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.