Figure 1.

Regulation of UPAR by MIR143

(A) Two putative MIR143-binding sites in the 3′ UTR of the uPAR-PLAUR gene are predicted by TargetScan (http://www.targetscan.org/vert_71/), located at 35–41 bp and 327–333 bp, respectively. (B) Luciferase reporter gene analysis. HEK293T cells were co-transfected with reporter gene constructs (top) and with the MIR143 expression vector or the empty vector (bottom). Firefly luciferase activity was normalized against the activity of Renilla luciferase. pMIR-UPAR-WT, vector with non-modified UPAR 3′ UTR; pMIR-UPAR MUT I, MUT II, and MUT I + II, vector containing the UPAR 3′ UTR with MIR143-binding sites I, II, and I + II, respectively, mutated by site-directed mutagenesis. (C) MIR143-mediated regulation of UPAR protein expression in vitro. PC-3 cells were transfected with synthetic MIR143 mimics or non-targeting control oligonucleotides for the indicated time. The expression of UPAR was analyzed by western blotting. The uPAR protein expression of each sample was normalized to GAPDH as the loading control. Bars represent changes from negative control-transfected cells; right, representative western blots. Data are presented as mean ± SEM; *p < 0.05; **p < 0.03.