miR-206 targets TIMP-3.

Lentivirus-mediated miR-206 overexpression in hypoxic CFs inhibited TIMP-3 mRNA (A) and protein (B) levels. TIMP-3 mRNA and protein were analyzed 3 and 24 hr after infection, respectively. Average results of densitometric analyses of western blot are also shown. (C) Representative western blot shows that the effect of HMGB1 to decrease TIMP-3 protein in hypoxic CFs was rescued by anti-miR-206. Cells were transfected with anti-miR206 and exposed to hypoxia for 24 hr either in the presence or in the absence of 100 ng/ml HMGB1. For western blot analysis (B,C), the same filter was probed with α-tubulin to normalize protein loading. (D) Upper panel: Conservation of miR-206 seed match sequences (in red and blue) in mammalian TIMP-3. Lower panel: Diagram of plasmid construction; the TIMP-3-3′UTR containing the 1683-1689 seed sequence (1651–1707) or the respective mutated segment were cloned downstream of the luciferase encoding sequence. (E) HEK293 cells were transfected with vector alone (pLUC) or firefly luciferase constructs that contain either the intact (pLUC-1683–1689) or the mutated (pLUC-M) miR-206 binging site. Each plasmid was cotransfected with a plasmid encoding Renilla luciferase along with miR-206 or scramble sequence. Firefly luciferase values were normalized to Renilla luciferase activity and the ratio of luciferase activity of each construct in the presence and in the absence of miR206 was calculated. Luciferase activity decreased upon pLUC-1683–1689 and miR-206 transfection whereas it was not modulated by cotransfection of pLUC-M and miR-206 (n = 6/group). These results indicate that miR-206 binds the 1683–1689 seed sequence in TIMP-3-3′UTR.