RNAi as a Tool for Mammalian Gene Analysis: Applications of siRNAs
Testing Hypotheses of Gene Function
* Al-Khalili et al (2) treated myotubes with serum and showed that increased glucose uptake correlated with increased cell-surface content of glucose transporter (GLUT1). To confirm that glucose transport depends on GLUT1 expression, cells were treated with GLUT1 siRNA and were shown to have reduced levels of serum-stimulated glucose transport.
* In another report, Chen and Barritt (3) used siRNAs to study the transient receptor potential canonical 1 (TRPC1) gene. The TRPC1 gene was thought to encode a non-selective cation channel activated by depletion of cellular storage and/or an intracellular messenger. When liver cells were treated with the TRPC1 siRNA, they exhibited increased cell volume and decreased inflow of Ca2+, Mn2+, and ATP in hypotonic solutions supporting the hypothesis.
* Filleur et al (4) showed that the antiangiogenic molecule thrombospondin-1 (TSP-1) could reduce vascularization and delay tumor onset. Over time, tumor cells producing active TSP1 began to form exponentially growing tumors. These tumors were composed of cells secreting unusually high amounts of the angiogenic stimulator, vascular endothelial growth factor (VEGF), which were sufficient to overcome the inhibitory TSP1. Treating tumor cells with a combination of TSP1 and a VEGF-specific siRNA caused a striking reduction in cell proliferation. This result suggested that using a combination of TSP1 and an anti-VEGF compound would slow or eliminate tumor growth.
* Based on the observation that fatty acid synthase (FASE) is over-expressed in human epithelial cells, De Schrijver et al (5) considered the gene to be an interesting target for antineoplastic therapy. The researchers used siRNAs to reduce the expression of FASE in lymph node carcinoma of the prostate (LNCaP) cells. The FASE siRNAs caused several phenotypes in the LNCaP cells, including induction of apoptosis. Interestingly, the FASE siRNAs had no effect on the growth rate or viability of nonmalignant cultured skin fibroblasts. These data point out the potential of cancer drugs that selectively inhibit FASE.
* Cyclin E is overexpressed in a number of tumor cells. To determine the potential value of the gene as a drug target, Li et al (6) used siRNAs to reduce cyclin E expression in hepatocellular carcinoma (HCC) cells. As expected, the cyclin E siRNA promoted apoptosis of HCC cells and blocked cell proliferation. In addition, the cyclin E siRNA inhibited HCC tumor growth in nude mice demonstrating the potential for creating drugs targeting cyclin E.
* Ramos-Nino et al (7) exposed RPM cells to crocidolite asbestos and monitored gene expression using arrays. Genes were categorized based on their response. The genes that were highly and quickly up-regulated included the proto-oncogene, fra-1. siRNA-induced reduction in fra-1 expression caused an increase in the expression of both cd44 and c-met, connecting fra-1 with genes governing cell motility and invasion in mesothelioma.
* Glucose levels are thought to be regulated by the family of Akt serine/threonine kinases. When Katome et al (8) reduced the expression of Akt2 with an siRNA, they noted a slight change in cellular glucose regulation. However, when they targeted the two isoforms of the Akt gene (Akt1 and Akt2), they noted a significant change in glucose regulation. Experiments with isoform-specific siRNA ultimately showed that Akt2, and Akt1 to a lesser extent, has an essential role in insulin-stimulated GLUT4 translocation and glucose uptake in two different cell lines, whereas Akt1 and Akt2 contribute equally to insulin-stimulated glycogen synthesis.
RNAi libraries targeting more than 10,000 genes have been used in C. elegans to identify genes that regulate fat (9), life expectancy (10), and mutation control (11). A similar RNAi library for Drosophila has been used to identify the genes responsible for regulating the phosphorylation of Down-Syndrome cell-adhesion molecule (12). In each of the screening applications, the keys to the experiments have been robust phenotypic assays and high quality RNAi libraries.
siRNAs as Therapeutics: The Next Frontier
As the RNAi field continues to develop, moving into animal models, therapeutics, and drug discovery and targeting, Ambion will continue to develop innovative products that harness the power of RNAi for applications to basic, applied and therapeutic research efforts.
