Gene expression profiles in HEK-293 cells with low or high store-operated calcium entry: can regulatory as well as regulated genes be identified?
Gene expression profiles in HEK-293 cells with low or high store-operated calcium entry: can regulatory as well as regulated genes be identified?
复制标题
具有低或高钙库操纵钙进入的 HEK-293 细胞中的基因表达谱：是否可以鉴定调节基因和被调节基因？
DOI：
10.1152/physiolgenomics.00099.2004
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发表时间：
2005
影响因子：
4.6
通讯作者：
Villereal,MitchelL
中科院分区：
文献类型：
--
作者：
Zagranichnaya,TatianaK;Wu,Xiaoyan;Danos,ArpadM;Villereal,MitchelL
利用基因芯片技术对具有高或低水平钙离子通道(SOCE)的人胚胎肾(HEK)-293细胞克隆进行基因表达谱分析。对5个高克隆、3个低克隆和对照HEK-293细胞，提取在血清存在下生长的细胞总RNA，复制Affymetrix U133A人基因芯片。在芯片上显示的22,000个∼基因中，58个基因的读数至少是对照细胞的两倍，而32个基因的读数至少降低了两倍，所有五个高SOCE值克隆细胞的读数都比对照细胞高。在低SOCE克隆中，92个基因的读数至少是HEK-293细胞的两倍，而58个基因的读数至少低两倍。实时定量RT-PCR分析证实了所选18个基因的微阵列结果；对于其中6个基因，通过另一种方法证实了低SOCE克隆的预测变化，通过小干扰(Si)RNA对典型的瞬时受体潜在蛋白-1的表达，降低了SOCE患者HEK-293中监测的mRNA水平。受SOCE调控的基因参与信号转导、转录、细胞凋亡、代谢和膜转运。这些数据为深入了解SOCE的生理作用提供了依据。此外，还发现了一种潜在的SOCE调节因子，胰岛素受体底物(IRS)-2。在两个高SOCE克隆中，通过siRNA方法降低IRS-2水平显著降低SOCE，而在低SOCE克隆中过表达IRS-2则提高SOCE。
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.