Impact of Glucocorticoids and PI3K Inhibition on Gene Expression in Myeloma. Homo sapiens

We previously noted that combination glucocorticoids and PI3K inhibition induces synergistic cell death. To identify genes involved in myeloma cell death we examined mRNA expression in each individual treatment and in the combination. The glucocoriticoid resistant cells (MM.1RL) are used as a negative control. Gene expression is assessed using Illumina Human HT-12v4 bead chips Overall design: For this experiment, the lab is using human multiple myeloma cell lines that were isolated from a patient. MM.1S cells display sensitivity to glucocorticoids. MM.1RL cells display resistance to glucocorticoid treatment due to spontaneous mutations that render the glucocorticoid receptor inoperable. In this experiment, there will be 4 conditions for the MM.1S cells, and 2 conditions for the MM.1RL cells. We will be treating the MM.1S cells with a glucocorticoid alone (Dexamethasone - 1 uM), a PI3K inhibitor alone (LY249002 - 25 uM), a combination of the two drugs, and a vehicle control. The MM.1RL cells will be treated with a PI3K inhibitor alone, and with a vehicle control. Since the PI3K inhibitor is diluted in DMSO, as a control an equal volume of DMSO will be added to all conditions not containing the PI3K inhibitor. Conditions within the MM.1S cell line will be compared with each other. Specifically, we hope to identify genes that are similarly affected by all three experimental conditions. The MM.1RL conditions will not be analyzed at this time. There will be 4 biological replicates of each condition.

我们此前已证实，糖皮质激素（glucocorticoid）联合磷脂酰肌醇3-激酶（PI3K）抑制剂可诱导协同性细胞死亡。为鉴定参与多发性骨髓瘤细胞死亡的基因，我们对单一药物处理及联合药物处理的样本开展了mRNA表达谱分析。糖皮质激素耐药细胞系MM.1RL作为阴性对照。基因表达水平通过Illumina Human HT-12v4微珠芯片进行检测。

### 总体实验设计
本实验所用细胞系为从患者体内分离得到的人多发性骨髓瘤细胞系：MM.1S细胞对糖皮质激素具有敏感性；MM.1RL细胞因发生自发突变导致糖皮质激素受体失活，因此对糖皮质激素处理产生耐药性。
本实验中，MM.1S细胞设置4组处理条件，MM.1RL细胞设置2组处理条件。其中，MM.1S细胞的处理方式分别为：单一糖皮质激素处理（地塞米松，1 μM）、单一PI3K抑制剂处理（LY249002，25 μM）、两药联合处理，以及溶剂对照；MM.1RL细胞的处理方式分别为：单一PI3K抑制剂处理，以及溶剂对照。由于PI3K抑制剂以二甲基亚砜（DMSO）作为溶剂配制，因此所有未添加PI3K抑制剂的实验组均需加入等体积的DMSO作为对照。
将对MM.1S细胞系的各组处理条件进行相互比较，以期鉴定出同时受三种实验处理条件调控的基因，本次暂不分析MM.1RL细胞系的处理组数据。每组处理条件均设置4次生物学重复。