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.     

Inhibition of cell proliferation in human breast tumor cells by antisense oligonucleotides against facilitative glucose transporter 5

In recent years, successful examples of antisense oligonucleotide (AS) therapy for genetic diseases have stimulated scientists to investigate its application on cancer diseases. AS can be used to down-regulate the mRNA and protein expression by annealing to specific region of the target mRNA which is responsible for the malignancy. Glucose transporter 5 (Glut5) is a tissue specific transporter that can be found on breast cancer tissues but not on normal breast tissues. Therefore, it is of clinical interest to investigate whether AS against Glut5 mRNA can tackle breast cancer. In this study, two cell lines, MCF-7 which is estrogen-receptor positive and MDA-MB-231 which is estrogen-receptor negative, were used to mimic breast cancer tissues at early and late stages, respectively. A 15-base sequence around the start codon of Glut5 was used. It was found that AS against Glut5 exerted anti-proliferative effect on both of these two breast tumor cell lines and seemed to exert its effect via the suppression of expression of Glut5 proteins in the cells. AS against Glut5 exhibited no effect on human hepatoma HepG2 cells which do not possess any Glut5. The results imply an alternative way in treating breast tumor as the AS against Glut5, unlike tamoxifen, takes effect on breast tumor cells via suppressing the expression of Glut5 that they specifically possess, and regardless whether the breast tumors are estrogen dependent or not.     

Prolonged culture in low glucose induces apoptosis of rat pancreatic beta-cells through induction of c-myc

Prolonged culture in low-glucose concentrations (相似文献