AR-V7 Targets in Castration-Resistant Prostate Cancer (CRPC) Cell Line

In order to define the genes responsible for the growth and survival of a human castration-resistant prostate cancer cell line, a short term (doxycycline inducible) knockdown system was developed and utilized. Three independent 22Rv1 cell isolates were derived for each of the following doxycycline-inducible shRNAs (shGFP, shAR3, and shVav3) (AR3 = AR-V7). The cells were grown in androgen depleted conditions, plus or minus doxycycline, for three days. RNA from the 18 samples was then sent to the University of Miami Genetics Core for RNA Integrity Number (RIN) evaluation and microarray analysis. Genes differentially regulated by AR-V7 knock-down or VAV3 knock-down were explored as downstream targets of AR-V7 or VAV3, respectively. Stable 22Rv1 cell lines were established for each doxycycline-inducible shRNA (tet-pKLO.1-shGFP, tet-pKLO.1-shAR3, and tet-pKLO.1-VAV3). Cells were grown in androgen depleted conditions (10% CSS) plus or minus doxycycline for three days, three replicates per condition. Knockdown was confirmed by parallel protein harvest. RNA was isolated and analyzed on Affymetrix Human Gene 1.0 ST Arrays v1. Arrays were analyzed using R (3.3.1, https://cran.r-project.org/), the Bioconductor suite (https://www.bioconductor.org/), and the oligo package(1.36.1). Arrays were rma normalized. Limma (3.28.21) was used to calaculate differential gene expression upon doxycycline induced knock-down.