediated gene silencing in a neuronal cell line.