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Identification of genes that regulate epithelial cell migration using an siRNA screening approach

Abstract

To provide a systematic analysis of genes that regulate epithelial cell migration, we performed a high throughput wound healing screen with MCF-10A breast epithelial cells, using siRNAs targeting 1,081 human genes encoding phosphatases, kinases and proteins predicted to influence cell migration and adhesion. The primary screen identified three categories of hits: those that accelerate, those that inhibit and those that impair migration with associated effects on cell proliferation or metabolism. Extensive validation of all the hits yielded 66 high confidence genes that, when downregulated, either accelerated or impaired migration; 42 of these high confidence genes have not been previously associated with motility or adhesion. Time-lapse video microscopy revealed a broad spectrum of phenotypic changes involving alterations in the extent and nature of disruption of cell–cell adhesion, directionality of motility, cell polarity and shape, and protrusion dynamics. Informatics analysis highlighted three major signalling nodes, β-catenin, β1-integrin and actin, and a large proportion of the genes that accelerated migration impaired cell–cell adhesion.

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Figure 1: Summary of the wound healing phenotypes and schematic representation of the results from the SMARTpool screen and validation studies.
Figure 2: Quantification and correlation of knockdown with phenotype for individual Accelerated bin siRNAs.
Figure 3: Analysis of cell morphology after transfection with HC Accelerated and Impaired migration siRNAs and quantification of the migration pattern determined by time-lapse imaging.
Figure 4: Analysis of the migration pattern of individual cells from representative HC Accelerated migration subgroups determined by time-lapse imaging.
Figure 5: Time-lapse imaging series of cells transfected with representative HC Accelerated migration siRNAs.
Figure 6: Time-lapse imaging series of cells transfected with representative HC Impaired migration siRNAs.
Figure 7: Network relationships for the major signalling nodes β-catenin, β1-integrin and actin.

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Acknowledgements

We wish to thank Stewart Rudnicki and Caroline Shamu (Institute for Chemical and Cell Biology) for screening assistance; Jim Horn (Department of Cell Biology) for manufacturing the wound healing pin; William Pearson for conceptualizing and generating the interactive website hosted by the Cell Migration Consortium; Sabina Winograd-Katz and Benjamin Geiger (Weizmann Institute) for collaborative creation of the MAR custom siRNA library; Rick Horwitz, Alan Hall, Gaudenz Danuser and Ghassan Mouneimne for stimulating and helpful discussions and critical reading of the manuscript and Lara Petrak (Nikon Imaging Centre) for assistance with time-lapse microscopy. We are also grateful to William Hahn, David Root and The RNAi Consortium for providing shRNA vectors. Funding was from the DOD W81XWH-04-1-0360 (K.J.S.) and the Cell Migration Consortium, supported by a grant from the NIH/NIGMS GM064346 (J.S.B.). A.R., D.L. and A.K. are employees of Thermo Fisher Scientific, which supported this work in part by supplying a subset of the siRNA reagents.

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Authors and Affiliations

Authors

Contributions

K.J.S. designed, performed and analysed experiments and wrote the manuscript; L.M.S. created and managed the screening database and performed the informatics analysis; J.B. performed experiments; A.R. performed the knockdown quantification under the supervision of D.L. and A.K.; J.S.B analysed and discussed data and wrote the manuscript.

Corresponding author

Correspondence to Joan S. Brugge.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures S1, S2, S3, S4, S5, S6, S7, S8 and Supplementary Discussion (PDF 2666 kb)

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Supplementary Table 4 (XLS 44 kb)

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Supplementary Table 5 (XLS 36 kb)

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Simpson, K., Selfors, L., Bui, J. et al. Identification of genes that regulate epithelial cell migration using an siRNA screening approach. Nat Cell Biol 10, 1027–1038 (2008). https://doi.org/10.1038/ncb1762

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