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Making a Difference Dharmacon SMARTpool® siRNA Reagents identify genes that regulate epithelial cell migrationKirsteen H. Maclean, Ph.D. Cell migration is a critical process in the development and maintenance of multicellular organisms. Wound healing, tissue formation (particularly during embryogenesis), immune responses, angiogenesis and tumor cell migration all require the orchestrated motility of cells both spatially and temporally to a specific location. Indeed, consequences of deregulated cell migration are known to contribute to many pathological disease states such as mental retardation, vascular disease, chronic inflammation and tumor formation and metastasis. The cell migratory cycle generally initiates with an extracellular stimulus that triggers a signal transduction cascade leading to polarized migration of the cell, meaning molecular processes at the front and at the back of a moving cell are different. The cellular response is to extend protrusions in the direction of migration which requires reorganization of actin filaments and microtubules. These protrusions are then stabilized by adhering to the extracellular matrix (ECM) and/or adjacent cells via transmembrane receptors linked to the actin cytoskeleton. Finally, tractile forces originating from the acto-myosin network pull the cell forward [1].Recently, research led by Joan Brugge’s group at Harvard [2] utilized a high throughput siRNA screening approach to identify genes involved in epithelial cell migration. Dharmacon SMARTpool siRNA reagents (Thermo Fisher Scientific, Lafayette, CO) were used to analyze genes predicted to affect cell migration and adhesion, including phosphatases and kinases. Previous RNAi based screens have adopted a less than optimal strategy of hit selection due to minimal target validation; the Brugge group has instead identified genes focused on the generation of a highly validated dataset. For their primary screen, the researchers utilized a classic wound healing assay with MCF-10A immortalized non-tumorigenic breast epithelial cells [3]. Typically, a wound is introduced to a cell monolayer by a fine needle. The monolayers recover and heal the wound in a well characterized process that can be observed by time-lapse microscopy [4]. This assay has been particularly useful in establishing the role of Rho family GTPases such as Rac and Rho in polarity and cell migration [5, 6]. The primary screen of siRNAs was binned on the basis of several migration specific criteria: area score; visual score and viability score and identified three categories of hits: those that accelerated; those that inhibited and those that impaired migration correlated with effects on proliferation and metabolism (Figure 1). Click to enlarge  Figure 1. Summary of the wound healing phenotypes. Representative wound healing images indicate the continuum of phenotypes, together with the normalized wound area and Alamar Blue scores. siRNAs were binned on the basis of the area score, visual score and Alamar Blue score. Accelerated bin (green) had an area score of <0.75 and visually was considered closed or closing. Impaired bin (red) had an area score of >2.0 and no effect on metabolism (Alamar Blue score >0.8). Low Alamar bin had poor Alamar Blue reduction (<0.8) and generally disrupted monolayer integrity. siRNAs that induced numerical scores bordering the cut-offs were visually inspected and binned appropriately. On occasion, the numerical score could inaccurately reflect wound closure due to small tears in the monolayer, variations in cell morphology or cell dissociation. From the SMARTpool screen, 11.8% of the Accelerated bin and 34% of the Impaired bin scores were visually overridden. The phalloidin channel montage is shown in grey scale. Figure reprinted with permission from Nature Cell Biology [2]. Extensive validation of genes from the primary screen focused on addressing the potential of off-target effects using Dharmacon ON-TARGETplus SMARTpool® technology together with deconvolution of the original siRNA SMARTpool® with their individual siRNAs. High confidence (HC) siRNAs were defined as those that were phenotypically consistent with those achieved using SMARTpool typically, 3/4 or 4/4. Similarly, moderate confidence (MC) siRNAs scored 2/4 and discordant siRNAs (0/4, 1/4, 0/2, 1/2). Of the 1,081 siRNAs initially screened, the group identified 66 HC genes affecting migration (either accelerating or impairing) to pursue further studies. Additional experiments using time-lapse microscopy distinguished several morphological features induced by the HC genes that were subdivided into several groups based on a variety of parameters that included lamellae and lamellipodia protrusion dynamics, speed, direction and the extent of cell-cell adhesion. Genes accelerating cell migration were divided into seven different phenotypic groups and genes impairing migration, divided into five different phenotypic groups. Interestingly, while several of the genes had an established role in cell migration studies (e.g. RHOA, NEDD9, VEGFC, VEGFB and VIM), many, 42/66 had no prior association with migration and adhesion processes as defined by their gene ontology. Arguably, the rate of wound healing as evidenced in some of the groups could be affected by several unrelated parameters including cell proliferation and apoptosis. To this end, the authors resolved these issues by also monitoring mitotic events and found no correlation between increased migration and cellular proliferation; however several siRNA phenotypes could in part be explained by suppression of proliferation. Further, to examine whether the low Alamar Blue phenotype could be attributed to apoptosis, the authors overexpressed Bcl-2, an anti-apoptotic member of the Bcl-2 family and found that this did not ameliorate the phenotype of impaired migration, though their was a rescue of the low viability. To identify the signaling pathways that potentially linked the HC genes and uncover a role for these novel 42 migration genes, the authors used informatics Ingenuity® software, a recent application for pathway retrieval. Interestingly, a network of both direct and indirect (generated from protein-protein interaction databases) interactions were centered on b-catenin, b1-catenin and actin, known genes in the regulation of cell migration. Collectively, these findings not only represent fertile ground for future studies to examine the regulatory mechanisms involved in cell migration but importantly, provide a better understanding of the genes involved in the cell migratory process which will hopefully lead to the development of novel therapeutic intervention strategies. For further information, please refer to the Cell Migration website. References
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