Hierarchical regulation in a KRAS pathway-dependent transcriptional network revealed by a reverse-engineering approach (RAS-ROSE and ROSE with siRNA)

RAS mutations are highly relevant for progression and therapy response of human tumours, but the genetic network that ultimately executes the oncogenic effects is poorly understood. Here we used a reverse-engineering approach in an ovarian cancer model to reconstruct KRAS oncogene-dependent cytoplasmic and transcriptional networks from perturbation experiments based on gene silencing and pathway inhibitor treatments. We measured mRNA and protein levels in manipulated cells by microarray, RT-PCR and Western Blot analysis, respectively. The reconstructed model revealed complex interactions among the transcriptional and cytoplasmic components, some of which were confirmed by double pertubation experiments. Interestingly, the transcription factors decomposed into two hierarchically arranged groups. To validate the model predictions we analysed growth parameters and transcriptional deregulation in the KRAS-transformed epithelial cells. As predicted by the model, we found two functional groups among the selected transcription factors. The experiments thus confirmed the predicted hierarchical transcription factor regulation and showed that the hierarchy manifests itself in downstream gene expression patterns and phenotype.RAS突变与人类肿瘤的进展及治疗反应高度相关，但最终介导致癌效应的遗传网络仍未被充分阐明。本研究以卵巢癌模型为研究对象，采用逆向工程策略，基于基因沉默与通路抑制剂处理的扰动实验，重构了KRAS致癌基因依赖的细胞质与转录调控网络。我们分别通过基因芯片、逆转录聚合酶链反应（RT-PCR）检测经干预处理细胞的mRNA水平，通过蛋白质印迹（Western Blot）检测其蛋白质水平。重构得到的模型揭示了转录组分与细胞质组分间的复杂相互作用，其中部分相互作用已通过双重扰动实验得到验证。值得注意的是，转录因子可被划分为两个层级排布的功能类群。为验证模型的预测结果，我们分析了KRAS转化的上皮细胞的生长参数与转录失调情况。正如模型预测所示，我们在所选的转录因子中发现了两个功能群组。本实验证实了预测的层级式转录因子调控机制，并表明该层级结构可通过下游基因表达模式与表型得以体现。