Combinatorial RNAi for quantitative protein network analysis

Abstract

The elucidation of cross-talk events between intersecting signaling pathways is one main challenge in biological research. The complexity of protein networks, composed of different pathways, requires novel strategies and techniques to reveal relevant interrelations. Here, we established a combinatorial RNAi strategy for systematic single, double, and triple knockdown, and we measured the residual mRNAs and proteins quantitatively by quantitative real-time PCR and reverse-phase protein arrays, respectively, as a prerequisite for data analysis. Our results show that the parallel knockdown of at least three different genes is feasible while keeping both untargeted silencing and cytotoxicity low. The technique was validated by investigating the interplay of tyrosine kinase receptor ErbB2 and its downstream targets Akt-1 and MEK1 in cell invasion. This experimental approach combines multiple gene knockdown with a subsequent quantitative validation of reduced protein expression and is a major advancement toward the analysis of signaling pathways in systems biology.combinatorial protein knockdown ͉ reverse-phase protein arrays ͉ signaling pathways ͉ cross-talk ͉ systems biology C omplex protein networks evoke an increasing demand for suitable methods to elucidate cross-talk between signaling pathways. For example, tumor invasion integrates three major pathways: the growth factor-signaling pathway, the integrinmediated pathway, and the Rho family GTPases (1). Overall, these pathways comprise Ϸ130 proteins (2-4), and currently little is known about how cross-talk events between these pathways impact tumor invasion. A common strategy to obtain insight into protein activities would be to knock down each of these proteins and to measure the induced effects on cell invasion. However, synergistic information about pathway crosstalk should be better attained through analyzing several proteins in parallel, e.g., via RNAi by multiple knockdown. Xia et al. (5) established small-scale multiple knockdown via a tetracyclineregulated pol II promoter construct that allows for the simultaneous expression of up to three short hairpin RNAs (shRNAs). In a different approach, Huang et al. (6) used siRNAs directed against different variants of Rab5 to understand their contribution to clathrin-dependent endocytosis. This work offered insights into the use of multiple siRNAs and revealed that systematic approaches for the production of double or triple knockdown by siRNA technology might be promising for the analysis of complex cellular processes. However, this approach was restricted to the use of siRNAs against different variants of the same protein, and the protein knockdown was not quantified. The validation of gene knockdown is a critical parameter in RNAi experiments, and current technologies suffer from the poor correlation between RNA and protein levels of expressed genes [quantitative real-time PCR (qRT-PCR)] (7) or a low dynamic range (Western blot) (8).Here, we describe a combinatorial RNAi strategy for a systematic knockdown of ...

miR-200bc/429 cluster targets PLCγ1 and differentially regulates proliferation and EGF-driven invasion than miR-200a/141 in breast cancer

“…In order to elucidate the molecular changes responsible for the different effects of the two clusters on the cell cycle, we examined the changes in several cell cycle components either with reverse phase protein arrays (Sahin et al, 2007) (Figure 4c), which could potentially explain the reduction of G1-phase after miR-200c mimic. Furthermore, we observed a significant decrease in CDK2 and increase in pCdc2 (T14) and pCdc25c (Ser 216), which refrain cells from transition from G2 to M phase, although we observed a strong increase in cyclin B1 and cyclin A levels in the miR-200c-transfected samples (Figures 4c and d).…”

Reverse‐phase protein arrays for application‐orientated cancer research

“…In most cases knowledge on the absolute amount of the protein is not required and biological results are discussed in relation to control samples. In this instance, ERBB2, MEK1, and AKT1 were silenced and analyzed by quantitative RT-PCR and RPPA [52]. In the case of the EGFR co-receptor, ERBB2, mRNA and protein expression correlated very well.…”

“…A recent study on the development of a combinatorial RNAi strategy addressed both issues by determining mRNA and protein abundance in a step-by-step combination of the silencing experiment [52]. The efficiency of gene silencing on mRNA as well as on protein abundance was determined by quantitative RT-PCR and by RPPA.…”

“…In particular, the extensive molecular cross-talk between different pathways requires suitable technologies to identify causative mechanisms leading to increased proliferation, cell migration, and invasion. Different genes can be targeted in parallel to downregulate the corresponding proteins in organisms ranging from Caenorhabditis elegans to human to study the cross-talk of signaling pathways [51][52][53][54][55]. The efficiency of a silencing reaction can be monitored by quantitative RT-PCR [51].…”

Integrating physical and genetic maps: from genomes to interaction networks

“…All of these methods allow the phenotypic consequences of double-mutant combinations to be assayed in high-throughput formats 15 . In worms and higher eukaryotes, genetic interactions are explored through the technique of combinatorial RNAi 98 and other RNAi-based screening approaches 99,100 . Other types of genetic interactions are non-symmetrical and establish a 'cause-and-effect' ordering…”