Whole genome pooled shRNA screen to characterize Raptinal-induced apoptosis in the MIA PaCa-2 cell line

The goal of the study was to characterize the mechanism by which Raptinal induces apoptosis using a genome-wide pooled shRNA screen. Following drug treatment, the abundance of the shRNA constructs were determined and compared between DMSO and Raptinal-treated samples. Constructs depleted in Raptinal samples versus DMSO control, are expected to potentiate Raptinal-induced apoptosis by down regulating anti-apoptotic proteins. In contrast, shRNA constructs enriched upon Raptinal treatment are expected to target pro-apoptotic proteins, whose down regulation confers protection from Raptinal-induced apoptosis. Follow up experiments were performed on enriched shRNAs in order to validate potentially novel pro-apoptotic proteins. Cells were treated with Raptinal (1.25-1.8 micromolar) or DMSO vehicle control over a 20 day period. After every 2-3 days of drug treatment, cells were allowed to recover in drug-free medium and treatment repeated for a total of 3 rounds over the 20 day period. Two biological replicates of Raptinal-treatment were performed and one biological replicate of DMSO vehicle control was performed. Cell passaging was performed on a scale sufficient to maintain >100 fold library representation. At the end of the 20 days, total RNA was extracted and shRNA abundances determined.

本研究旨在借助全基因组混合shRNA（pooled shRNA）筛选，解析Raptinal诱导细胞凋亡(apoptosis)的分子机制。药物处理完成后，分别检测二甲基亚砜（DMSO）与Raptinal处理样本中shRNA构建体的丰度并进行对比。相较于DMSO对照组，Raptinal处理样本中丰度降低的shRNA构建体，可通过下调抗凋亡蛋白增强Raptinal诱导的细胞凋亡效应。与之相反，经Raptinal处理后丰度升高的shRNA构建体，其靶向的应为促凋亡蛋白；这类蛋白的敲低会使细胞免受Raptinal诱导的细胞凋亡。针对丰度升高的shRNA构建体开展了后续实验，以验证潜在的新型促凋亡蛋白。实验期间，将细胞置于浓度为1.25~1.8微摩尔的Raptinal或DMSO溶剂对照中处理，时长共计20天。每经过2~3天的药物处理后，将细胞置于无药培养基中恢复培养，如此重复给药处理，在20天周期内共计完成3轮处理。Raptinal处理组设置2次生物学重复，DMSO溶剂对照组设置1次生物学重复。细胞传代的规模需保证shRNA文库的覆盖度维持在100倍以上。20天处理结束后，提取细胞总RNA并检测shRNA的丰度。