Involvement of SIRT1-regulated transcription profiles in prostate cancer. Involvement of SIRT1-regulated transcription profiles in prostate cancer

In order to understand the mechanism associated with SIRT1-mediated development of prostate cancer progression, we conducted RNA-sequencing analysis in SIRT1 suppressed hormone sensitive prostate cancer cells (LNCaP) under conditions of androgen sufficiency, deprivation, androgen stimulation or AR suppression. Overall design: Hormone-sensitive LNCaP cells transduced with MOI of 10 lentiviral particles carrying shRNA against non-targeted control (NTC) or SIRT1 (shSIRT1 or 10) were selected with 1 μg/ml puromycin. Stable knockdown (KD) clones were cultured and maintained in culture medium in the presence of 0.5 μg/ml puromycin. To identify SIRT1 target genes in LNCaP cells, LNCaP-NTC and SIRT1 KD (shSIRT1) cells were cultured in androgen supplemented conditions (FBS-supplemented medium). To identify SIRT1 target genes involved in cancer progression, LNCaP-NTC and SIRT1 KD (10) cells were subjected to androgen deprived condition (charcol-stripped serum (CSS)-supplemented medium for 24 hours following 48 hours in FBS-supplemented medium. Cells were then cultured in CSS-supplemented medium with vehicle control (VC), 1 nM Dihydrotestosterone stimulation (D) in the absence or presence of 10 µM Enzalutamide treatment (DE) for 24 hours. Total RNA prepared from cells follwoing treatment was used in RNA-Seq analysis using Illumina Illumina HiSeq 3000Sequencing System.

为阐明沉默信息调节因子1（SIRT1）介导的前列腺癌进展相关分子机制，我们在雄激素充足、雄激素剥夺、雄激素刺激或雄激素受体（Androgen Receptor, AR）抑制条件下，对SIRT1敲低的激素敏感性前列腺癌细胞（LNCaP）开展了RNA测序分析。
整体实验设计如下：将感染复数（Multiplicity of Infection, MOI）为10的靶向非靶向对照（NTC）或SIRT1的短发夹RNA（short hairpin RNA, shRNA）慢病毒颗粒（分别为shSIRT1与sh10）感染激素敏感性LNCaP细胞，随后以1 μg/ml嘌呤霉素进行筛选。将获得的稳定敲低（KD）细胞克隆置于含0.5 μg/ml嘌呤霉素的培养基中培养传代。
为鉴定LNCaP细胞中SIRT1的靶基因，我们将LNCaP-NTC与SIRT1 KD（shSIRT1）细胞培养于添加胎牛血清（Fetal Bovine Serum, FBS）的雄激素充足培养基中。
为探究参与癌症进展的SIRT1靶基因，我们将LNCaP-NTC与SIRT1 KD（sh10）细胞先在FBS培养基中培养48小时，随后转移至活性炭吸附血清（charcoal-stripped serum, CSS）培养基中培养24小时以构建雄激素剥夺模型。此后，将细胞分别置于添加载体对照（VC）、1 nM二氢睾酮（Dihydrotestosterone, DHT）刺激的CSS培养基中培养24小时；同时设置10 µM恩扎卢胺（Enzalutamide）共处理组（DE）。
提取各组细胞的总RNA，采用Illumina HiSeq 3000测序系统进行RNA测序分析。