Short interfering RNA (siRNA) is widely used for studying gene function and holds great promise as a tool for validating drug targets and treating disease. A critical assumption in these applications is that the effect of siRNA on cells is specific, i.e., limited to the specific knockdown of the target gene. In this article, we characterize the specificity of siRNA by applying gene expression profiling. Several siRNAs were designed against different regions of the same target gene for three different targets. Their effects on cells were compared by using DNA microarrays to generate gene expression signatures. When the siRNA design and transfection conditions were optimized, the signatures for different siRNAs against the same target were shown to correlate very closely, whereas the signatures for different genes revealed no correlation. These results indicate that siRNA is a highly specific tool for targeted gene knockdown, establishing siRNA-mediated gene silencing as a reliable approach for large-scale screening of gene function and drug target validation.DNA microarray ͉ RNA interference ͉ gene knockdown T he use of RNA interference for inhibiting gene expression represents a powerful tool for exploring gene function (1-7), identifying and validating new drug targets, and treating disease (8, 9). The process of RNA interference is mediated by doublestranded RNA, which is cleaved by the enzyme DICER into 21-to 23-nt duplexes containing a 2-nt overhang at the 3Ј end of each strand. These duplexes are incorporated into a protein complex called the RNA-induced silencing complex (RISC). Directed by the antisense strand of the duplex, RISC recognizes and cleaves the target mRNA (for recent reviews, see refs. 1 and 10-13). Although long double-stranded RNAs (Ͼ30 nt) invoke an interferon response, short interfering RNAs (siRNAs) that resemble the products produced by DICER have been reported to specifically inhibit gene expression in many different mammalian cell lines (1-7). It has been shown that even single nucleotide mismatches between the antisense strand of the siRNA and target mRNA can abolish RNA interference (14). In addition, mapping of mRNA cleavage sites has revealed no cleavage sites outside of the region of complementarity (10). However, the specificity of siRNA at the cellular level remains to be comprehensively studied.For siRNAs to be a useful tool in gene knockdown experiments, it is critical that siRNA-mediated transcriptional silencing be specific. It is not enough to simply show that a control siRNA with a scrambled nucleotide sequence fails to knock down the protein of interest or produce the same cellular phenotype. Ideally, the siRNA must not cause any effects other than those related to the knock down of the target gene. There are several types of nonspecific effects that siRNA could potentially display. In addition to the possibility for cross-hybridization of the antisense strand of the siRNA to different mRNAs, siRNAs could bind in a sequencedependent manner to various cellular proteins. I...
Non-small-cell lung cancer (NSCLC) is the most deadly type of cancer in the United States and worldwide. Although new therapy is available, the survival rate of NSCLC patients remains low. One hallmark of cancer cells is defects in the apoptotic cell death program. In this study, we investigate the role of B-cell lymphoma 2 (Bcl-2) family members Bcl-2, Bcl-
Validating potential targets is an important step in the drug discovery process. In this study, we tested the feasibility of using inducible RNA interference (RNAi) in vivo to obtain an unbiased evaluation on the efficacy of inhibiting hypoxiainducible factor-1A (HIF-1A) in established tumors. We showed that HIF-1A inhibition resulted in transient tumor stasis or tumor regression, and inhibiting HIF-1A in earlystage tumors was found to be more efficacious than inhibiting HIF-1A in more established tumors. A differential requirement of HIF-1A for tumor growth was also observed among different tumor types. Examination of tumors resistant to HIF-1A inhibition suggested that the resistance might result from a less hypoxic tumor environment and the level of HIF-1A expression in tumors may be a useful marker for predicting tumor response to HIF-1 inhibition. This study shows that inducible RNAi is a versatile tool for evaluating cancer targets in vivo. In addition to broad implications on in vivo validation of cancer targets, results from this study will also be instructive for practical applications of HIF-1-based cancer therapeutics. (Cancer Res 2005; 65(16): 7249-58)
Many established cancer therapies involve DNA-damaging chemotherapy or radiotherapy. Gain of DNA repair capacity of the tumor represents a common mechanism used by cancer cells to survive DNA-damaging therapy. Poly(ADPribose) polymerase-1 (PARP-1) is a nuclear enzyme that is activated by DNA damage and plays a critical role in base excision repair. Inhibition of PARP represents an attractive approach for the treatment of cancer. Previously, we have described the discovery and characterization of a potent PARP inhibitor, ABT-888. ABT-888 potentiates the activity of DNA-damaging agents such as temozolomide (TMZ) in a variety of preclinical models. We report here the generation of HCT116 cells resistant to treatment with TMZ and ABT-888 (HCT116R cells). HCT116R cells exhibit decreased H2AX phosphorylation in response to treatment with TMZ and ABT-888 relative to parental HCT116 cells. Microarray and Western blot studies indicate that HCT116R cells have decreased PARP-1 and elevated Rad51 expression levels. HCT116R cells are dependent on Rad51 for proliferation and survival, as shown by inhibition of proliferation and induction of apoptosis upon treatment with Rad51 small interfering RNA. In addition, HCT116R cells are more resistant to radiation than the parental HCT116 cells. Our study suggests that cancer cells upregulate the homologous recombination DNA repair pathway to compensate for the loss of base excision repair, which may account for the observed resistance to treatment with TMZ and ABT-888.
The task of specific gene knockdown in vitro has been facilitated through the use of short interfering RNA (siRNA), which is now widely used for studying gene function, as well as for identifying and validating new drug targets. We explored the possibility of using siRNA for dissecting cellular pathways by siRNA-mediated gene silencing followed by gene expression profiling and systematic pathway analysis. We used siRNA to eliminate the Rb1 gene in human cells and determined the effects of Rb1 knockdown on the cell by using microarray-based gene expression profiling coupled with quantitative pathway analysis using the GenMapp and MappFinder software. Retinoblastoma protein is one of the key cell cycle regulators, which exerts its function through its interactions with E2F transcription factors. Rb1 knockdown affected G1/S and G2/M transitions of the cell cycle, DNA replication and repair, mitosis, and apoptosis, indicating that siRNA-mediated transient elimination of Rb1 mimics the control of cell cycle through Rb1 dissociation from E2F. Additionally, we observed significant effects on the processes of DNA damage response and epigenetic regulation of gene expression. Analysis of transcription factor binding sites was utilized to distinguish between putative direct targets and genes induced through other mechanisms. Our approach, which combines the use of siRNA-mediated gene silencing, mediated microarray screening and quantitative pathway analysis, can be used in functional genomics to elucidate the role of the target gene in intracellular pathways. The approach also holds significant promise for compound selection in drug discovery.
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