Figure 3.
Downregulation of AR by miR-31. A, AR protein level was examined by immunoblot. LNCaP and VCaP cells were transfected with miR-31 or miR-NC (n=3). B, expression of PSA and TMPRSS2 evaluated by qPCR (n = 3). LNCaP cells transfected with siCTL, siAR, miR-NC, miR-31, and miR-31 with AR-CDS for 48 hours, followed by treatment with 1nM R1881 or vehicle (ethanol) for 24 hours. C, schematic graph illustrating predicted locations of three miR-31 MREs within the transcript of AR variant 1. Numbers in parenthesis correspond to the position in the whole transcript (NM_000044). Perfect matches are shown by a line; G:U pairs by a colon (:). D, previously reported mutations are shown in red and the original sequence in bold. Three point mutations, A > G, G > A, and G > T were located within MRE2 and one deletion, ΔG, was located within MRE3. E, luciferase activity of LNCaP cells co-transfected with reporter constructs containing WT, mutant (mt), or empty vector (v) and either miR-31 or miR-NC (n = 3). F, AR expression levels in HEK293 cells co-transfected with AR-CDS WT or mutant containing the G > T mutation in MRE2 and either miR-31 or miR-NC, evaluated by qPCR (n = 3). G, AR expression in PC3AR cells transfected with miR-31, miR-NC, inhibitor negative control (IN-NC), or miR-31 inhibitor (IH-miR-31), evaluated by qPCR and immunoblot (n = 3). **p < 0.01, all bar graphs are shown with mean ± SEM.