Inferring drug-induced gene regulatory relationships in primary human hepatocytes

Statins are widely used cholesterol-lowering drugs that inhibit HMG-CoA reductase, a key enzyme in cholesterol synthesis. In some cases, however, these drugs may cause a number of toxic side effects in hepatocytes and skeletal muscle tissue. Currently, the specific molecular mechanisms that cause these adverse effects are not sufficiently understood. In this work, genome-wide RNA expression changes in primary human hepatocytes of six individuals were measured at five time points upon atorvastatin treatment. A novel systems-level analysis workflow was applied to reconstruct regulatory mechanisms based on these drug-response data and available knowledge about transcription factor binding specificities, protein-protein interactions and protein-drug interactions. Several previously unknown transcription factors, regulatory cofactors and signaling molecules were found to be involved in atorvastatin-responsive gene expression. Some novel relationships, e.g., the regulatory influence of nuclear receptor NR2C2 on CYP3A4, were successfully validated in wet-lab experiments. Whole-genome Affymetrix U133 Plus 2.0 (Affymetrix, Santa Clara, CA) microarray measurements were conducted using samples of primary human hepatocytes cultured from six individuals (i.e., hh62, hh65, hh67, hh79, hh80 and hh81). Each sample was treated with atorvastatin and dimethylsulfoxide (DMSO), which was used as a control substance. Microarray measurements were performed at five time points (6 h, 12 h, 24 h, 48 h and 72 h) after the drug stimulus.

他汀类（Statins）是一类广泛应用的降胆固醇药物，可通过抑制胆固醇合成通路中的关键酶——羟甲基戊二酰辅酶A还原酶（HMG-CoA reductase）发挥药效。然而在部分临床场景中，该类药物可对肝细胞与骨骼肌组织引发多种毒性不良反应。目前，此类不良反应背后的特异性分子机制尚未得到充分阐明。
本研究针对6名个体的原代人肝细胞（primary human hepatocytes），在阿托伐他汀（atorvastatin）处理后的5个时间点开展全基因组RNA表达变化检测。研究采用一套全新的系统级分析流程，基于该药物响应数据以及已公开的转录因子结合特异性（transcription factor binding specificities）、蛋白质-蛋白质相互作用（protein-protein interactions）、蛋白质-药物相互作用（protein-drug interactions）相关知识，重构药物调控的分子机制。研究发现了多个此前未被报道的、参与阿托伐他汀响应基因表达调控的转录因子（transcription factors）、调控辅因子（regulatory cofactors）与信号分子（signaling molecules）。部分全新的调控关系——例如核受体NR2C2（nuclear receptor NR2C2）对CYP3A4的调控作用——已通过湿实验得到成功验证。
本研究使用全基因组Affymetrix U133 Plus 2.0（Affymetrix U133 Plus 2.0）微阵列平台，对6名个体（即hh62、hh65、hh67、hh79、hh80与hh81）来源的原代人肝细胞样本进行检测。所有样本均接受阿托伐他汀处理，同时以二甲基亚砜（dimethylsulfoxide, DMSO）作为对照底物。微阵列检测分别在药物处理后的6 h、12 h、24 h、48 h与72 h共5个时间点完成。