siRNA-mediated gene silencing: a global genome view.
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.
Global Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA. Dimitri.Semizarov@abbott.com
Dimitri
D
Semizarov
Paul
P
Kroeger
Stephen
S
Fesik
D001665:Binding Sites; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D050684:E2F Transcription Factors; D020869:Gene Expression Profiling; D023281:Genomics / Q000379:methods*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D011518:Proto-Oncogene Proteins; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D020218:Response Elements; D016160:Retinoblastoma Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D012984:Software; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D016160:Retinoblastoma Protein; D014157:Transcription Factors; C495900:CDK4 protein, human; C495911:CDK6 protein, human; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases