Transient strong reduction of PTEN expression by specific RNAi induces loss of adhesion of the cells
Transient strong reduction of PTEN expression by specific RNAi induces loss of adhesion of the cells
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DOI：
10.1016/j.bbrc.2005.01.066
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发表时间：
2005-03-25
影响因子：
3.1
通讯作者：
Doi, TS
中科院分区：
文献类型：
--
作者：
Mise-Omata, S;Obata, Y;Doi, TS
抑癌基因pten编码一种脂质磷酸酶，可使磷脂酰肌醇（3，4，5）三磷酸的D3去磷酸化，产生磷脂酰肌醇（4，5）二磷酸。尽管PTEN与细胞粘附和迁移有关，但其潜在的分子机制尚不清楚。为了研究PTEN在细胞粘附中的作用，我们设计了三种不同的siRNA（siRNA PTEN-a、siRNA PTEN-b和siRNA PTEN-c）并转染到293 T细胞中。两天后，只有用siRNA PTEN-b转染的细胞变圆并从培养皿中分离，而用针对GFP的对照siRNA或针对PTEN的另外两种siRNA转染的细胞则没有。RNAi效果的评估显示siRNA PTEN-b抑制>95%的PTEN表达，这是三种siRNA中最有效的。为了检查非特异性效应，如干扰素反应和脱靶基因的抑制，我们随后使用定量PCR分析和DNA微阵列分析。没有检测到，表明RNAi系统是高度特异性的。使用PTEN敲低的HeLa细胞的免疫荧光研究表明，粘附的损失伴随着减少粘着斑的数量和肌动蛋白细胞骨架的解体。PTEN表达的瞬时和几乎完全丧失诱导细胞粘附的丧失。(C)2005年爱思唯尔公司All rights reserved.
The tumor suppressor gene pten encodes a lipid phosphatase that dephosphorylates D3 of phosphatidylinositol(3,4,5)trisphosphate, producing phosphatidylinositol(4,5)bisphosphate. Although PTEN has been implicated in cell adhesion and migration, the underlying molecular mechanism is unknown. To investigate the role of PTEN in cell adhesion, we designed three different siRNAs (siRNA PTEN-a, siRNA PTEN-b, and siRNA PTEN-c) and transfected into 293T cells. Two days later, only the cells transfected with siRNA PTEN-b became round and detached from the culture dishes, whereas cells transfected with a control siRNA against GFP or the two other siRNAs against PTEN did not. Evaluation of the RNAi effect revealed that siRNA PTEN-b inhibited >95% of PTEN expression, the most effective among the three siRNAs. To check for non-specific effects such as interferon response and inhibition of off-target genes, we then used quantitative PCR analysis and DNA microarray analysis. None was detected, indicating that the RNAi system was highly specific. Immunofluorescence studies using PTEN-knockdown HeLa cells revealed that the loss of adhesion was accompanied by a reduction in the number of focal adhesion plaques and disorganization of the actin cytoskeleton. Transient and near-complete loss of PTEN expression induces loss of adhesion of the cells. (C) 2005 Elsevier Inc. All rights reserved.