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Gene expression profiles in HEK-293 cells with low or high store-operated calcium entry: can regulatory as well as regulated genes be identified?

Department of Neurobiology, Pharmacology and Physiology, The University of Chicago, Chicago, Illinois

Gene expression profiles were generated using cDNA microarray technology for clones of human embryonic kidney (HEK)-293 cells selected to have either high or low levels of store-operated Ca²⁺ entry (SOCE). For five high clones, three low clones, and control HEK-293 cells, duplicate Affymetrix U133A human gene arrays were run after extraction of total RNA from cells growing in the presence of serum. Of the 22,000 genes represented on the microarray, 58 genes had readings at least twofold higher, while 32 genes had readings at least twofold lower, in all five high SOCE clones compared with control HEK-293 cells. In the low SOCE clones, 92 genes had readings at least twofold higher, while 58 genes had readings at least twofold lower, than in HEK-293 cells. Microarray results were confirmed for 18 selected genes by real-time RT-PCR analysis; for six of those genes, predicted changes in the low SOCE clone were confirmed by an alternative method, monitoring mRNA levels in HEK-293 with SOCE decreased by expression of small interfering (si)RNA to canonical transient receptor potential protein-1. Genes regulated by SOCE are involved in signal transduction, transcription, apoptosis, metabolism, and membrane transport. These data provide insight into the physiological role of SOCE. In addition, a potential regulator of SOCE, insulin receptor substrate (IRS)-2, has been identified. A reduction of IRS-2 levels by siRNA methods in two high clones dramatically reduced SOCE, whereas overexpression of IRS-2 in a low SOCE clone elevated SOCE.

cDNA microarray; fluorescence-activated cell sorting analysis; thapsigargin; insulin receptor substrate-2; small interfering RNA; real-time PCR; canonical transient receptor potential protein-1

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