Fig. 6

TSP-2 promotes migration and MMP-2 expression by down-regulating microRNA-376c expression in human PCa cells. a Starved PC-3 and DU145 cells were treated with or without 30 ng/ml of TSP-2 for 24 h, and the expressions of nine miRNAs which targets to MMP-2 3′UTR were measured by real-time PCR in PC-3 (open bar) and DU145 (closed bar) cells. Results were shown as multiple of untreated control. b PC-3 and DU145 cells were transfected with miR-376c mimic, and the MMP-2 protein expression was then determined by immunoblotting assay. c Starved PC-3 and DU145 cells were treated with 10, 30, and 100 ng/ml of TSP-2 for 24 h, and miR376c expression was then assessed by real-time PCR. The PC-3 and DU145 cells were transfected with miR-376c mimic, followed by treatment with TSP-2 for 24 h. MMP-2 mRNA expression (d), as well as the abilities of migration (e) and invasion (f) were then assessed. g PC-3 and DU145 cells were transfected with wild-type or mutant 3′UTR plasmid of MMP-2 containing a miR-376c binding site (upper row). The relative luciferase/renilla activities were then measured following 10, 30, and 100 ng/ml of TSP-2 stimulation. h PC-3 and DU145 cells were co-transfected with miR-376c and plasmids harboring with wild-type or mutant 3′UTR of MMP-2, followed by stimulation of TSP-2 for 24 h. The relative luciferase/renilla activities were then measured. Starved PC-3 and DU145 were pretreated with (i) SSO (500 and 5000 nM), RAD control peptide (200 nM), RGD peptide (200 nM), and BIO1211 (5 and 500 nM), or (j and k) SB203580, U0126, and SP600125 (2 μM) for 1 h, or (l and m) transfected with p38, ERK, and JNK siRNA, followed by incubation of TSP-2 for another 24 h. The miR-376c expression and the relative luciferase/renilla activities were then assessed. Cells without treatments were used as a control (set to 1 or 100), and data were shown as multiples of that. Results are shown as the mean ± SEM (n ≥ 3, *p < 0.05 when compared to untreated control; ^# p < 0.05 when compared to the TSP-2-treated group). UT untreated control, WT wild type, MUT mutant