siRNA-mediated gene silencing: a global genome view

Abstract

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.

Figure 1

siRNA sequences used in this work.

Figure 2

Elimination of the target mRNA (a) and protein (b) by siRNAs 1–5 against Rb1. In control assays, no siRNA was used (N) or a random-sequence control siRNA was used (C).

Figure 3

The pathway associations of the Rb knockdown signature. The Gene Ontology nodes strongly affected by the Rb1 silencing are shown, along with the number of genes affected, number of genes present on the chip (in the affected/present format), and the Z-score, which indicates the relatedness of the gene expression signature to the process. The color of the box reflects the Z-score for the node (red, Z ≥ 5; orange, 2 ≤ Z ≤ 5; yellow, Z ≤ 2).