Fig. 6.

MiR-346, -361-3p and 197-3p repress known tumour suppressors and upregulate the oncogene, YWHAZ in prostate cancer (PC). a Luciferase 3′UTR reporter assay analysis of C42 cells transfected with pLightSwitch (i), pLightSwitch-random-3′UTR (ii), pLightSwitch-ARHGDIA-3′UTR (iii), pLightSwitch-TAGLN2-3′UTR (iv) or pLightSwitch-YWHAZ-3′UTR (v) ± miR-346, -361-3p or -197-3p mimics and/or inhibitors (20 nM) for 48 h. b Luciferase 3′UTR reporter assay analysis of C42 cells transfected with wild-type or miR binding site-mutant pLightSwitch-ARHGDIA-3′UTR (i), pLightSwitch-TAGLN2-3′UTR (ii) or pLightSwitch-YWHAZ-3′UTR (iii) ± miR-346, -361-3p or -197-3p mimic, as appropriate, for 48 h. a, b Luciferase activity was normalised to β-galactosidase activity to correct for transfection efficiency, and data are displayed relative to negative control miR-transfected cells. Columns: mean relative luminescence from three independent experiments performed in duplicate ± SEM. c Western blot analysis of TAGLN2, ARHGDIA and YWHAZ protein levels in C42 cells transfected with miR-346, -361-3p or -197 mimic (20 nM) for 96 h. β-actin was used as a control for loading and replicate blots are shown (Fig. S10c). d AGO2/biotin-miR RNA-IP analysis of miR-197 and miR-346 association with (i) ARHGDIA, (ii) TAGLN2 and (iii) YWHAZ 3′UTRs. 22RV1 cells were transfected with biotin-labelled miR (200 pmol) for 24 h, followed by two-step immunoprecipitation with AGO2 antibody- and streptavidin-coated beads. RNA was extracted from input and IP samples and qRT-PCR performed for ARHGDIA, TAGLN2 and YWHAZ. Data are presented relative to input values. Columns: mean pulldown relative to input from three independent experiments ± SEM. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0001. See also Figs. S11, S11 and S13, and Supplementary Tables 3, 4, 5 and 6