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

• 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
• ISSN: 0022-3549; 1520-6017
• DOI: 10.1002/jps.21398

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.