Fig 1: DHA inhibits proteins involved in the polycomb repressive complex.a Immunoblot analysis of protein extracts obtained from DU145 and PC-3 treated with DHA (5 μM) using anti-phospho EZH2, and anti-JARID2, anti-EZH2 and anti-GAPDH. b DHA Treatment of PCa cells inhibits JARID2-EZH2 interaction. DU145 cells were treated with 5 µM of DHA and DMSO (0.05%) and protein extracts were immunoprecipitated using anti-JARID2 or anti-EZH2 antibodies. The interaction between the proteins was detected by anti-EZH2 (right) or anti-JARID2 antibodies. c Inhibition of JARID2 protein regulates miR-34a and miR-7 expression and Axl expression. RT-PCR analysis of miR-34a, miR-7, and Axl expression levels of in DU145 cells lacking JARID2 expression. DU145 were transfected with JARID2 siRNA or GFP siRNA duplexes (50 nM). Total RNA was collected and was analyzed 24 h post transfection. ΔCt values graphed are relative to the endogenous control RNU6B small RNA (for miR-34a and miR-7) and GAPDH for Axl. Data are representative of three independent experiments and all values shown are mean ± SD from a representative experiment; *p < 0.05, two-tailed Student’s t-test. d ChIP-qPCR analysis demonstrating the effect of DHA treatment on DU145 and C4-2 cells on H3K27me3 at specific gene loci. PCa cell lines were treated with 5 uM of DHA or DMSO and subjected to ChIP analysis using antibodies against H3K27me3 or control IgG. The signals of H3K27me3 at the indicated genomic locations were normalized to that obtained from IgG ChIP at the same location to calculate their enriched signal. e Schematic representation of DHA inhibition of Axl expression via regulation of microRNAs and proteins of the polycomb repressive complex 2
Fig 2: DHA regulates the expression of miRNAs in PCa cell lines.Cells were treated with DHA (5 μM) or DMSO (0.05%) for 24 h. a Heat map of miRNA microarray expression data. Cluster analysis classified the samples into groups based on miRNA expression levels in each sample. Red indicates high expression and green indicates relatively low expression of miRNA. b Schematic representation of the location of the putative miR-7 target site is shown in the Axl 3′-UTR. c RT-PCR analysis of miR-34 expression levels (left) and miR-7 (right) expression levels in PCa cell lines. ΔCt values graphed are relative to the endogenous control RNU6B small RNA and data were normalized to normal cell PNT1A. Data are representative of three independent experiments and all values shown are mean ± SEM from a representative experiment; *p < 0.05, two-tailed Student’s t-test. d RT-PCR analysis of expression levels of miR-34a, miR-7, and Axl in human PCa samples. Total RNA was collected from human tissue consisting of paired normal and tumor samples and was analyzed for miR-7, miR-34a, and Axl expression levels. ΔCt values graphed are relative to the endogenous control RNU6B small RNA (for miR-34a and miR-7) and GAPDH for Axl. Data are shown as the triplicate independent experiments; *p < 0.05, two-tailed, nonparametric Mann–Whitney test. e RT-PCR analysis of expression levels of miR-34a (right), miR-7 (left) and Axl (bottom) in tumor extracted from mice treated with 40 mg/kg of DHA or DMSO (2 mL/kg). Total RNA was collected from tumor and analyzed for miR-7 and miR-34a expression levels. ΔCt values graphed are relative to the endogenous control RNU6B small RNA. Data are representative of three independent experiments and all values shown are mean ± SD from a representative experiment; *p < 0.05, two-tailed, nonparametric Mann–Whitney test
Fig 3: Expression of miR-7 and miR-34a inhibits Axl protein expression as well as proliferation and migration of in PCa cells.a Western blot analysis of Axl expression in DU145 and PC-3 cells transfected with miR-7 or miR-34a expression vectors and a vector control. b Proliferation assay of DU145 and PC-3 cells transfected with miR-7 or miR-34a expression vectors. Proliferation was measured 48 h post transfection. Data are representative of three independent experiments and all values shown are mean ± SD from a representative experiment; *p < 0.05, two-tailed Student’s t-test. c Migration assay of DU145 and PC-3 cells transfected with miR-7 or miR-34a expression vectors. Migration was measured 24 h post transfection. Migrating cells were fixed and stained, and three to five random microscopic fields were counted. Data are representative of three independent experiments and all values shown are mean ± SD from a representative experiment; *p < 0.05, two-tailed Student’s t-test
Supplier Page from OriGene Technologies for MIR34a Human MicroRNA Expression Plasmid (MI0000268)