Fig. 4: 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