KRAS-induced transcription analysis in immortalized pancreatic cells. Homo sapiens

We utilized non-transformed, human pancreatic ductal epithelial (HPDE) cells, previously engineered with the E6 and E7 proteins of the HPV16 virus to emulate loss of p53 and inactivation of the Rb pathway, respectively. Given the frequent activation of KRAS (>90% PDAC tumors) and its early role in pancreatic neoplasia, we sought to engineer HPDE cells containing KRASG12D to provide the appropriate context in which to screen for novel drivers that might represent KRAS effectors. Overall design: The KRAS-induced transcription analysis was conducted using RNAs extracted from HPDE cells transduced with either control, wild-type KRAS or KRASG12D(pInducer) with or without DOX (100ng/ml) for 72 h, followed by hybridization of labeled cDNA onto Agilent arrays (Agilent G3 Human GE 8x60K) by the Baylor College of Medicine Genome Profiling Core Facility. multi-group comparison

本研究使用了未转化的人胰腺导管上皮细胞（human pancreatic ductal epithelial, HPDE），该细胞此前已通过导入人乳头瘤病毒16型（HPV16）的E6和E7蛋白，分别模拟p53功能缺失与Rb通路失活。
鉴于KRAS在胰腺导管腺癌（pancreatic ductal adenocarcinoma, PDAC）肿瘤中的激活率超过90%，且其在胰腺肿瘤发生中发挥早期关键作用，本研究构建了携带KRASG12D突变的HPDE细胞，以搭建合适的实验模型，用于筛选可能作为KRAS效应因子的新型驱动基因。
整体实验设计：本研究开展KRAS诱导的转录组分析，具体流程为：提取经空载体对照、野生型KRAS或KRASG12D(pInducer)慢病毒感染的HPDE细胞的总RNA，将细胞分别在添加或不添加100ng/ml多西环素（doxycycline, DOX）的培养基中培养72小时，随后由贝勒医学院基因组分析核心实验室将标记的cDNA与安捷伦G3 Human GE 8x60K基因芯片（Agilent G3 Human GE 8x60K）进行杂交。本研究采用多组比较实验设计。