RNAi analysis of the IGF pathway

已经报道了一些与胰岛素样生长因子(IGF)信号通路相关的抗癌分子靶点，包括IGF-1受体(IGF-1R)及其一些定义和定义较少的结合伙伴。到目前为止，我们已经鉴定了与IGF-1R及其相关受体的下游或直接相关的35个基因对应的合成siRNAs介导的稳态mRNA水平的下降。RNA分析用于表征所有的RNAi效应器，定量Western印迹分析用于分析一些靶蛋白的蛋白质水平的下降。在所有病例中，至少有一种合成的siRNA被鉴定为显著降低了相应基因的稳定状态mRNA水平，在研究中，这与蛋白质水平的显著下降相匹配，并持续了至少96小时。为了研究一些与IGF途径相关的基因被敲除的功能后果，我们在2D和3D细胞培养中研究了RNAi对一些途径成员的影响。到目前为止，大多数基因的敲除对细胞活力的影响微乎其微，但在3D中已经观察到了对细胞生长的实质性影响。我们现在正在与MTP和MTDP合作，将这些特有的RNAi资源应用于其他功能分析和模型系统

Several putative anti-cancer molecular targets associated with the Insulin like growth factor (IGF) signally pathway have been reported including the IGF-1 receptor (IGF-1R) and some of its defined and less defined binding partners. To date we have characterized the decrease in steady state mRNA levels mediated by synthetic siRNAs corresponding to 35 genes associated directly with or down stream of IGF-1R and its related receptors. RNA analysis was used to characterize all of the RNAi effectors, with quantitative Western blot analysis used to analyze the decrease in protein levels of some of the target proteins. In all cases at least one synthetic siRNA was identified that reduced steady-state mRNA levels of the corresponding gene significantly, and where studied this was matched with a significant decrease in protein levels that was sustained at least 96 hours. To investigate the functional consequence of the knockdown of some of the genes associated with the IGF pathway we have investigated the effect of RNAi against some of the pathway members in 2D and 3D cell culture. To date the knockdown of most genes show only a minimal effect on cell viability but a substantial effect on cell growth in 3D has been observed. We are now initiating a collaborative effort with MTP and MTDP to applying these characterized RNAi resources in additional functional assays and model systems