Identification of TAL1/SCL Target Genes through siRNA and Microarray Expression Analysis.
Identification of TAL1/SCL Target Genes through siRNA and Microarray Expression Analysis.
复制标题
通过 siRNA 和微阵列表达分析鉴定 TAL1/SCL 靶基因。
DOI：
10.1182/blood.v104.11.4294.4294
复制
发表时间：
2004
期刊：
影响因子：
20.3
通讯作者：
A. Look
中科院分区：
文献类型：
--
作者：
J. O'neil;A. Ferrando;Teresa Palomero;A. Look
TAL1是一种基本的螺旋-环-螺旋(BHLH)蛋白，在造血过程中起重要作用。多达60%的儿童和成人T细胞急性淋巴细胞白血病(T-ALL)患者存在TAL1/SCL基因的激活。小鼠模型已经证明TAL1通过抑制E蛋白的功能而转化，然而TAL1导致白血病的转录靶点尚不清楚。在这项研究中，我们试图通过siRNA介导的敲除Jurkat T-ALL细胞系中高水平表达TAL1的TAL1表达来确定TAL1促进胸腺细胞转化的靶点。我们已经建立了几个克隆细胞系，它们稳定表达针对TAL1的siRNA，在RNA和蛋白质水平上对TAL1的抑制率高达90%。这些细胞的表型特征表明，TAL1表达降低的Jurkat细胞表现出增殖能力降低。在野生型和siRNA控制Jurkat细胞快速增殖的血清浓度较低时，这种表型会增强。利用Affymetrix U133 Plus 2.0阵列进行的微阵列表达分析表明，TAL1是基因表达的全球调节因子，许多途径都受到TAL1基因敲除的影响，包括那些与细胞生长和增殖有关的途径。这些数据将使我们能够通过击倒或过度表达这些TAL1靶标(取决于它们在没有TAL1的情况下是上调还是下调)来剖析TAL1的下游通路，并评估它们是否影响T-ALL细胞的增殖率或存活率。我们期望通过这两种方法的结合，确定并验证TAL1诱导白血病的潜在治疗靶点。
TAL1 is a basic helix-loop-helix (bHLH) protein that is essential for hematopoiesis. Activation of the TAL1/SCL gene occurs in as many as 60% of patients with pediatric and adult T cell acute lymphoblastic leukemia (T-ALL). Mouse models have demonstrated that TAL1 transforms by inhibiting the function of E proteins, however the transcriptional targets of TAL1 that contribute to leukemia are not known. In this study we sought to identify targets of TAL1 that contribute to the transformation of thymocytes through siRNA-mediated knockdown of TAL1 expression in the Jurkat T-ALL cell line that expresses high levels of TAL1. We have generated several clonal cell lines that stably express a siRNA directed against TAL1 with up to 90% knockdown of TAL1 at both the RNA and protein level. Phenotypic characterization of these cells revealed that Jurkat cells with decreased TAL1 expression display a decreased proliferative capacity. This phenotype is augmented in lower serum concentrations in which wild-type and siRNA control Jurkat cells proliferate rapidly. Microarray expression analysis using Affymetrix U133 Plus 2.0 arrays has revealed that TAL1 is a global regulator of gene expression and that many pathways are affected by TAL1 knockdown, including those implicated in cell growth and proliferation. This data will enable us to dissect the downstream pathways of TAL1 by knocking-down or overexpressing these TAL1 targets (depending on whether they are up- or down-regulated in the absence of TAL1), and assessing whether they affect the proliferative rate or survival T-ALL cells. We anticipate that we will identify and validate potential therapeutic targets for TAL1-induced leukemia using this combination of approaches.