Abstract
BackgroundCell migration plays a major role in a variety of normal biological processes, and a detailed understanding of the associated mechanisms should lead to advances in the medical sciences in areas such as cancer therapy. Previously, we developed a simple chip, based on transfected-cell microarray (TCM) technology, for the identification of genes related to cell migration. In the present study, we used the TCM chip for high-throughput screening (HTS) of a kinome siRNA library to identify genes involved in the motility of highly invasive NBT-L2b cells.ResultsWe performed HTS using TCM coupled with a programmed image tracer to capture time-lapse fluorescence images of siRNA-transfected cells and calculated speeds of cell movement. This first screening allowed us to identify 52 genes. After quantitative PCR (qPCR) and a second screening by a conventional transfection method, we confirmed that 32 of these genes were associated with the migration of NBT-L2b cells. We investigated the subcellular localization of proteins and levels of expression of these 32 genes, and then we used our results and databases of protein-protein interactions (PPIs) to construct a hypothetic but comprehensive signal network for cell migration.ConclusionsThe genes that we identified belonged to several functional categories, and our pathway analysis suggested that some of the encoded proteins functioned as the hubs of networks required for cell migration. Our signal pathways suggest that epidermal growth factor receptor (EGFR) is an upstream regulator in the network, while Src and GRB2 seem to represent nodes for control of respective the downstream proteins that are required to coordinate the many cellular events that are involved in migration. Our microarray appears to be a useful tool for the analysis of protein networks and signal pathways related to cancer metastasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-015-0170-7) contains supplementary material, which is available to authorized users.
Highlights
Cell migration plays a major role in a variety of normal biological processes, and a detailed understanding of the associated mechanisms should lead to advances in the medical sciences in areas such as cancer therapy
We decided to search for kinases that regulate cell migration using a kinome siRNA library printed on a transfected-cell microarray (TCM) chip
We used a line of NBT-L2b cells that is a subpopulation of cells isolated from parent NBT-II cells derived from a rat bladder carcinoma [8] because of their rapid (2 μm/min) and unidirectional movement on type I collagen-coated substrates [5]
Summary
Cell migration plays a major role in a variety of normal biological processes, and a detailed understanding of the associated mechanisms should lead to advances in the medical sciences in areas such as cancer therapy. We developed a simple chip, based on transfected-cell microarray (TCM) technology, for the identification of genes related to cell migration. We used the TCM chip for high-throughput screening (HTS) of a kinome siRNA library to identify genes involved in the motility of highly invasive NBT-L2b cells. We developed a simple cell microarray chip for high-throughput phenotypic screening (HTS) that could be used to identify genes that are required for cell migration [5,6]. Our chip is based on the transfected cell microarray (TCM) technology [7]. Our methodology has advantages over earlier methods, such as the wound-healing and Boyden chamber assays, in that (i) it can be used for efficient HTS of siRNAs and cDNAs and (ii) it excludes the possibility of contact inhibition and the release of inflammatory cytokines from wounded cells
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