RNAi Analysis of the IGF Pathway

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，我们研究了RNAi对多个细胞系中与该途径成员相关的32个基因的影响，重点是乳腺上皮细胞和乳腺癌细胞系。已经建立了这些细胞系的RNAi图谱，作为与NCI分子靶标开发计划合作努力的一部分，后续工作正在进行中。

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 32 genes associated with the pathway members in multiple cell lines with a focus on mammary epithelial and breast cancer cell lines. An RNAi profile for these cell lines has been established and follow up work is now being conducted as part of collaborative effort with the Molecular Target Development Program, CCR, NCI.