Fig. 5

EZH2 coupled with HOTAIR to repress miR-193a expression. (a1-a2) Expression of miR-193a was measured by qRT-PCR in EZH2 knock-down PC3 and DU145 cells (b1-b2) or in PC3 and DU145 cell lines with overexpression of EZH2 by transfection of pcDNA3.1-EZH2. U6 snRNA were used as internal controls. (c1-c4) qRT-PCR assessing the knocking down efficiency of si-HOTAIR and consequent miR-193a expression change in EZH2-overexpressiong PC3 and DU145 cell lines, GAPDH and U6 snRNA were used as an endogenous control, respectively. (d-e) Western blot assays were performed to measure EZH2 and H3K27me3 expression level in PCa cell lines after depletion of EZH2 by LV-shEZH2 or overexpression of EZH2 by pcDNA3.1-EZH2. Total H3 was used as endogenous control. (f1-f2) Enrichment of EZH2 and H3K27me3 was measured by western blot in the HOTAIR knock-down PC3 and DU145 cells. (g) A diagram of miR-193a gene showing the positions of the mature miR-193a, transcriptional start site (TSS), CpG island and the primer flanking region of the ChIP primer. (h1-h2) Promoter luciferase reporter assay indicated that depletion of EZH2 by si-EZH2 elevated the promoter activity of miR-193a gene, while upregulation of EZH2 decreased the promoter activity of miR-193a gene in PC3 and DU145 cells. (i1-i2) ChIP assay showed that knock-down of HOTAIR in PC3 and DU145 cells reduced the enrichment level of EZH2 and H3K27me3 at miR-193a promoter region. (j) A hypothetical model illustrating the coupling of EZH2 and HOTAIR to silence miR-193a in PCa cells. Each bar represents the mean ± SD of three independent experiments. *P < 0.05