Quantitative and Temporal Analysis of Gene Silencing in Tumor Cells Induced by Small Interfering RNA or Short Hairpin RNA Expressed from Plasmid Vectors

Vector-based RNA interference (RNAi) has attracted great interest, because of its more prolonged gene silencing effect compared with small interfering RNA (siRNA). However, the intensity and duration of vector-based RNAi effect has received little attention. In this study, the gene silencing kinetic...

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Bibliographic Details
Published in:Journal of pharmaceutical sciences 2009-01, Vol.98 (1), p.74-80
Main Authors: Takahashi, Yuki, Yamaoka, Kiyoshi, Nishikawa, Makiya, Takakura, Yoshinobu
Format: Article
Language:English
Subjects:
Adenoviridae - genetics
Animals
Biological and medical sciences
bootstrap method
Cell Line, Tumor
DNA, Viral - genetics
Gene Expression Regulation, Neoplastic - genetics
Gene Silencing - physiology
General pharmacology
Genetic Vectors - biosynthesis
Genetic Vectors - genetics
Luciferases, Firefly - genetics
Luciferases, Renilla - genetics
Medical sciences
Melanoma, Experimental
Mice
moment analysis
Pharmaceutical technology. Pharmaceutical industry
Pharmacology. Drug treatments
RNA interference
RNA polymerase III promoter
RNA, Small Interfering - biosynthesis
RNA, Small Interfering - genetics
RNA, Small Nuclear - chemistry
Time Factors
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Summary:Vector-based RNA interference (RNAi) has attracted great interest, because of its more prolonged gene silencing effect compared with small interfering RNA (siRNA). However, the intensity and duration of vector-based RNAi effect has received little attention. In this study, the gene silencing kinetics of short hairpin RNA (shRNA)-expressing plasmid DNA (pDNA) driven by U6, H1 or tRNA promoter (pU6-shLuc, pH1-shLuc, and ptRNA-shLuc) was studied in melanoma cells expressing firefly luciferase. A bootstrap method-based moment analysis was performed to statistically and quantitatively evaluate the profile of gene silencing. The analysis showed that pU6-shLuc induced a significantly greater and longer gene silencing than that produced by other promoter-driven shRNA expression vectors. In addition, it was found that pU6–shLuc was at least 100-fold more potent in gene silencing than siRNA targeting the same gene on a numerical basis. These statistical considerations demonstrated that U6 promoter-driven shRNA expressing pDNA is the most effective in inducing gene silencing effect as far as the intensity and duration of RNAi effect is concerned.
ISSN:0022-3549
1520-6017
DOI:10.1002/jps.21398