Figure 5. HYOU1 and HMGB1 are direct targets of miR-193a-3p.

(A) Validation of protein expression of HMGB1 and HYOU1 in control and mimic treated CB ECFC-derived cells using Western blot. The values presented are the mean ± S.E.M of three independent experiments (*p < 0.05; Student’s t-test). Cropped blots were used here and uncropped images of blots are shown in Supplementary Fig. S6. (B) Luciferase constructs were used to test whether miR-193a-3p binds to HMGB1 and HYOU1. The 3′ UTRs of HMGB1 and HYOU1 were subcloned into the CMV-driven pMir-target luciferase vector (Origene). (C) Luciferase activity of plasmid containing firefly luciferase associated with 3′ region of HMGB1 co-transfected separately with miR-193a-3p mimic (10 nM) was reduced. Co-transfection of HMGB1 with a non-targeted miR-126 did not cause a significant decrease in firefly luciferase activity. Data were normalised to the activity of RFP expressed by the HMGB1 plasmid. (D) Luciferase activity of plasmid containing firefly luciferase associated with 3′ region of HYOU1 co-transfected separately with miR-193a-3p mimic (10 nM) was reduced. Co-transfection of HYOU1 with a non-targeted miR-126 did not cause a significant decrease in firefly luciferase activity. Data were normalised to the activity of RFP expressed by the HYOU1 plasmid. The values presented are the mean ± S.E.M of three independent experiments (*p < 0.05; **p < 0.01; one-way ANOVA, Dunnett’s multiple comparison). (Abbreviations: RPP, red fluorescent protein).