Silencing of antiapoptotic survivin gene by multiple approaches of RNA interference technology.
Silencing of mammalian gene expression by RNA interference (RNAi) technology can be achieved using small interfering RNA (siRNA) or short hairpin RNA (shRNA). However, the relative effectiveness of these two approaches is not known. It is also not clear whether gene-specific shRNA transcribed from an RNA polymerase II (Pol II)-directed promoter in a fusion form can disrupt the targeted gene expression. Here, we report that using both luciferase and antiapoptotic survivin genes as targets, both siRNA and shRNA approaches significantly silenced the targeted gene expression in cancer cells. We further demonstrated that shRNAs transcribed from an RNA Pol II-mediated promoter in a green fluorescent protein (GFP) fusion form at the 3'-untranslated region silenced luciferase and survivin expression as well, suggesting that the extra RNA sequence outside of the shRNA hairpin does not disrupt shRNA function. We also showed that silencing of survivin expression selectively induces apoptosis in transfected cells. Together, we have validated multiple approaches of RNAi technology using both survivin and luciferase genes as targets and demonstrated for the first time that GFP-shRNAs transcribed from an RNA Pol II-mediated promoter could mediate gene silencing, which may lead to new directions for the application of RNAi technology.
Ling
Xiang
X
Roswell Park Cancer Institute, Buffalo, NY, USA.
Li
Fengzhi
F
D017209:Apoptosis / Q000235:genetics*; D001483:Base Sequence; D020868:Gene Silencing / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009363:Neoplasm Proteins; D015202:Protein Engineering / Q000379:methods*; D034622:RNA Interference / Q000502:physiology*; D000077022:Survivin
C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D000077022:Survivin