LincRNA PVT1 associates with EZH2 and with the androgen receptor in LNCaP prostate cancer cells and inhibits the expression of genes involved with cell death, cell differentiation and cell adhesion

Long intergenic non-coding RNA (lincRNA) PVT1 is an oncogene known to be overexpressed in various types of cancer. PVT1 high expression is associated with increased prostate cancer (PCa) risk while androgen-independent PCa progression is correlated with increased androgen receptor (AR) expression. However, the mechanism of PVT1 and AR involvement in the development of prostate cancer is still unclear. Here, we tested the hypothesis that PVT1 participates along with AR and the methyltransferase EZH2 from the Polycomb repressive complex 2 in the repression of gene expression in LNCaP prostate cancer cells. Native RNA-binding proteins immunoprecipitation followed by quantitative PCR of co-precipitated RNAs (RIP-qPCR) revealed that in LNCaP, PVT1 lincRNA is associated both with AR (10 – 12 % of PVT1 input) and EZH2 (36 – 42 % of input) in the presence or absence of androgen. PVT1 knockdown in LNCaP in the presence of androgen increased the expression of 160 genes whose expression was repressed by androgen, including genes involved in regulation of cell differentiation, in inhibition of cell migration/invasion and in triggering apoptosis. Analysis by chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) of the histone marks occupancy at the promoter region of the tumor suppressor gene NOV, one of the genes that had an increased expression upon PVT1 silencing, showed a significant epigenetic remodeling at its promoter and enhancer regions upon PVT1 knockdown. We provide evidence for a genome-wide transcriptional repressive role of PVT1 lincRNA on tumor suppressor genes in prostate cancer cells. Two-color hybridization design. Aliquots of total RNA isolated from LNCaP cells transfected with siCTRL or siPVT1 and treated with androgen R1881 or vehicle controls were labeled with Cy3 or Cy5 and hybridized to Agilent DNA SurePrint G3 Human Gene Expression 8x60k v3 Oligoarray (design ID # 039494) . Biological replicates: 4. No technical replicates.