Identification of an SRF- and androgen-dependent gene signature in prostate cancer. Homo sapiens

The androgen receptor (AR) is the principal target for treatment of non-organ confined prostate cancer (PCa). Systems and bioinformatics approaches suggest that considerable variation exists in the mechanisms by which AR regulates expression of effector genes and point towards a role for secondary transcription factors (TFs) therein. We identified a novel indirect mechanism of androgen action in which effects of androgens on PCa cells are mediated by Serum Response Factor (SRF). To identify and characterize genes and cellular processes that are androgen-regulated in an SRF-dependent manner in PCa, Affymetrix HG-U133 Plus 2.0 GeneChip Array analysis was performed starting from RNA obtained from LNCaP cells in which androgen stimulation was combined with siRNA-mediated SRF silencing. To this end, LNCaP cells were seeded in 60 mm dishes at a density of 550,000 cells per dish in antibiotic-free medium. The next day, cells were transfected with siGenome SmartPool siRNA targeting SRF (Dharmacon, Lafayette, CO) or a custom-made control SmartPool targeting luciferase (LUC condition) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Forty-two hours after transfection, cells were treated with 5nM R1881 or ethanol vehicle. 3 biological triplicates were included per treatment group. Forty-eight hours later, cells were harvested in Trizol reagent (Invitrogen). RNA was isolated, purified on RNeasy columns (Qiagen, Germantown, MD) and checked for integrity by Agilent testing (Affymetrix, Santa Clara, CA). cDNA was generated and hybridized to Human Genome U133 Plus 2.0 arrays (Affymetrix) according to the manufacturer’s instructions at the Mayo Clinic Advanced Genomics Technology Microarray Shared Resource core facility. Overall design: Two-factor factorial design with three biological replicates (12 total samples) using LNCaP or SRF Silenced cells treated with either R1881 or ethanol vehicle.

雄激素受体(androgen receptor, AR)是治疗非器官局限性前列腺癌(prostate cancer, PCa)的核心靶点。系统生物学与生物信息学研究方法提示，AR调控效应基因表达的机制存在显著异质性，并暗示次级转录因子(transcription factors, TFs)在此过程中发挥作用。我们发现了一种全新的雄激素作用间接机制：雄激素对前列腺癌细胞的调控效应由血清反应因子(Serum Response Factor, SRF)介导。为鉴定并表征前列腺癌细胞中以SRF依赖方式受雄激素调控的基因及细胞生物学过程，本研究从经雄激素刺激联合siRNA介导的SRF沉默的LNCaP细胞中提取RNA，开展Affymetrix HG-U133 Plus 2.0基因芯片阵列分析。具体而言，将LNCaP细胞以每皿55万个细胞的密度接种于60 mm培养皿，采用无抗生素培养基培养。次日，按照产品说明书，使用Lipofectamine 2000（Invitrogen，美国加利福尼亚州卡尔斯巴德）将靶向SRF的siGenome SmartPool小干扰RNA（Dharmacon，美国科罗拉多州拉斐特）或靶向荧光素酶的定制对照SmartPool（LUC对照组）转染至细胞中。转染42小时后，分别用5nM R1881或乙醇溶剂处理细胞。每个处理组设置3次生物学重复。转染后48小时，使用Trizol试剂（Invitrogen）收集细胞。提取总RNA后，通过RNeasy柱（Qiagen，美国马里兰州日耳曼敦）进行纯化，并采用Agilent检测（Affymetrix，美国加利福尼亚州圣克拉拉）评估RNA完整性。随后按照产品说明书，在梅奥诊所高级基因组技术微阵列共享资源核心设施完成cDNA合成及人类基因组U133 Plus 2.0基因芯片（Affymetrix）杂交。整体实验设计：采用双因素析因设计，设置3次生物学重复，共包含12个样本，处理组为经R1881或乙醇溶剂处理的LNCaP细胞或SRF沉默细胞。