Figure 6.
HOTAIR regulates DR5 expression via EZH2-mediated modulation of histone H3 lysine 27 trimethylation (H3K27me3) on DR5 gene. A, DZNeP inhibited H3K27me3 in pancreatic cancer cells. PANC-1 cells were exposed to DZNeP (5 μm) for 24 h. H3K27me3 and DR5 expression was determined by Western blot analysis. The expression of β-actin was used as a loading control. Representative blots from three independent experiments are shown. B, HOTAIR knockdown inhibited H3K27me3 on DR5 gene. PANC-1 cells stably expressing scrambled shRNA (shScr) or shRNA for HOTAIR (shHOTAIR) were selected by puromycin. Panel a, Western blot analyses of EZH, DR5, and H3K27me3. Panels b and c, ChIP was performed using mouse anti-trimethyl-histone 3 (lysine 27) antibody or control mouse IgG. H3K27me3-bound DR5 gene was determined by PCR (panel b) and quantitative PCR (panel c). Representative results of three independent experiments are shown. *, p < 0.05. C, HOTAIR overexpression increased H3K27me3 on DR5 gene. MiaPaCa-2 cells stably expressing HOTAIR cDNA or control vector were selected by puromycin. Panel a, Western blot analysis of the expression of EZH2, DR5, and H3K27me3. The expression of β-actin was used as a loading control. Panels b and c, ChIP analysis of H3K27me3 on DR5 gene, determined by PCR (panel b) and quantitative PCR (panel c). Representative results of three independent experiments are shown. *, p < 0.05. D, schematic model of HOTAIR/EZH2-mediated H3K27me3 and DR5 expression in pancreatic cancer cells.