- S M Elbashir, J Harborth, W Lendeckel, A Yalcin, Klaus Weber, T Tuschl (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in mammalian cell culture. Nature 411: 494498.
- Al-Khalili L, Cartee GD, Krook A (2003) RNA interference-mediated reduction in GLUT1 inhibits serum-induced glucose transport in primary human skeletal muscle cells. Biochem Biophys Res Commun. 307(1): 12732.
- Chen J, Barritt GJ. (2003) Evidence that TRPC1 (transient receptor potential canonical 1) forms a Ca(2+)-permeable channel linked to the regulation of cell volume in liver cells obtained using small interfering RNA targeted against TRPC1. Biochem J. 373(Pt 2): 32736
- Filleur S, Courtin A, Ait-Si-Ali S, Guglielmi J, Merle C, Harel-Bellan A, Clezardin P, Cabon F (2003) siRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth. Cancer Res. 63(14): 391922.
- De Schrijver E, Brusselmans K, Heyns W, Verhoeven G, Swinnen JV (2003) RNA interference-mediated silencing of the fatty acid synthase gene attenuates growth and induces morphological changes and apoptosis of LNCaP prostate cancer cells. Cancer Res. 63(13): 3799804.
- Li K, Lin SY, Brunicardi FC, Seu P. (2003) Use of RNA interference to target cyclin E-overexpressing hepatocellular carcinoma Cancer Res. 63(13): 35937.
- Ramos-Nino ME, Scapoli L, Martinelli M, Land S, Mossman BT (2003) Microarray analysis and RNA silencing link fra-1 to cd44 and c-met expression in mesothelioma. Cancer Res. 63(13): 353945.
- Katome T, Obata T, Matsushima R, Masuyama N, Cantley LC, Gotoh Y, Kishi K, Shiota H, Ebina Y (2003) Use of RNA interference-mediated gene silencing and adenoviral overexpression to elucidate the roles of AKT/protein kinase B isoforms in insulin actions. J Biol Chem. 25;278(30): 2831223.
- Ashrafi K, Chang FY, Watts JL, Fraser AG, Kamath RS, Ahringer J, Ruvkun G (2003) Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature. 421(6920): 26872.
- Lee SS, Lee RY, Fraser AG, Kamath RS, Ahringer J, Ruvkun G (2003) A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity. Nat Genet. 33(1): 408.
- Pothof J, Van Haaften G, Thijssen K, Kamath RS, Fraser AG, Ahringer J, Plasterk RH, Tijsterman M (2003) Identification of genes that protect the C. elegans genome against mutations by genome-wide RNAi. Genes Dev. 15;17(4): 4438.
- Muda M, Worby CA, Simonson-Leff N, Clemens JC, Dixon JE (2002) Use of double-stranded RNA-mediated interference to determine the substrates of protein tyrosine kinases and phosphatases. Biochem J. 366(Pt 1): 737.
- Caplen NJ (2003) RNAi as a gene therapy approach. Expert Opin Biol Ther. 3(4): 57586.
- McCaffrey AP, Meuse L, Pham TT, Conklin DS, Hannon GJ, Kay MA (2002) RNA interference in adult mice. Nature 418(6893): 3839.
- Song E, Lee SK, Dykxhoorn DM, Novina C, Zhang D, Crawford K, Cerny J, Sharp PA, Lieberman J, Manjunath N, Shankar P (2003) Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages. J Virol. 77(13): 717481.
- Banerjea A, Li MJ, Bauer G, Remling L, Lee NS, Rossi J, Akkina R (2003) Inhibition of HIV-1 by lentiviral vector-transduced siRNAs in T lymphocytes differentiated in SCID-hu mice and CD34+ progenitor cell-derived macrophages. Mol Ther. 8(1): 6271.
- Klein C, Bock CT, Wedemeyer H, Wustefeld T, Locarnini S, Dienes HP, Kubicka S, Manns MP, Trautwein C (2003) Inhibition of hepatitis B virus replication in vivo by nucleoside analogues and siRNA. Gastroenterology. 125(1): 918.
- Daude N, Marella M, Chabry J (2003) Specific inhibition of pathological prion protein accumulation by small interfering RNAs. J Cell Sci. 116: 27759.