Construction And The Antitumor Efficacy Of ShRNA Expression Vector Against NRas And C-Myc

RNA interference (RNAi) is a potential tool of cancer gene therapy. There are two RNA interfering agents for the therapeutics. One is synthetic small interfering RNAs (siRNAs), and the other is shRNAs expression vectors. ShRNA expression vector is more stable in vivo and easier to be used for investigating the gene functions compared with synthetic siRNAs. ShRNA expression vectors include viral and non-viral vectors. Viral vector shows high transfection efficiency and low safety. So far, non-viral vector is more attractive because of adequate safety profile.1. Design and construction of a vector with the ability to express several shRNAsPlasmid DNA is hard to penetrate nuclear membrane from cytoplasm to nucleus, and the reduction of its size will improve the mobility. High dosage of plasmid DNA can induce an intense interferon response after transfection into cells, and in contrast the low dosage reduces the induction of interferon response. In this study, we designed a therapeutic shRNA expression plasmid pCSH1. There were two points considered in this design: high efficiency and low non-specific toxicity. This vector had small size and consisted of the basic sequence of pUC19 and H1 promoter identified by RNA polymerase III. To reduce the dosage, several shRNA expression cassettes were assembled into one plasmid.To investigate whether pCSH1 induced effective and specific inhibition of the target mRNA, we designed shRNAs against NRas and c-Myc gene and constructed them into pCSH1, named pCSH1-shN2 and pCSH1-shMyc3. RT-PCR and Western Blot were adopted to detect the mRNA and protein levels of NRas and c-Myc after transient transfection of these plasmids. The results showed that pCSH1-shN2 and pCSH1-shMyc3 specifically inhibited the target gene expression both in mRNA and protein levels.