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.
Nahreini
Piruz
P
Department of Radiology, School of Medicine, Center for Vitamins and Cancer Research, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA. piruz.nahreini@uchsc.edu
Hanson
Amy J
AJ
Andreatta
Cynthia P
CP
Koustas
William T
WT
Prasad
Kedar N
KN
D000818:Animals; D001483:Base Sequence / Q000235:genetics; D045744:Cell Line, Tumor; D020868:Gene Silencing / Q000502:physiology*; D051379:Mice; D009447:Neuroblastoma / Q000235:genetics*; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000235:genetics; D034622:RNA Interference / Q000502:physiology; D016337:RNA, Catalytic / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics*
D016376:Oligonucleotides, Antisense; D016337:RNA, Catalytic; C427204:hammerhead ribozyme