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.

Retinoblastoma Tumor Suppressor Gene Expression Determines the Response to Sequential Flavopiridol and Doxorubicin Treatment in Small-Cell Lung Carcinoma

“…The effect of sequential administration on the cytotoxic interaction between flavopiridol and doxorubicin was tested with three different schedules: 24 hours of flavopiridol and then 24 hours of doxorubicin exposure, doxorubicin then flavopiridol exposure in reverse order, and 24 hours of flavopiridol followed by 48-hour washout period and then 24 hours of doxorubicin exposure. For combination studies, fixed drug IC x ratios were as follows: flavopiridol IC 10 and doxorubicin IC 50 , or flavopiridol IC 25 and doxorubicin IC 50 . Figure 2 presents the results for flavopiridol IC 25 and doxorubicin IC 50 fixed ratios but the other drug combination ratio (flavopiridol IC 10 and doxorubicin IC 50 ) had similar results (data not shown).…”

“…The H865 cell line was transduced with Rb-specific siRNA coding pFIV U6/H1 vector (System Biosciences) to generate stable pRb-negative H865 clones. Two different siRNA sequences known to down-regulate Rb mRNA >90% were chemically synthesized (25). The sequences for the siRNA template were as follows: (a) 5 ¶-aaagGATACCAGATCATGTCAGA-3 ¶, 3 ¶-CTATGGTCTAGTACAGTCTaaaa-5 ¶ and (b) 5 ¶-aaagCCCAGCAGTTC-GATATCTA-3 ¶, 3 ¶-GGGTCGTCAAGCTATAGATaaaa-5 ¶.…”

“…The schedules tested were as follows: (a) flavopiridol for 24 h and then doxorubicin for 24 h; (b) doxorubicin for 24 h and flavopiridol for 24 h; and (c) flavopiridol for 24 h, 2 d of washout period and then doxorubicin for 24 h. Fixed ratios were flavopiridol IC 10 and doxorubicin IC 50 , or flavopiridol IC 25 …”

“…Single drug exposures were for 24 h and then cells were transferred into fresh growth medium. For combination studies, flavopiridol and doxorubicin doses were scheduled as IC 25 In vivo studies. Athymic nude male mice were purchased from The Jackson Laboratory at 5 to 6 wk age.…”

A Novel siRNA-Based Approach to Study Mechanisms of Resistance/Action of a New Drug in Treatment of Breast Cancer. Addendum

“…MCF-7 cells were seeded in twenty 10-cm dishes at a density of 2x10 6 cells/plate (total cell number 40x10 6 cells). To tranduce cells, 5 ml of pseudoviral supernatant containing 4ug/ml polybrene (Sigma) was added to each plate.…”