Effects of mTOR Knockdown by RNA Interference

描述（由申请人提供）：雷帕霉素是一种细菌大环内酯类药物，正在成为一种治疗癌症的有前途的药物。哺乳动物雷帕霉素靶蛋白（mTOR）控制生长、增殖和细胞周期进展的蛋白质翻译。我们假设mTOR也可能具有不依赖雷帕霉素的功能。利用RNA干扰（RNAi）在乳腺癌细胞中敲低mTOR的模型，我们的具体目的如下：1。以确定mTOR敲低和2。获得mTOR敲低的全面分子图谱。为了解决第一个目标，我们将使用细胞活力测定法测量增殖，使用颗粒计数器评估细胞大小，并使用caspase和TUNEL测定法检查细胞凋亡。为了解决第二个目标，我们将使用西方分析来检查已知的mTOR下游靶点，并使用基因表达阵列来获得全局表达谱。我们期望发现RNAi敲低mTOR和雷帕霉素抑制mTOR之间的显著差异，这将导致进一步研究雷帕霉素不依赖的mTOR功能。这些功能可能被证明是癌症化疗的新靶点，特别是在雷帕霉素耐药的情况下。

DESCRIPTION (provided by applicant): Rapamycin, a bacterial macrolide, is emerging as a promising agent for the treatment of cancer. The mammalian Target of Rapamycin (mTOR) controls translation of proteins for growth, proliferation, and progression of the cell cycle. We hypothesize that mTOR may have rapamycin-independent functions as well. Using a model of mTOR knockdown by RNA interference (RNAi) in breast cancer cells, our specific aims are as follows: 1. To determine functional effects of mTOR knockdown and 2. To obtain a comprehensive molecular profile of mTOR knockdown. To address the first aim, we will measure proliferation using cell-viability assays, assess cell size using particle counters, and check apoptosis using caspase and TUNEL assays. To address the second aim, we will use Western analysis to examine known downstream targets of mTOR, and gene-expression arrays to obtain global expression profiles. We anticipate finding significant differences between mTOR knockdown by RNAi and mTOR inhibition by rapamycin, which would lead to further investigation of rapamycin-independent mTOR functions. Such functions may prove to be novel targets in cancer chemotherapy, particularly in the setting of rapamycin-resistance.