Fig. 3

miR-346 Associates with NORAD to Inhibit its Genome Integrity-, DNA Replication- and DNA Repair-Promoting Activity in Prostate Cancer. A qRT-PCR analysis of NORAD expression in LNCaP cells transfected with miR-346 ± miR-346 inhibitor (both 10 nM) for 72 h. B Correlation between NORAD expression and PUM2 expression in SU2C mCRPC patient data set. C qRT-PCR analysis of NORAD transcript levels in PUM2 and IgG immunoprecipitates from C42 cells transfected ± miR-346 (10 nM) and miR-346 inhibitor (10 nM) for 24 h. D miR-346 binding sites within NORAD identified from PC AGO-PAR-CLIP-seq [51], IntaRNA (http://rna.informatik.uni-freiburg.de/IntaRNA/Input.jsp) and by seed complementarity. E Western blot analysis of PUM2 protein levels in biotin-NORAD 1950–2110 (WT or mutant, as indicated) immunoprecipitates from C42 cells. F Western blot analysis of PUM2 protein levels in C42 cells transfected with miR-346 or negative control miR (20 nM) for 72 h. β-actin was used as a loading control and a representative image is shown. G qRT-PCR analysis of NORAD target gene [29, 30] expression in C42B cells transfected ± miR-346 (20 nM) for 72 h. L19 was used as a normalisation gene. Columns: mean ± SEM for three independent experiments performed in triplicate. H) Proposed mechanism of miR-346 regulation of NORAD:PUM2 DNA damage response axis. J, K Gene ontology (i) and KEGG (ii) pathway analysis of differentially-expressed genes identified by RNA-seq of C42/miR-346 (J) and C42/shNORAD (K) cells (three independent monoclones) ± dox. Top 20 up- and down-regulated transcripts are shown (Jiii, Kiii). miR-346 BS mut = miR-346 binding site-mutant NORAD, PRE mut = Pumilio recognition element-mutant NORAD, NC = negative control NORAD region. * P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.0001. See also Fig. S8, S9, S10 and Tables S2–6