RNAi analysis of the IGF pathway

已经报道了与胰岛素样生长因子（IGF）信号通路相关的几种推定的抗癌分子靶点，包括IGF-1受体（IGF-1 R）及其一些确定的和不太确定的结合配偶体。迄今为止，我们已经表征了由合成siRNA介导的稳态mRNA水平的降低，所述合成siRNA对应于与IGF-1 R及其相关受体直接相关或下游的35个基因。RNA分析用于表征所有RNAi效应物，定量Western印迹分析用于分析一些靶蛋白的蛋白质水平的降低。在所有情况下，鉴定出至少一种合成siRNA，其显著降低相应基因的稳态mRNA水平，并且在研究的情况下，这与蛋白质水平的显著降低相匹配，其持续至少96小时。为了研究与IGF途径相关的一些基因的敲低的功能后果，我们研究了RNAi对2D和3D细胞培养中的一些途径成员的影响。迄今为止，大多数基因的敲除仅显示出对细胞活力的最小影响，但已经观察到对3D中的细胞生长的实质性影响。我们现在正与MTP和MTDP合作，将这些特征化的RNAi资源应用于其他功能测定和模型系统中

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