Figure 3: TIMP3 is the direct target of miR-712.

Panel a shows TIMP3 as a potential target of miR-712 and its link to putative downstream metalloproteinase targets. Panel b shows the seed sequence of miR-712 and complementary 3′-UTR sequence of TIMP3. (c) iMAECs transfected with dual-luciferase reporter plasmids containing wild-type (WT) or mutant TIMP3-3′-UTR were treated with pre-miR-712 or control pre-miR. Firefly luciferase activity (normalized to control Renilla luciferase) indicating TIMP3 expression was determined using Luc-Pair miR Luciferase Assay Kit (n=3 each, data shown as mean±s.e.m.; *P<0.05 as determined by Student’s t-test). (d) TIMP3 expression in iMAECs determined by qPCR was decreased by exposure to OS compared with LS or ST for 24 h (n=6, data shown as mean±s.e.m.; *P<0.05 as determined by Student’s t-test). (e–g) Representative western blots show modulation of TIMP3 expression by shear stress in the presence of pre-miR-712 or anti-miR-712 for 24 h in iMAECs. (e) TIMP3 expression decreased by OS compared with LS for 24 h. (f) Treatment with pre-miR-712 (20 nM) downregulated TIMP3 expression under LS condition. (g) anti-miR-712 (400 nM) treatment rescued OS-induced loss of TIMP3. β-actin was used as an internal loading control. Full sized scans of all western blots are provided in Supplementary Figs S19 and S20.