Fig. 6

MicroRNA-96 directly targets and regulates RARG in prostate cells. a Cross-correlation matrices depicting the relationships between RARG and miR-96 cluster member expression in PCa samples from MSKCC and TCGA-PRAD cohorts (corrplot). b Relative expression of miR-96 (bottom) and RARG (top) across 10 prostate cell lines representing different stages of PCa progression. The cell models examined comprised immortalized RWPE-1 and HPr1-AR non-malignant prostate epithelial cells, LNCaP, LAPC4 and EAA006 androgen-sensitive PCa cells, MDAPCa2b, 22Rv1 and LNCaP-C42 CRPC cells, as well as PC3 and DU145 cells derived from distant metastases. c Correlation analyses of RARG with miR-96 expression over the course of palpable tumor (PT) development in TRAMP. The strength and significance of the correlation is indicated. Tumors from 5 mice were examined at each time point and compared to the mean of 10 6-week-old wild-type mice; 2 6-week tumors were dropped due to technical failure to generate high-quality RNA. d RARG mRNA (left) and RARγ protein expression (right) in RWPE-1 cells after 48 h of transfections with miR-96 mimics or siRNA targeting RARG. Significance of difference between targeting and control cells are noted (*p < 0.05). e Luciferase assay assessing direct targeting of miR-96 to the full-length (FL) RARG 3′UTR or individual predicted target sites (t1, t2) within the RARG 3′UTR. Either miR-96 or miR-CTL mimics (30 nM) were transfected into RWPE-1 cells for 48 h along with indicated RSV-pGL3 constructs and pRL-CMV Renilla luciferase expressing vectors, and luciferase activity measured by Dual-Glo Luciferase Assay System in triplicate. Significance of difference between luciferase construct with and without RARG 3′UTR sequences cells are noted (*p < 0.05). f RWPE-1 cells were pretreated with miR-CTL, miR-96 mimic (30 nM), or combination of miR-96 mimic and antagomiR-96 for 48 h prior to CD437 exposure (10 nM) for 24 h, and candidate transcripts measured by RT-qPCR in triplicate. Induction relative to untreated control for each condition is shown, and significance of CD437 induction/repression between miR-CTL and miR-96 groups are indicated (*p < 0.05). g Cell viability of RWPE-1 (left) and LNCaP (right) cells for up to 120 h post transfection with either miR-96 or non-targeting control (NC) mimics was measured in triplicate. Significance of differences between viability of indicated experimental groups at the end of the study is noted